Optical sources for fluorescent lifetime analysis

This application relates to devices and methods for generating short and ultrashort optical pulses for time domain applications, including fluorescence lifetime and time-of-flight applications.

Ultrashort optical pulses (i.e., optical pulses less than about 100 picoseconds) are useful in a variety of research and development fields and commercial applications, including time-domain analysis. For example, ultrashort optical pulses can be useful for time-domain spectroscopy, optical ranging, time-domain imaging (TDI), and lifetime-resolved fluorescence detection for optical coherence tomography (OCT). Ultrashort pulses can also be useful in optical communication systems, medical applications, and commercial applications, including testing of optoelectronic devices and materials.

<p>Conventional mode-locked lasers have been developed to generate ultrashort optical pulses, and a variety of such lasers are currently commercially available. For example, several solid-state and fiber lasers have been developed to deliver pulses having durations well below 200 femtoseconds. However, for some applications, these pulse durations may be shorter than required to obtain useful results, and the cost of these lasing systems may be prohibitively high. In addition, these lasing systems have a fairly large footprint (e.g., 0.0929 m²(1ft²) or more) and significant weight, and may be stand-alone systems that may not be easily portable. Such lasing systems and their driving electronics may be difficult to incorporate into an instrument as a replaceable module, or even impossible to incorporate into a handheld device. As a result, ultrashort pulse lasers are often manufactured as separate stand-alone instruments from which the output beam can be coupled into another instrument for a specific application.</p>

<p>The technology described herein relates to an apparatus and method for generating short and ultrashort optical pulses using a laser diode (LD) or a light emitting diode (LED). A short pulse is a pulse with a full width at half maximum (FWHM) temporal profile between about 100 picoseconds and about 10 nanoseconds. An ultrashort pulse is a pulse with a FWHM temporal profile less than about 100 picoseconds. Gain switching techniques and associated circuitry are described that can be implemented in compact, low-cost laser systems to generate pulses with durations less than about 2 nanoseconds in some embodiments, and in some cases less than about 100 picoseconds. The inventors have recognized and come to appreciate that compact, low-cost pulsed laser systems can be incorporated into instruments (e.g., fluorescence lifetime imaging devices, bioanalytical instruments utilizing lifetime resolved fluorescence detection, time-of-flight instruments, optical coherence tomography instruments) that can enable such instruments to be easily portable and manufactured at a much lower cost than is possible for such systems using conventional ultrashort pulsed laser systems. High portability can make such devices more useful for research, development, clinical, commercial, and home applications.</p><p>Some embodiments relate to a pulsed light source comprising a semiconductor diode configured to emit light and a driver circuit including a transistor coupled to terminals of the semiconductor diode, the driver circuit configured to receive a unipolar pulse and to apply a bipolar electrical pulse to the semiconductor diode in response to receiving the unipolar pulse.</p><p>Some embodiments relate to a method of generating an optical pulse, the method including receiving at least one clock signal, generating an electrical pulse from the at least one clock signal, driving a gate terminal of a transistor with the electrical pulse, a current carrying terminal of the transistor being connected to a semiconductor diode configured to emit light, and applying a bipolar current pulse to the semiconductor diode to generate an optical pulse in response to activation of the transistor by the electrical pulse.</p><p>Some embodiments relate to a fluorescence lifetime analysis system comprising a semiconductor diode configured to emit light, a drive circuit configured to apply a bipolar current pulse to the semiconductor diode to generate a light pulse, an optical system configured to deliver the light pulse to a sample, and a photodetector configured to discriminate photon arrival times into at least two time bins during a single charge accumulation interval of the photodetector.</p><p>Some embodiments relate to a pulsed light source comprising a semiconductor diode configured to emit light, a first logic gate configured to form a first pulse at an output of the first logic gate, and a driver circuit coupled to the first logic gate, the driver circuit configured to receive the first pulse and apply a bipolar electrical pulse to the semiconductor diode to generate a light pulse in response to receiving the first pulse.</p><p>Some embodiments relate to a pulsed light source comprising a semiconductor diode configured to emit light and a driver circuit including a transistor coupled to a terminal of the semiconductor diode, the driver circuit configured to receive a unipolar pulse and to apply a bipolar electrical pulse to the semiconductor diode in response to receiving the unipolar pulse, the transistor being connected in parallel with the semiconductor diode between a current source and a reference potential.</p><p>Some embodiments relate to a pulsed light source comprising a semiconductor diode configured to emit light and a plurality of first circuit branches connected to a first terminal of the semiconductor diode, each circuit branch comprising a transistor having a current-carrying terminal connected between a reference potential and the first terminal of the semiconductor diode.</p><p>Some embodiments relate to a pulsed light source comprising a radio frequency amplifier providing a signal and an inverted signal, a logic gate configured to receive the signal and the phase-shifted inverted signal and output a pulse and an inverted pulse, a combiner configured to combine the pulse and the inverted pulse onto a common output, and a semiconductor diode coupled to the common output and configured to generate an optical pulse in response to receiving the pulse and the inverted pulse.</p><p>Some embodiments relate to a pulsed light source that includes a radio frequency logic gate configured to receive a first signal and an inverted version of the first signal and to output a pulse and an inverted version of the pulse, and a semiconductor diode connected to the radio frequency logic gate, the semiconductor diode configured to receive the pulse at a first terminal of the semiconductor diode and the inverted version of the pulse at a second terminal of the semiconductor diode and to emit a light pulse.</p><p>The above and other aspects, implementations, operations, features, functions, features and embodiments of the present teachings can be more fully understood from the following description taken in conjunction with the accompanying drawings.</p><p>Those skilled in the art will appreciate that the figures described herein are for illustrative purposes only. It should be understood that in some instances, various aspects of the present invention may be shown exaggerated or enlarged to facilitate understanding of the present invention. In the drawings, like reference characters generally refer to like features, functionally similar and/or structurally similar elements throughout the various views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the present teachings. The drawings are not intended to limit the scope of the present teachings in any way.</p>

<figref num="1-1">FIG. 1 illustrates a pulse lasing system incorporated into an analytical instrument according to some embodiments.</figref><figref num="1-2">1 illustrates a train of ultrashort optical pulses in accordance with some embodiments.</figref><figref num="2-1A">1 illustrates an optical pump and output pulse for gain switching according to some embodiments.</figref><figref num="2-1B">FIG. 1 illustrates relaxation oscillations, according to some embodiments.</figref><figref num="2-1C">4A-4C are diagrams illustrating optical output pulses exhibiting tails according to some embodiments.</figref><figref num="2-2A">FIG. 1 illustrates a pulsed semiconductor laser diode according to some embodiments.</figref><figref num="2-2B">1 is a schematic diagram of a pulser circuit for pulsing a laser diode or light emitting diode, according to one embodiment.</figref><figref num="2-2C">13A-13C illustrate improvements in current delivered to a laser diode according to some embodiments.</figref><figref num="2-3">4A-4C illustrate current drive waveforms for gain switching a laser diode in accordance with some embodiments.</figref><figref num="2-4A">FIG. 2 illustrates a pulser circuit for driving a laser diode or a light emitting diode, according to some embodiments.</figref><figref num="2-4B">1 is a schematic diagram of a pulser circuit for driving a laser diode or a light emitting diode, according to some embodiments.</figref><figref num="2-4C">1 is a schematic diagram of a pulser circuit for driving a laser diode or a light emitting diode, according to some embodiments.</figref><figref num="2-4D">FIG. 2 illustrates an RF driver for pulsing a laser diode or a light emitting diode, according to some embodiments.</figref><figref num="2-4E">2-4D show drive waveforms generated by the circuits of FIGS. 2-4D according to some embodiments.</figref><figref num="2-4F">FIG. 2 illustrates an RF driver for pulsing a laser diode or a light emitting diode, according to some embodiments.</figref><figref num="2-4G">2-4F show drive waveforms generated by the circuits of FIGS. 2-4F in accordance with some embodiments.</figref><figref num="2-4H">1 is a schematic diagram of a pulser circuit for driving a laser diode or a light emitting diode, according to some embodiments.</figref><figref num="2-4I">4 illustrates the efficiency of power coupling into a laser diode in accordance with some embodiments.</figref><figref num="2-4J">FIG. 2 illustrates a pulser and driver circuit for pulsing optical emission from a laser diode or light emitting diode, according to some embodiments.</figref><figref num="2-4K">FIG. 2 illustrates a pulser circuit for generating a train of pulses, according to some embodiments.</figref><figref num="2-4L">FIG. 2 illustrates data inputs to logic gates in a pulser circuit according to some embodiments.</figref><figref num="2-4M">FIG. 2 illustrates a driver circuit for driving a laser diode or a light emitting diode with electrical pulses according to some embodiments.</figref><figref num="2-5A">FIG. 2 illustrates a pulser circuit for gain switching a laser diode according to some embodiments.</figref><figref num="2-5B">4A-4C are diagrams illustrating drive voltages for a pulser circuit according to some embodiments.</figref><figref num="2-5C">1 illustrates an example measurement of ultrafast optical pulses generated from a gain-switched laser diode, according to some embodiments.</figref><figref num="2-5D">1 illustrates an example measurement of ultrafast optical pulses generated from a gain-switched laser diode, according to some embodiments.</figref><figref num="2-6A">1 illustrates a slab-coupled optical waveguide semiconductor laser that can be gain-switched or Q-switched, according to some embodiments.</figref><figref num="2-6B">4 illustrates an optical mode profile in a slab-coupled optical waveguide laser according to some embodiments.</figref><figref num="2-6C">1 illustrates an integrated gain-switched semiconductor laser and associated saturable absorber in accordance with some embodiments.</figref><figref num="3-1">FIG. 1 illustrates a system for synchronizing timing of light pulses to instrument electronics, according to some embodiments.</figref><figref num="3-2">FIG. 1 illustrates a system for synchronizing timing of light pulses to instrument electronics, according to some embodiments.</figref><figref num="3-3">FIG. 1 illustrates a system for synchronizing the timing of light pulses from two pulse sources to an instrument electronics device, according to some embodiments.</figref><figref num="3-4A">FIG. 1 illustrates a system for synchronizing interleaved timing of light pulses from two pulse sources to instrument electronics, according to some embodiments.</figref><figref num="3-4B">FIG. 1 illustrates interleaved and synchronized pulse trains from two pulsed light sources in accordance with some embodiments.</figref><figref num="4-1">FIG. 1 illustrates an apparatus for analyzing the fluorescence lifetime of a sample according to some embodiments.</figref><figref num="4-2">FIG. 1 shows the emission probability of fluorescent molecules with different emission lifetimes.</figref><figref num="4-3">FIG. 1 shows time-binned detection of fluorescence emission from fluorescent molecules.</figref><figref num="4-4">1 illustrates a time-binning photodetector according to some embodiments.</figref><figref num="4-5A">FIG. 1 illustrates multiple excitation pulses and subsequent fluorescence emissions and corresponding binned signals according to some embodiments.</figref><figref num="4-5B">FIG. 13 shows a histogram generated from the binned signal for a particular fluorophore, according to some embodiments.</figref>

The features and advantages of the present invention will become more apparent from the detailed description set forth below when taken together with the drawings. When describing the embodiments with reference to the drawings, directional references (such as "above," "below," "top," "bottom," "left," "right," "horizontal," "vertical," etc.) may be used. Such references are intended only to assist the reader in viewing the drawings in a normal orientation. These directional references are not intended to describe a preferred or only orientation of an embodied device. The device may be embodied in other orientations. I. Introduction The inventors have recognized and come to appreciate that conventional ultrashort pulse light sources having pulse repetition rates below 1 GHz are generally large, expensive, and not suitable for many mobile applications. For example, conventional ultrashort pulse lasers cannot be integrated into compact portable devices. The inventors have come to realize and appreciate that small short-pulse or ultrashort-pulse light sources can enable new and useful devices for a wide range of time-domain applications. Such applications include, but are not limited to, time-of-flight imaging, ranging, fluorescence and fluorescence lifetime analysis, biological or chemical analysis, optical coherence tomography (OCT), and medical point-of-care (POC) instrumentation. In some cases, the POC instrumentation can include devices for detecting fluorescent emissions from biological samples and analyzing the fluorescent emissions to determine properties of the biological samples. Pulsed light sources can be used to excite the fluorescent emissions in such instrumentation. The inventors have conceived compact short-pulse and ultrashort-pulse light sources and systems according to some embodiments that can generate optical pulses at various wavelengths with pulse durations below about 2 nanoseconds and even below 100 picoseconds.

In overview, FIG. 1-1 shows a pulsed light source 1-110 that can be incorporated into an analytical instrument 1-100, such as a POC or OCT instrument or a time-of-flight imaging instrument that excites and detects fluorescent emissions. The instrument can include an optical system 1-140 and an analytical system 1-160. The optical system 1-140 can include one or more optical components (e.g., lenses, mirrors, optical fibers, optical filters, attenuators) and can be configured to operate on the light pulses from the light source 1-110 and/or deliver the light pulses to the analytical system 1-160. The analytical system can include one or more components (e.g., lenses, mirrors, optical filters, attenuators, photodetectors) configured to receive an optical signal (e.g., fluorescent emissions, backscattered radiation) from a sample 1-170 to be analyzed and generate an electrical signal representative of the received optical signal. In some embodiments, the analytical system 1-160 can further include electronics configured to process the electrical signal.

According to some embodiments, the pulsed light source 1-110 can include at least one gain-switched laser diode (LD). In some embodiments, the pulsed light source 1-110 can include at least one light-emitting diode (LED) driven by short current pulses. A pulser circuit 1-112 that generates nanosecond-scale or shorter current pulses can be included in the analytical instrument 1-100 to drive the light source 1-110.

When configured as a laser diode, the pulsed light source 1-110 may comprise a gain medium 1-105 (e.g., any suitable semiconductor junction that may or may not include multiple quantum wells) and at least two cavity mirrors 1-102, 1-104 (or reflective facets of a laser diode) that define the ends of an optical laser cavity. In some embodiments, there may be one or more additional optical elements in the laser cavity for beam shaping, polarization control, wavelength selection, and/or pulse shaping. Collection optics may be included in the laser diode and configured to focus the emission from the laser diode into a beam. The beam from the laser diode may or may not be collimated by the collection optics. When the laser operates in a gain-switching mode, an optical pulse may be built up in the laser cavity between the cavity end mirrors 1-102, 1-104 in response to application of a current pulse through the diode junction of the laser. One of the cavity mirrors 1-104 (often referred to as the output coupler) may partially transmit a portion of the pulse, causing an optical pulse 1-122 to be emitted from the pulsed laser 1-110. When a current drive pulse is repeatedly applied to the laser diode, a train of pulses 1-122 (only one shown) may be emitted in rapid succession from the laser cavity. This train of pulses may be referred to as a laser beam, which may be characterized by a beam waist w. The laser beam may be collimated (indicated by the parallel dashed lines), partially collimated, or uncollimated. The beam waist is the lateral dimension of the emitted laser beam (e.g., ±1/e of the lateral intensity profile of a Gaussian beam).²value or other transverse intensity beam/profile full width at half maximum (FWHM) value) and may vary with distance from the output coupler. The collimation and waist of the beam may depend on the geometry and optical properties of the laser cavity and whether any optical elements (e.g., collimating lenses) are included in the laser cavity.

When configured as a light emitting diode, the pulsed light source 1-110 may comprise any suitable semiconductor junction configured to emit incoherent or partially coherent light. Collection optics may be included with the LED and configured to focus the light emission from the LED into an output beam. The beam from the LED may or may not be collimated by collection optics. In operation, the LED generates a light pulse of primarily spontaneously emitted photons in response to application of a current pulse across the LED junction, although some stimulated emission may be present in the output as amplified spontaneous emission. Typically, the spectral bandwidth emitted from an LD may be less than 2 nanometers, while the spectral band emitted from an LED is on the order of tens of nanometers.

The specific wavelength emitted from an LD or LED can be selected by the choice of semiconductor material and/or impurities added to the semiconductor material. For longer wavelengths in the red and infrared regions of the spectrum, indium phosphide based semiconductors and their alloys can be used. For shorter wavelengths to the yellow region of the spectrum, gallium arsenide phosphide based semiconductors and their alloys can be used. For the green and blue regions of the spectrum, aluminum gallium phosphide or gallium nitride and their alloys can be used.

According to some embodiments, a particular semiconductor material can be selected for a pulsed light source 1-110 of an instrument for exciting and detecting fluorescent emission (e.g., a POC fluorescence lifetime imaging instrument) to generate pulses having one or more of the following characteristic wavelengths: 270 nm, 280 nm, 325 nm, 340 nm, 370 nm, 380 nm, 400 nm, 405 nm, 410 nm, 450 nm, 465 nm, 470 nm, 490 nm, 515 nm, 640 nm, 665 nm, 808 nm, and 980 nm. In some embodiments, a semiconductor can be selected for a pulsed light source 1-110 of an instrument for generating pulses having a wavelength range or spectral distribution that falls within one of the following wavelength ranges: about 270 nm to about 370 nm, about 340 nm to about 400 nm, about 380 nm to about 490 nm, and about 410 nm to about 470 nm.

For reference, the phrase "characteristic wavelength" or "wavelength" may refer to a central or dominant wavelength within a limited emission bandwidth. In some cases, it may refer to a peak wavelength within the emission bandwidth. The phrase "characteristic energy" or "energy" may refer to the energy associated with a characteristic wavelength. The term "optical" may refer to ultraviolet, visible, near infrared, and short wavelength infrared spectral bands.

In some embodiments, the optical system 1-140 can operate on the beam of pulses 1-122 emitted from the pulsed light source 1-110. For example, the optical system may include one or more lenses to reshape the beam and/or change the divergence of the beam. Reshaping the beam may include increasing or decreasing the value of the beam waist and/or changing the cross-sectional shape of the beam (e.g., from elliptical to circular, from circular to elliptical, etc.). Changing the divergence of the beam may include increasing or decreasing the divergence of the beam. In some embodiments, the optical system 1-140 can include an attenuator or optical amplifier to change the amount of beam energy. In some cases, the optical system can include wavelength filtering elements. In some embodiments, the optical system can include pulse shaping elements, such as a pulse stretcher and/or a pulse compressor. In some embodiments, the optical system can include one or more nonlinear optical elements, such as a saturable absorber to reduce the pulse length or a nonlinear crystal to convert the pulse wavelength to a shorter wavelength via frequency doubling or to a longer wavelength via parametric amplification. According to some embodiments, the optical system 1-140 can include one or more elements that modify, select, and/or control the polarization of the pulses from the light source 1-110.

Although the pulsed light source 1-110 and the optical system 1-140 are shown in FIG. 1-1 as separate elements of the analysis system 1-160, the pulsed light source and the optical system may be fabricated as a compact, replaceable module that can be housed within the analysis system 1-160, according to some embodiments. In some embodiments, the pulser circuit 1-112 and the pulsed light source 1-110 may be integrated on the same board (e.g., the same printed circuit board) or the same substrate (e.g., the same semiconductor substrate).

In various embodiments, the pulses 1-122 emitted from the pulsed light source may have a temporal intensity profile as shown in FIG. 1-2. In some embodiments, the peak intensity values of the emitted pulses may be approximately equal and the profile may have a Gaussian temporal profile, but the second peak intensity profile may be approximately equal.²Other profiles, such as the profile, may be possible. In some cases, the pulses may not have a symmetrical temporal profile and may have other temporal shapes. In some embodiments, the gain and/or loss dynamics in the light source 1-110 may result in pulses with an asymmetrical profile, as described below in connection with FIG. 2-1C. The duration of each pulse may be characterized by a full width at half maximum (FWHM) value, as shown in FIG. 1-2. Ultrashort optical pulses may have FWHM values of less than 100 picoseconds. Short optical pulses may have FWHM values of less than about 10 nanoseconds.

The pulses emitted from the light source 1-110 can be spaced apart in time by a periodic interval T, sometimes referred to as the pulse separation interval. In some embodiments, T can be determined by the active gain and/or loss modulation rate in the laser. For example, the repetition rate at which a laser diode is gain switched or the current applied to a junction of a light emitting diode can determine the pulse separation interval T. According to some embodiments, the pulse separation interval T can be between about 1 ns and about 100 ns. In some implementations, the pulse separation interval T can be longer, for example, to repeat at the frame rate of an imaging device. In some cases, the pulse separation interval T can be between about 100 ns and about 50 ms.

The transverse spatial profile of the pulse 1-122 may be single mode Gaussian in some embodiments, although the invention is not limited to such a profile. In some embodiments, the transverse spatial profile of the pulse 1-122 may be multimode, e.g., having multiple distinct intensity peaks. For a multimode source, the optical system 1-140 may include a diffusing optical element that homogenizes the transverse intensity profile of the pulse. By allowing the use of a multimode source, higher pulse energies may be obtained from a laser diode. For example, the active area of the laser diode may be enlarged in a direction transverse to the optical axis of the laser to increase its optical output.

When used to excite fluorescent emission, pulses 1-122 from a pulsed light source may be referred to as "excitation pulses."

The term "fluorescent molecule" may be used to refer to fluorescent tags, fluorescent markers that may be attached to molecular probes, fluorophores, and autofluorescent molecules. The term "fluorescence emission" may be used to refer to light emanating from fluorescent tags, fluorescent markers that may be attached to molecular probes, fluorophores, and autofluorescent molecules.

II. Pulsed Light Sources The inventors have conceived a pulser circuit and techniques for generating short and ultrashort optical pulses from laser diodes and light emitting diodes. The pulsing circuit and techniques have been utilized in some embodiments to gain switch a semiconductor laser and generate a train of about 85 picosecond (ps) pulses (FWHM) with a peak power of about 1 W at repetition rates up to 100 MHz (T as short as 10 nanoseconds). In some embodiments, a unipolar or bipolar current waveform can be generated by the pulser circuit and used to drive the gain medium of the laser diode to excite the optical pulse and suppress the emission in the tail of the pulse. In some embodiments, a unipolar or bipolar current waveform can be generated by the pulser circuit and used to drive one or more light emitting diodes to output short or ultrashort optical pulses.

For purposes of explaining gain switching in laser diodes, Figures 2-1A through 2-1C are included to illustrate the laser dynamics associated with gain switching. Figure 2-1A illustrates a pump power curve 2-110 representing the pump power applied to the gain medium of a gain-switched laser according to some embodiments. As illustrated, the pump power can be applied to the gain medium in the laser cavity for a short duration (shown as approximately 0.6 microseconds). For semiconductor laser diodes, the application of the pump power can include applying a bias current across a pn junction or multiple quantum well (MQW) of the laser diode. The pump power pulses can be applied repeatedly at periodically spaced time intervals, for example, a pulse separation interval or pulse repetition time T.

While the pump power pulse is applied, the optical gain in the laser cavity increases until the gain begins to exceed the optical losses in the cavity. After this point, the laser can begin to lase (i.e., amplify the photons passing through the gain medium by the process of stimulated emission). As a result of the amplification process, the laser light increases rapidly and the excited states in the gain medium are depleted to produce at least one output pulse 2-130 as shown. In some embodiments, the pump power pulse 2-110 is timed to be turned off at approximately the same time that the peak of the output pulse occurs. The turning off of the pump power pulse terminates further lasing, thereby causing the output pulse 2-130 to disappear. In some embodiments, the output pulse 2-130 may have a shorter duration than the pump pulse 2-110, as shown in the drawings. For example, the output pulse 2-130 produced by gain switching may be less than 1/5 the duration of the pump pulse 2-110.

If the pump power pulse is not turned off, the dynamics shown in FIG. 2-1B may occur. In this case, the pump power curve (shown as pump current density) 2-140, shown as a step function, represents the current density applied to the semiconductor laser. The graph shows that the gain medium is excited by the pumping current density, which creates a carrier density N in the gain region of the laser diode. The lasing threshold current density I_thA pump current density I of about twice the current density I is applied at time t=0 and then left on. The graph shows that the carrier density N of the semiconductor gain region increases until the optical gain of the laser exceeds the losses in the cavity. After this point, a first pulse 2-161 is increased and emitted, which depletes the carrier density and optical gain to a value below the cavity losses. A second pulse 2-162 is then increased, depletes the carrier density N, and is emitted. The increase and depletion of the carrier density repeats for several cycles until the laser is stable in continuous wave operation (e.g., after about 7 nanoseconds in this example). The cycle of pulses (pulse 2-161, pulse 2-162, and subsequent pulses) is referred to as the relaxation oscillation of the laser.

The inventors have come to realize and appreciate that a challenge when gain switching a laser to generate ultrashort pulses is to avoid the deleterious effects of continuing relaxation oscillations. For example, as shown in FIG. 2-1C, if the pump power pulse 2-110 is not terminated quickly enough, at least a second light pulse 2-162 (due to relaxation oscillations) may begin to build up in the laser cavity and add a tail 2-172 to the gain-switched output pulse 2-170. The inventors have come to realize and appreciate that such a tail may be undesirable for some applications, such as applications aiming to distinguish fluorescent molecules based on their fluorescence lifetime. If the tail of the excitation pulse is not reduced quickly enough, the excitation radiation may overwhelm the detector unless wavelength filtering is utilized. Alternatively or additionally, the tail on the excitation pulse may continue to excite fluorescent molecules, complicating the detection of fluorescence lifetimes.

If the tail of the excitation pulse is reduced quickly enough, the excitation radiation present in the fluorescence emission may be negligible. In such an embodiment, filtering the excitation radiation during the detection of the fluorescence emission may not be necessary to detect the fluorescence emission and distinguish the fluorescent molecular lifetime. In some cases, eliminating the excitation filtering can greatly simplify and reduce the cost of the analysis system 1-160, and allow the system to be configured more compactly. For example, if a filter is not required to suppress the excitation wavelength during the fluorescence emission, the excitation source and the fluorescence detector can be located in close proximity (e.g., on the same circuit board or integrated device, or even within a few micrometers (microns) of each other).

The inventors have also come to realize and appreciate that in some cases, a tail on the excitation pulse can be tolerated. For example, the analysis system 1-160 can have an optical configuration that allows a wavelength filter to be easily incorporated into the detection optical path. The wavelength filter can be selected to reject the excitation wavelength so that the detector receives quantifiable fluorescence from the biological sample. As a result, the excitation radiation from the pulsed light source does not overwhelm the fluorescence being detected.

In some embodiments, the emission lifetime τ of a fluorescent molecule can be characterized by a 1/e intensity value, according to some embodiments, although in some embodiments other metrics may be used (e.g., 1/e², emission half-life, etc.). The accuracy of determining the lifetime of a fluorescent molecule is improved when the excitation pulse used to excite the fluorescent molecule has a duration shorter than the lifetime of the fluorescent molecule. Preferably, the excitation pulse has a FWHM duration at least three times shorter than the emission lifetime of the fluorescent molecule. An excitation pulse with a longer duration, or a tail 2-172 with significant energy, may continue to excite the fluorescent molecule while the decaying emission is being evaluated, complicating the analysis of the fluorescent molecule lifetime. To improve the fluorescence lifetime determination in such cases, deconvolution techniques can be used to unconvolute the excitation pulse profile from the fluorescence being detected.

In some cases, it may be preferable to use ultrashort pulses to excite fluorescent molecules to reduce quenching of the fluorescent molecules or samples. It has been found that extended pumping of fluorescent molecules may bleach and/or damage them over time, while higher intensity for shorter durations may not damage the fluorescent molecules as much as longer exposure at lower intensity (even if the total amount of energy applied to the molecule is the same). By reducing the exposure time, light-induced damage to the fluorescent molecules can be avoided or reduced, and the time or number of measurements for which the fluorescent molecules are used in the analysis system 1-160 can be increased.

In some applications, the inventors have found that it is desirable to terminate the pump pulse quickly (e.g., within about 250 ps of the peak of the pulse) to a power level that is at least about 40 dB below the peak power level of the pulse. Some embodiments can tolerate a smaller amount of power reduction, for example, between about 20 dB and about 40 dB within about 250 ps. Some embodiments may require a similar or larger amount of power reduction within 250 ps, for example, between about 40 dB and about 80 db in some embodiments, or between about 80 dB and about 120 db in some embodiments. In some embodiments, these power reduction levels may be required within about 100 ps of the peak of the pump pulse.

According to some embodiments, the pulse separation interval T (see Fig. 1-2) can also be an important aspect of the pulsed laser system. For example, when using a pulsed laser to evaluate and/or differentiate the luminescence lifetime of fluorescent molecules, the time between excitation pulses is preferably longer than any luminescence lifetime of the fluorescent species being tested to allow the luminescence lifetime to be determined sufficiently accurately. For example, a subsequent pulse should not arrive before the excited fluorescent molecule or collection of excited fluorescent molecules from the previous pulse has had a reasonable time to fluoresce. In some embodiments, the interval T needs to be long enough to determine the time between the excitation pulse exciting the fluorescent molecule and the subsequent photon emitted by the fluorescent molecule after the end of the excitation pulse and before the next excitation pulse.

The interval T between excitation pulses should be long enough to determine the decay characteristics of the fluorescent species, but it is also desirable for the pulse separation interval T to be short enough to allow many measurements to be made in a short period of time. By way of example and not limitation, the emission lifetimes (1/e values) of fluorescent molecules used in some applications may be in the range of about 100 picoseconds to about 10 nanoseconds. Thus, depending on the fluorescent molecule used, pulse separation intervals as short as about 200 ps may be used, while for longer-lived fluorescent molecules, pulse separation intervals T longer than about 20 nanoseconds may be used. Thus, the excitation pulses used to excite the fluorescent emission for fluorescence lifetime analysis may have a FWHM duration between about 25 picoseconds and about 2 nanoseconds, according to some embodiments.

In some applications where an integrated time domain imaging array is used to detect fluorescent emissions and provide data and visual display for lifetime analysis, the pulse separation interval T may not need to be shorter than the frame rate of the imaging system. For example, if there is sufficient fluorescent signal after a single excitation pulse, signal accumulation over multiple excitation pulses for an imaging frame may not be necessary. In some embodiments, the pulse repetition rate R of the pulsed light source 1-110 may be 1/2._pis the frame rate of the imaging system, R_f, whereby the pulse rate can be as slow as about 30 Hz. In other embodiments, the pulse rate can be much higher than the frame rate, and the fluorescence delay signal for each pixel in the image can be an integral after multiple excitation pulses.

An example of a pulsed light source 2-200 is shown in FIG. 2-2A. According to some embodiments, the pulsed light source 2-200 can include a commercially available or custom semiconductor laser diode 2-201 (or one or more LEDs) formed on a substrate 2-208. The laser diode or LED can be packaged in a housing 2-212 that includes an electrical connector 2-224. There can be one or more optical elements 2-205 (e.g., one or more lenses) included in the package to reshape and/or change the divergence of the output beam from the laser or LED. The laser diode 2-201 (or one or more LEDs) can be driven by a pulser circuit 2-210 that can provide a train of current pulses to the diode 2-201 via a connecting cable 2-226 and at least one wire 2-220. The drive current from the pulser circuit 2-210 can generate a train of light pulses 2-222 that are emitted from the laser diode or LED.

One advantage of using LEDs is that their cost is lower compared to laser diodes. In addition, LEDs provide a broader, generally incoherent spectral output that may be better suited for imaging applications (e.g., LEDs may produce fewer optical interference artifacts). For laser diodes, the coherent radiation may introduce speckle unless measures are taken to avoid speckle in the collected image. LEDs can also extend the excitation wavelength into the ultraviolet (e.g., down to about 240 nm) and can be used to excite autofluorescence in biological samples.

According to some embodiments, the laser diode 2-201 may include a semiconductor junction including a first layer 2-202 having a first conductivity type (e.g., p-type) and a second layer 2-206 having an opposite conductivity type. One or more intermediate layers 2-204 may be formed between the first and second layers. For example, the intermediate layer may include a multiple quantum well (MQW) layer in which carriers injected from the first and second layers recombine to generate photons. In some embodiments, the intermediate layer may include an electron and/or hole blocking layer. The laser diode may include inorganic and/or organic semiconductor materials in some implementations. The materials may be selected to obtain a desired emission wavelength. For example, for inorganic semiconductors, III-nitride compositions may be used for lasers emitting at wavelengths less than about 500 nm, and III-arsenide or III-phosphide compositions may be used for lasers emitting at wavelengths greater than about 500 nm. Any suitable type of laser diode 2-201 may be used, including, but not limited to, a vertical cavity surface emitting laser (VCSEL), an edge emitting laser diode, or a slab coupled optical waveguide laser (SCOWL).

According to some embodiments, one or more LEDs may be used instead of a laser diode. Multiple LEDs may be used because LEDs may have lower intensity than LDs. Because LEDs do not undergo relaxation oscillations or dynamics associated with lasing action, their output pulses may be longer in duration and have a wider spectral bandwidth than occurs for lasers. For example, the output pulses may be between about 50 ps and about 2 ns, and the spectral bandwidth may be about 20 nm or more. In some embodiments, the output pulses from the LEDs may be between about 100 ps and about 500 ps. For fluorescent molecules with longer decay times, longer excitation pulses may be acceptable. In addition, LEDs can generate unpolarized or partially polarized output beams. In some embodiments of the pulsed light source, the pulser circuit embodiments described below can be used to drive one or more LEDs.

The present inventors have come to realize that some conventional laser diode systems include a current driver circuit that can be modeled as shown in FIG. 2-2B. For example, a current driver 2-210 can include a pulsed voltage source 2-230 configured to deliver a current pulse to the laser diode. The connection to the laser diode is typically made through a cable 2-226, an adapter or connector 2-224, and a single wire 2-220 that is bonded to a contact pad on the laser diode 2-210. The connection between the adapter 2-224 and the laser diode can include a series inductance L1 and a series resistance R1. The connection can also include a small junction capacitance (not shown) associated with the contact and/or diode junction.

The present inventors have recognized and appreciated that by increasing the number of wire bonds (e.g., between the connector 2-224 and the laser diode 2-201), the inductance and/or resistance of the connection to the laser diode 2-201 can be reduced. Such a reduction in inductance and/or resistance can allow for faster current modulation of the laser diode and shorter output pulses. According to some embodiments, to improve the speed of the laser diode, the single wire bond 2-220 can be replaced with multiple parallel wire bonds. For example, the number of wire bonds can be increased to three or more. In some embodiments, there can be up to 50 wire bonds to the laser diode.

The inventors investigated the effect of increasing the number of wire bonds 2-220 on a commercial laser diode. One commercial laser considered was an Oclaro laser diode, model HL63133DG, currently available from Ushio (Cypress, Calif., USA). The results of a numerical simulation of increasing the number of wire bonds are shown in FIG. 2-2C. The simulation increased the number of wire bonds from a single bond (curve 2-250) on the commercial device to three wire bonds (curve 2-252) and 36 wire bonds (curve 2-254). The average drive current delivered to the laser diode for a fixed 18V pulse was determined over a range of frequencies for three different cases. The results show that a higher number of wire bonds allows more current to be delivered to the laser diode at higher frequencies. For example, at 1 GHz, using only three wire bonds (curve 2-252) allows more than four times the amount of current to be delivered to the laser diode than with a single wire bond. Because short and ultrashort pulses require a wider bandwidth (higher frequency components to form a short pulse), adding multiple wire bonds allows higher frequency components to drive the laser diode in shorter pulses than with a single wire bond. In some embodiments, multiple wire bonds can extend between a single contact pad or multiple contact pads on the laser diode and an adapter or connector 2-224 on the laser diode package. The connector can be configured to connect to an external standard cable (e.g., to a 50 ohm BNC or SMA cable).

In some embodiments, the number of wire bonds and the wire bond configuration can be selected to match the impedance of the adapter and/or cable connected to the laser diode. For example, the impedance of the wire bonds can be matched to the impedance of the connector 2-224 to reduce power reflection from the laser diode to the current driver according to some embodiments. In other embodiments, the impedance of the wire bonds can be selectively mismatched to generate negative pulses during positive current drive pulses. By selecting the packaging method of the laser diode (e.g., selecting the number of wire bonds from the adapter to the laser diode), the current modulation provided to the laser diode at higher frequencies can be improved. This can increase the response of the laser diode to fast gain switching signals, allowing for shorter optical pulses, faster reduction in optical power after the pulse peak, and/or increased pulse repetition rates.

Now, with reference to FIG. 2-3, the inventors have further realized and appreciated that by applying a bipolar pulse waveform 2-300 to a laser diode, the undesired emission tail 2-172 (see FIG. 2-1C) on the generated light pulse can be suppressed. A bipolar pulse can also be used to shorten the light pulse from an LED. A bipolar pulse can include a first pulse 2-310 of a first polarity followed by a second pulse 2-312 of the opposite polarity. The magnitude of the second pulse 2-312 can be different from the magnitude of the first pulse. In some embodiments, the second pulse can have a magnitude approximately equal to or less than the first pulse 2-310. In other embodiments, the second pulse 2-312 can have a magnitude greater than the first pulse 2-310.

In some embodiments, the magnitude of the second pulse may be between about 10% of the magnitude of the first pulse and about 90% of the magnitude of the first pulse. In some embodiments, the magnitude of the second pulse may be between about 25% of the magnitude of the first pulse and about 90% of the magnitude of the first pulse. In some cases, the magnitude of the second pulse may be between about 50% of the magnitude of the first pulse and about 90% of the magnitude of the first pulse. In some embodiments, the energy content of the second pulse may be between about 25% of the energy content of the first pulse and about 90% of the energy of the first pulse. In some embodiments, the energy content of the second pulse may be between about 50% of the energy content of the first pulse and about 90% of the energy of the first pulse.

The first drive pulse forward biases the laser diode junction, thereby generating carriers in the diode active region, which can recombine to generate a light pulse. A second drive pulse 2-312 of opposite polarity can reverse bias the diode junction, accelerating the removal of carriers from the active region, terminating photon generation. If the second electrical pulse 2-312 is timed to occur approximately simultaneously with or just prior (e.g., within about 200 ps) to the second relaxation oscillation pulse (see pulse 2-162 in FIG. 2-1B), the carrier concentration that would otherwise generate the second light pulse is reduced such that the emission tail 2-172 is suppressed.

A variety of circuit configurations can be used to generate a bipolar pulse waveform. FIG. 2-4A shows just one example of a circuit that can be used to drive a laser diode or one or more LEDs with a bipolar pulse waveform. In some embodiments, a transmission line 2-410 (e.g., a strip line or coaxial conductor assembly) can be configured in the pulse circuit 2-400 to deliver the bipolar pulse to the semiconductor laser diode 2-420 or at least one LED. The transmission line 2-410 can be formed in a U-shaped configuration and includes a charging resistor R on a first conductor._ChThrough the DC voltage source V_DDThe transmission line may be biased by . The transmission line, according to some embodiments, may have an impedance that approximately matches the impedance of the laser diode. In some embodiments, the impedance of the transmission line may be about 50 ohms. In some implementations, the impedance of the transmission line may be between about 20 ohms and about 100 ohms. In some implementations, the impedance of the transmission line may be between about 1 ohm and about 20 ohms.

Pulser 2-400 is a terminating resistor Z connected between the second conductor of the transmission line at one end of the transmission line and a reference potential (e.g., ground in the illustrated example)._termThe other end of the second conductor of the transmission line can be connected to a laser diode 2-420. The end of the first conductor of the transmission line can be connected to a switch M1 (e.g., a field effect transistor or a bipolar junction transistor) that can be activated to periodically shunt the end of the first conductor to a reference potential (e.g., ground).

In some cases, the termination impedance Z_termcan be approximately equal to the impedance of the transmission line 2-410 to reduce reflections back into the line. Alternatively, the termination impedance Z_termmay be smaller than the impedance of the line to reflect the negative pulse back into the line (after shunting by switch M1) and to the laser diode 2-420. In some embodiments, the termination impedance Z_termmay include capacitive and/or inductive components selected to control the shape of the reflected negative pulse. A transmission line pulser such as that shown in FIG. 2-4A may be used to generate electrical bipolar pulses having repetition rates in the range of about 30 Hz to about 200 MHz. According to some embodiments, the transmission line 2-410 for the transmission line pulser may be formed on a printed circuit board (PCB) as shown in FIG. 2-5A.

FIG. 2-4B illustrates an embodiment of a driver circuit 2-401 connected to a semiconductor photodiode 2-423 (e.g., a laser diode or one or more LEDs) that can be formed using discrete components and integrated on a substrate (such as a chip or PCB). In some embodiments, the circuit may be integrated on the same substrate as the laser diode or LEDs 2-423. The laser driver circuit 2-401 may include a control input 2-405 connected to a gate or base of a transistor M1. The transistor may be a CMOS FET, a bipolar junction transistor, or a high electron mobility transistor (such as a GaN pHEMT), although other high speed, high current handling transistors may be used. The transistor may be connected between a current source 2-430 and a reference potential (e.g., ground potential, although other reference potential values may be used). The transistor M1 couples the laser diode 2-423 (or one or more LEDs) and a resistor R connected in series with the laser diode between the current source 2-430 and the reference potential.₁According to some embodiments, the driver circuit 2-401 may include a resistor R between the laser diode and a reference potential.₁A capacitor C is connected in parallel with₁Although transistor M1 is described, any suitable controllable switch having a high conduction state and a low conduction state may be used.

In operation, the driver circuit 2-401 can provide current that bypasses the laser diode 2-423 when the transistor M1 is on, i.e., in a conducting state. Therefore, there is no light output from the laser diode. When the transistor M1 is off, current can flow through the laser diode due to an increased resistance path in the transistor. The current turns the laser diode on until the transistor is turned on again. Light pulses can be generated by modulating the control gate of the transistor between on and off states to provide current pulses to the laser diode. This approach can reduce the amount of voltage on the power supply and voltage on the transistor required to drive the laser compared to some pulse techniques, which is an important aspect for implementing such high speed circuits.

Resistor R₁and parallel capacitor C₁Due to the presence of resistor R, charge builds up on the capacitor when the diode is forward conducting. This can occur when transistor M1 is in an "off" state, e.g., a low or non-conducting state. When the transistor is turned on, the voltage stored across the capacitor reverse biases the laser diode. The reverse bias effectively creates a negative pulse across the laser diode, which can reduce or eliminate the emission tail 2-172 that would otherwise occur in the absence of a negative pulse.₁The value of time constant t can be selected such that substantially all of the charge on the capacitor is discharged before the switch is subsequently opened and/or a subsequent light pulse is generated by the laser diode.₁=R₁C₁may be designed to be less than about one-half or one-third of the pulse repetition interval T. In some embodiments, the time constant t₁=R₁C₁may be between about 0.2 ns and about 10 ns.

In some embodiments, the transistor M1 can be configured to switch to a conductive state after the first peak of the output light pulse from the laser diode. For example, referring to FIG. 2-1B, the photodetection and logic circuit can sense the decaying intensity of the first pulse 2-161 and trigger the transistor M1 to switch to a conductive state. In some embodiments, the transistor M1 can be triggered to switch to a conductive state based on a stable clock signal (e.g., triggered with respect to a synchronous clock edge). In some embodiments, the transistor M1 can be triggered to switch to a conductive state according to a predetermined delay time measured from the time when the transistor M1 switches to a non-conductive state. By switching the transistor M1 to a conductive state at a selected time, the laser power can be reduced immediately after the peak light pulse, the laser pulse can be shortened, and/or the tail emission of the pulse can be reduced.

Although the drive circuit shown in FIG. 2-4B shows a current source 2-430 located on the anode side of the laser, in some embodiments the current source may alternatively or additionally be located on the cathode side of the laser (e.g., transistor M1, resistor R₁, and a reference potential such as ground).

Other embodiments of the driver circuit for generating ultrashort pulses are possible. For example, a laser diode or LED current pulse driver circuit 2-402 may include multiple current driver branches connected to the nodes of the laser diode as shown in FIG. 2-4C. The driver circuit 2-402 may be formed using discrete or integrated components and may be integrated on a substrate (e.g., an ASIC chip or PCB). In some embodiments, the driver circuit may be integrated on the same substrate as one or more semiconductor photodiodes 2-425 (e.g., a laser diode or one or more light emitting diodes). Although the drawings show the driver circuit as being connected to the anode of the laser diode 2-425, in some embodiments, a similar driver circuit may alternatively or additionally be connected to the cathode of the laser diode. The driver circuit connected to the cathode side of the laser diode may utilize transistors of the opposite type and voltage sources of the opposite polarity to those used on the anode side of the laser diode.

According to some embodiments, there may be N circuit branches (e.g., circuit branches 2-432, 2-434, 2-436) configured to apply N forward bias current pulses to the laser diode 2-425 or LED, and M circuit branches (e.g., circuit branch 2-438) configured to apply M reverse bias current pulses to the laser diode. In FIG. 2-4C, N=3 and M=1, although other values may be used. Each forward bias current branch is connected to a voltage source V configured to deliver a forward bias current to the laser diode._iEach reverse bias current branch may include a voltage source V configured to deliver a reverse bias current to the laser diode._jEach circuit branch may include a resistor R₁Each circuit branch may further include a transistor M1 and a resistor R₁and a capacitor C connected to a fixed reference potential on the other side._iIn some embodiments, the capacitor C_imay be a junction capacitance (e.g., source-body capacitance) associated with transistor M1, and a separate discrete capacitor may not be provided. In some embodiments, at least one additional resistor may be included in series with diode 2-425 to limit the total amount of current delivered from the circuit branch.

In operation, timed pulse control signals are applied to the control input S of a switch or transistor Mi to generate a series of current pulses from each of the circuit branches that are summed and applied across the laser diode junction._iThe components in each branch (V_i, V_j, R_i, C_i) and control input S_iThe timing and pulse duration values of the control pulses applied to V can be independently selected to produce a desired bipolar current pulse waveform applied to the laser diode 2-425.₁, V₂and V₃The value of R can be chosen to have different values.₁, R₂, and R₃The values of may be the same, and C₁, C₂, and C₃In this example, a pulse signal is applied to the control input S_iBy offsetting the forward bias pulses relative to the forward bias circuit branch, a series of offset overlapping current pulses from the forward bias circuit branch can be generated having similar pulse durations but different pulse amplitudes. A timed pulse from the reverse bias circuit branch can generate a current pulse of opposite polarity that can extinguish or quickly turn off the forward bias pulse and can further generate a reverse bias pulse that can suppress tail emission from the laser diode. The reverse bias pulses can be carefully timed to at least partially overlap in time with one or more of the forward bias pulses. Thus, the circuit shown in FIG. 2-4C can be used to synchronize bipolar current pulses as shown in FIG. 2-3.

FIG. 2-4D shows another embodiment of a pulse driver 2-403 that can be fabricated using radio frequency (RF) components. The RF components can be designed to process signals at frequencies between about 50 MHz and about 1 GHz, according to some embodiments. In some implementations, the pulse driver 2-403 can include an input DC block 2-435 that AC-couples an input waveform (e.g., a square wave or a sine wave) to the driver. The DC block can be followed by an amplifier 2-440 that generates non-inverted and inverted output waveforms that travel along separate circuit paths 2-440a, 2-440b, respectively. The first circuit path 2-440a can include one or more adapters 2-442. A variable phase adjuster 2-445 can be included in the second circuit path 2-440b to selectively phase shift the signal in the second path relative to the signal in the first path.

The first and second circuit paths may be connected to a non-inverting input of an RF logic gate 2-450 (e.g., an AND gate or other logic gate). The inverting input of the logic gate 2-450 may be terminated by an appropriate impedance matching terminator 2-446 to avoid spurious power reflections at the gate. The non-inverting and inverting outputs of the logic gate 2-450 may be connected to a combiner 2-460 along two circuit paths 2-450a, 2-450b. The inverting circuit path 2-450b may include a delay element 2-454 and an attenuator 2-456, either or both of which may be adjustable. The delay element may be used to delay the inverted signal relative to the non-inverted signal, and the attenuator may be used to adjust the amplitude of the inverted signal.

The inverted and non-inverted signals resulting from the logic gates are then summed in a combiner 2-460. The output from the combiner 2-460 can be connected to an RF amplifier 2-470 that provides an output bipolar pulse for driving a laser diode or one or more LEDs. The output bipolar pulse can have a waveform as shown in FIG. 2-4E.

In operation, an input square wave or sine wave can be AC-coupled to the driver and split into two circuit paths 2-440a, 2-440b as a non-inverted and inverted version. The first amplifier 2-440 can be a limiting amplifier that shapes the sine waveform, according to some embodiments. In the second circuit path 2-440b, the inverted waveform can be phase shifted by an adjustable phase adjuster 2-445 to delay the inverted waveform in time relative to the non-inverted waveform. The resulting waveform from the first amplifier 2-440 can then be processed by an RF logic gate 2-450 (e.g., an AND gate) to generate short RF pulses at the non-inverted and inverted outputs of the logic gate. The duration of the short RF pulse can be adjusted using the phase adjuster 2-445, according to some embodiments. For example, the phase adjuster can adjust the period during which both the non-inverted and inverted waveforms at the inputs to the logic AND gate 2-450 are simultaneously in the "on" state, which determines the length of the output pulse.

2-4E, the short inverted pulse 2-417 from logic gate 2-450 may be delayed by an amount δ relative to the non-inverted pulse 2-415 by delay element 2-454 and attenuated to a desired amplitude by attenuator 2-456 before being combined with the non-inverted pulse. In some embodiments, the negative pulse amplitude |V_P-| is the positive pulse amplitude V_p+The pulse separation interval T may be less than 1/2. The pulse separation interval T may be determined by the frequency of the sine wave or square wave input to the pulse driver 2-403. The output pulse waveform may or may not include a DC offset. Although the output waveform is shown as having a square wave waveform, capacitance and inductance in the RF components and/or cables may produce output pulses having a rounder waveform rather closer to the waveform shown in FIG. 2-3.

As previously described in connection with Figures 2-4C and 2-4B, the application of current or voltage to a laser diode or LED may be to both the anode and the cathode in some embodiments. A radio frequency pulse driver circuit 2-404 is shown in Figure 2-4F that can apply split or differential voltage or current pulses to both the cathode and the anode. The front end of the circuit may be similar to the front end of the pulse driver circuit 2-403 shown in Figure 2-4D, according to some embodiments. However, in the pulse driver circuit 2-404, the non-inverting and inverting outputs from the logic gate 2-450 may not be combined, but instead may be applied as a differential drive to the anode and cathode of the laser diode. For simplicity, the circuitry associated with generating the subsequent negative or reverse bias pulse is not shown in Figure 2-4F.

An example of a split or differential drive generated by the differential pulse driver circuit 2-404 is shown in FIG. 2-4G. The first output from the logic gate 2-450 has an amplitude of +V_pA positive pulse 2-416 of opposite amplitude -V can be generated, and a second inverted output from logic gate 2-450 can be generated of opposite amplitude -V_pA negative pulse 2-418 of 2V can be generated. The pulse train may or may not have a small DC offset in some embodiments. The presence of the positive pulse 2-416 and the negative pulse 2-418 results in an effective amplitude of 2V._pA forward bias pulse having a bias voltage +V is generated across the laser diode. By splitting the bias across the laser diode and applying a partial bias to the anode and cathode, the amplitude of the voltage pulse processed by the pulse driver 2-404 can be effectively reduced by a factor of two. Thus, the pulse driver 2-404 can operate at a higher frequency and generate shorter pulses than might otherwise be achievable for larger amplitude pulses. Alternatively, the pulse driver circuit 2-404 can generate a bias pulse +V_pIn one embodiment, the amplitude of the drive pulse applied across the laser diode can be effectively doubled as compared to a drive circuit that only provides a pulse width to the anode of the laser diode. In such an embodiment, the power output from the laser diode can be increased.

Another way in which the power and/or drive speed applied to the laser diode can be increased is shown in FIG. 2-4H. According to some embodiments, multiple pulse driver outputs 2-470 can be connected to the anode of the laser diode 2-425 or LED. In this example, four pulse drivers are connected to the anode of the laser diode. In some embodiments where a differential pulse driver circuit is used, there may also be multiple drivers connected to the cathode of the laser diode. Each driver and its associated cable has an impedance Z₀and the laser diode 2-425 can have an impedance Z_LBecause they are connected in parallel, the output impedance of the drivers is divided by the number of drivers connected to the laser diode. The power delivered to the diode can be increased when the combined impedance of the pulse drivers closely matches the impedance of the laser diode 2-425, or vice versa.

The graph in FIG. 2-4I shows the increase in efficiency of power coupled into the laser diode 2-425 for four drive sources as a function of the impedance of the laser diode and the laser diode circuit. In this example, the four pulse drivers each have a line impedance of about 50 ohms and are configured to deliver an output pulse of 5V amplitude with a maximum current of about 100mA. The plot shows that the power coupled into the laser diode reaches a maximum value when the impedance of the laser diode is about 10 ohms. This value is approximately equal to the parallel output impedance of the four pulse driver outputs 2-470. Thus, the impedance of the laser diode 2-425 and its associated circuitry can be designed to approximately match the combined impedance of one or more pulse drivers used to drive the laser diode, according to some embodiments.

Other circuit driver configurations may be used to pulse the laser diode or light emitting diode. According to some embodiments, current injection into the light emitting diode may be performed using the same method as described in "A simple sub-nanosecond ultraviolet lightpulse generator with high repetition rate and peak power," P. H. Binh et al., Rev. Sci.Instr. Vol. 84, 083102 (2013) or "An ultraviolet nanosecond light pulse generator using a light emitting diode for test of photodetectors," T. Araki et al., Rev. Sci.Instr. Vol. 68, 1365 (1997) to generate subnanosecond pulses.

Another example of a pulser circuit is shown in FIG. 2-4J. According to some embodiments, the pulser circuit can include a pulse generator 2-480, which can receive one or more clock signals, for example, from a system clock, and output a train of electrical pulses to a driver circuit 2-490, which injects current pulses into the laser diode or light emitting diode in response to receiving the electrical pulses from the pulse generator. Thus, the output light pulses can be synchronized to the system clock. The system clock can also be used to operate the detection electronics (e.g., the imaging array).

According to some embodiments, the pulse generator 2-480 can be formed from a combination of passive and digital electronic components and can be formed on a first circuit board. In some cases, the pulse generator can include analog circuit components. In other embodiments, a portion of the pulse generator can be formed on the same board as the driver circuit 2-490, and a portion of the pulse generator can be formed on a separate board remote from the driver circuit. The driver circuit 2-490 can be formed from passive, analog, and digital electronic components and can be formed on the same or different circuit board as the pulse generator or a portion of the pulse generator. The light source (laser diode or light emitting diode) can be included on the circuit board with the driver circuit, or can be located in the system and connected to the driver circuit 2-490 by a high speed cable (e.g., SMA cable). In some embodiments, the pulse generator 2-480 and the driver circuit 2-490 can include emitter coupled logic elements. According to some embodiments, the pulse generator 2-480, the driver circuit 2-490, and the semiconductor photodiode 2-423 may be integrated on the same printed circuit board, laminate, or integrated circuit.

An example of a pulse generator 2-480 is shown in FIG. 2-4K. In some embodiments, the pulse generator may include a first stage that generates two differential clock outputs, one delayed with respect to the other. The first stage may receive a clock input and may include a fan-out 2-481 and a delay 2-483. The fan-out may include logic drivers and logic inverters configured to generate two copies of the clock signal and two inverted copies of the clock signal. According to some embodiments, the clock may have a symmetric duty cycle, although in other embodiments an asymmetric duty cycle may be used. One copy and one inverted copy are output as differential clock outputs (CK1,

<math num="1"><img file="JP7631258B2_D0001.tif" /></math>) can be formed, and a second copy and a second inverted copy (CK2,

<math num="2"><img file="JP7631258B2_D0002.tif" /></math>) may be delayed relative to the first pair of clock signals (CK1,

<math num="3"><img file="JP7631258B2_D0003.tif" /></math>) is a second pair of clock signals (CK2,

<math num="4"><img file="JP7631258B2_D0004.tif" /></math>) by at least a fraction of a clock cycle. The delay may include one or more full cycles in addition to the partial cycle. Within each pair of clock signals, the inverted signal may be synchronized to its corresponding one such that the rising and falling edges of the clocks occur essentially simultaneously.

The inventors have found that by adjusting the length of the current drive pulse from the pulse generator 2-480 and maintaining a fixed amplitude rather than adjusting the amplitude of the ultrashort current drive pulse, the ultrashort pulsing of the laser diode or LED can be more reliably controlled. By adjusting the length of the current drive pulse, the amount of energy delivered to the laser diode per pulse is adjusted. In some embodiments, high speed circuitry allows for high resolution control of the signal phase (e.g., by adjusting the delay or phase with an analog or digital delay element 2-483), which can be used to achieve high resolution control of the pulse length, according to some implementations.

In some cases, the first stage of the pulse generator 2-480 may include a dual output clock instead of the fan-out 2-481 and delay 2-483. The dual output clock can generate two differential clock signals and can provide an adjustable phase delay between the two differential clock signals. In some embodiments, the adjustable phase delay can have a corresponding time resolution of as little as 3 ps.

Regardless of how the delayed clock signals CK1, CK2 and their inverted signals are generated, the signals can be transmitted to the high-speed logic gate 2-485 via high-speed transmission lines. For signal transmission via cables between boards, the clock pulses may be degraded due to the cables. For example, the limited bandwidth of the transmission lines may distort the clock pulses differently, resulting in uneven timing. In some embodiments, the same type of cable or transmission line may be used for all the clock signals so that transmission distortions affect the four clock signals equally. For example, when the signal distortions and timing offsets are essentially the same for the four clock signals, the resulting drive pulses generated by the receiving logic gate 2-485 will be essentially the same as if there were no signal distortion from the transmission of the clock signals. Thus, transmission of the clock signals over distances of several feet can be tolerated without affecting the drive pulse duration. This may be useful for generating ultra-short drive pulses that are synchronized to a system clock and have precisely adjustable pulse durations (e.g., adjustable in increments of about 3 ps). If the clock signal is generated locally (eg, on the same substrate as the driver circuit 2-490), signal distortion associated with the transmission of the clock signal may not be significant, and the transmission lines may differ to some extent.

According to some embodiments, the clock signal is coupled to a capacitor C₁and can be AC-coupled to the data input of the high-speed logic gate 2-485.₁can have a capacitance between about 10 nF and about 1 μF. According to some embodiments, the logic gate may include an emitter coupled logic (ECL) two-input differential AND/NAND gate. An example of a logic gate 2-485 is the 2-485 available from ON Semiconductor (East Greenwich, Rhode Island, USA). The logic gates may include model MC100EP05 available from Logic Corporation, Greenwich, NC. The AC-coupled signal at the data input to the logic gate may look similar to the signal shown in FIG. 2-4L, where the horizontal dashed line indicates the zero voltage level. The illustration in FIG. 2-4L does not include distortion introduced by the transmission line. Distortion may round off and change the shape of the signal profile, but may not affect the relative phase of the clock signals when the same type and length of cable is used for each clock signal. Delay element 2-483 may provide a delay Δt, indicated by the vertical dashed line, which may be adjustable in increments as small as 3 ps. In some embodiments, delay element 2-483 may provide an adjustable delay in increments having values between 1 ps and 10 ps. Logic gate 2-485 may process the received clock signal and generate an output signal at output port Q that corresponds to the delay introduced by delay element 2-483. With a small delay, the output includes a short or ultrashort pulse sequence. With the high speed logic gate 2-485, the pulse duration may be between about 50 ps and about 2 ns (FWHM) in some embodiments, between about 50 ps and about 0.5 ns in some embodiments, between about 50 ps and about 200 ps in some embodiments, and even between about 50 ps and about 100 ps in some embodiments. The drive pulse from port Q may have a substantially square profile due to the fast slew rate of the ECL logic gate 2-485. A bias circuit 2-487 may be connected to the output port and may provide a voltage V₁can be applied to the output terminal P of the pulse generator 2-480._outThe output pulse provided from may include a DC offset according to some embodiments.

In some embodiments, two or more high speed logic gates 2-485 are connected to a capacitor C₁and bias circuit 2-487. These logic gates may be the same and can operate in parallel to provide a larger current drive capability at the output of the pulse generator. The inventors have recognized and appreciated that the logic gate 2-485 or gates must be capable of fast switching (e.g., fast rise and fall times to generate ultra-short drive pulses) and provide sufficient output current to drive the high current transistor M1 in the driver circuit 2-490. In some embodiments, connecting the logic gates 2-485 in parallel can improve the performance of the pulse circuit, making it possible to generate optical pulses of less than 100 ps.

FIG. 2-4M shows an embodiment of a driver circuit 2-490 that can be connected to a laser diode or LED 2-423. The driver circuit includes a resistor R₃and a capacitor C in series₂It may include an AC-coupled input having C₂may be between about 0.1 μF and about 10 μF, according to some embodiments, and R₃may have a value between about 10 ohms and about 100 ohms. Transistor M1, according to some embodiments, may include a high electron mobility field effect transistor (HEMT FET) capable of switching a high current (e.g., at least 1 amp, and in some cases up to 4 amps or more). Transistor M1 may be a high speed transistor capable of switching such high current at multi-gigahertz speeds. According to some embodiments, transistor M1 may switch currents in excess of 1 amp for electrical pulse durations between about 50 ps and about 2 ns at repetition rates between 30 Hz and about 200 MHz. An example of transistor M1 includes model ATF-50189-BLK available from Avago Technologies, San Jose, Calif. Bias and filtering circuitry (e.g., resistor R₄, R₇, and C₃) into the capacitor C₂and the gate of transistor M1. The drain of transistor M1 can be directly connected to the cathode of a laser diode or light emitting diode 2-423, and the source of transistor M1 can be connected to a reference potential (e.g., ground). The anode of diode 2-423 can be connected to a diode voltage source V_LDIt can be connected to resistor R₆and a capacitor₄may be connected in parallel across diode 2-423. According to some embodiments, resistor R₆may have a value between about 50 ohms and about 200 ohms, and C₄may have a capacitance between about 5 pF and about 50 pF.₅A diode voltage source V (having a value between about 1 μF and about 5 μF) is also provided in parallel with diode 2-423 and transistor M1._LDand a reference potential (eg, ground).

In some embodiments, a protection diode (not shown) may be reverse connected across the cathode and anode of the laser diode 2-423. The protection diode can protect the laser diode from excessive reverse bias potentials that could destroy the laser diode junction.

In operation, a pulse from the pulse generator 2-480 momentarily turns on transistor M1, allowing current to be injected into the active region of the laser diode or light emitting diode 2-423. In some embodiments, a large amount of forward current (e.g., up to 4 amps) flows momentarily through transistor M1. The forward current injects carriers into the laser diode junction, generating a short or ultrashort pulse of optical radiation. When transistor M1 turns off, parasitic inductances continue to pass current across the light emitting diode or laser diode, causing charge to build up on the cathode side of the diode until it can be dissipated by an RC network connected in parallel with the laser diode. This momentary buildup of charge at the cathode provides a reverse bias pulse to the laser diode, accelerating the removal of carriers from the active region. This accelerates the termination of the optical pulse.

The inventors have found that the optical pulsing techniques described for the embodiments of Figures 2-4M are superior to pulsing techniques based on the derivative of a square wave pulse because they can result in higher, shorter current pulses that may be required to turn on a laser diode.

The inventors have fabricated various pulse drive circuits and used them to drive laser diodes. FIG. 2-5A shows another embodiment of the assembled pulser circuit 2-500. This embodiment implements the pulser 2-400 as shown in FIG. 2-4A. In the assembled circuit, the transmission line 2-410 is formed as a parallel plate strip line patterned in a U-shaped configuration on a printed circuit board as shown. A GaN pHEMT transistor was used as a shunt switch M1 to short the two ends of the U-shaped transmission line. The pulser circuit 2-500 can operate at a repetition rate of up to 100 MHz and can be used to drive a 50 ohm load. In some embodiments, the pulser circuit can operate at a repetition rate between about 10 MHz and about 1 GHz.

The measured waveform from Pulser 2-500 is shown in FIG. 2-5B. The waveform shows a positive pulse with an amplitude of about 19.5V, followed by a negative pulse that reaches an amplitude of about -5V following the positive pulse. The duration of the positive pulse is about 1.5 nanoseconds. Referring again to FIG. 2-4A, Pulser 2-500 is connected to a termination resistor Z of about 50 ohms._termand a pull-up or charging resistor R of approximately 200 ohms._chIt was constructed to have Z_termThe value of was chosen to reduce power reflections from the termination resistor back to the transmission line. The bias applied to the transmission line 2-410 was 100V, and switch M1 was driven at a repetition rate of 100MHz. A DC bias of about -1.3V was coupled to the diode through a bias tee to adjust the relative offset from the 0V bias. The drive pulse for switch M1 was a square wave signal oscillating between about 0V and about 2V.

A commercially available test bed driver was used to drive a commercially available laser diode (Ushio Model HL63133DG) to generate optical pulses of less than 100 ps. Optical pulse measurements are shown in Figures 2-5C and 2-5D. Pulses with reduced tail emission were generated at a repetition rate of 100 MHz, as shown in Figure 2-5C. The average power from the laser diode was measured to be about 8.3 milliwatts. The pulse duration, shown in Figure 2-5D, was measured to be about 84 picoseconds. The intensity of the optical emission from the laser diode was found to be reduced by about 24.3 dB about 250 ps after the peak of the pulse. Even though the laser diode had a single bond wire, pulses of less than 100 ps were generated. With multiple bond wires or further improvements to the pulse circuitry, shorter pulses (e.g., between about 25 ps and about 75 ps) can be generated.

FIG. 2-6A shows an example of a semiconductor laser 2-600 that can be used to generate optical pulses by gain switching according to any of the gain switching devices and techniques described above. The laser and pulse drive circuitry can be mass produced and manufactured at low cost. For example, the laser can be microfabricated as an edge-emitting device using planar integrated circuit technology. Such lasers are sometimes referred to as slab-coupled optical waveguide lasers (SCOWLs). The drawing shows an elevational cross-sectional view of the laser. The laser may be formed from a GaAs/AlGaAs material system (e.g., to emit radiation in the green, red, or infrared regions of the optical spectrum), although other material systems (such as GaN/AlGaN) may be used in other embodiments (e.g., to emit radiation in the green, blue, or ultraviolet regions of the spectrum). The laser diode may be manufactured from other semiconductor material systems, including, but not limited to, InP, AlInGaP, InGaP, and InGaN.

According to some embodiments, the SCOWL may be formed on an n-type substrate or buffer layer 2-627 (e.g., a GaAs substrate or layer containing Al). For example, the buffer layer may be Al_xGa_1-xThe refractive index of the substrate or base layer may include As, and x is between about 0.25 and about 0.30. The refractive index of the substrate or base layer may include a first value n₁An electron transport layer 2-617 of lightly doped n-type semiconductor material can be formed on the substrate 2-627. In some embodiments, the electron transport layer 2-617 is epitaxially grown on Al_xGa_1-xAs, x is between about 0.20 and about 0.25, and about 5×10¹⁶cm^-3The thickness h of the electron transport layer may be between about one micrometer (1 micron) and about two micrometers (2 microns).₁A second reflectance value n that is greater than₂A multiple quantum well region 2-620 may then be formed on the electron transport layer 2-617. The multiple quantum well region may include alternating layers of materials (e.g., alternating layers of AlGaAs/GaAs) having different doping concentrations that modulate the energy band gap in the MQW region. The layers in the quantum well region 2-620, which may have a thickness between about 20 nm and about 200 nm, may be deposited by epitaxy, atomic layer deposition, or a suitable vapor deposition process. The multiple quantum well region may include n₂The effective third reflectance value n is greater than₃A hole transport layer 2-615 of p-type doped material may be formed adjacent to the quantum well region,₂Reflectance value less than n₄In some embodiments, the reflectance values of different regions of the SCOWL may be as shown in Figures 2-6B according to some embodiments. In some embodiments, the SCOWL may include GaN semiconductors and alloys thereof, or InP semiconductors and alloys thereof.

The term "adjacent" may refer to two elements positioned in close proximity to one another (e.g., within a distance of less than about one-fifth of the larger lateral or vertical dimension of the two elements). In some cases, there may be intervening structures or layers between adjacent elements. In some cases, adjacent elements may be directly adjacent to one another without intervening structures or elements.

After the layers of the laser device are deposited, a trench 2-607 may be etched into the layers to form an active region of the laser having a width w that is between about 0.25 micrometers (0.25 microns) and about 1.5 micrometers (1.5 microns). An n-contact 2-630 may be formed on a first surface of the device, and a p-contact 2-610 may be formed on the p-type transport layer 2-615 adjacent to the active region. Exposed surfaces of the semiconductor layers may be passivated with an oxide or other electrically insulating layer according to some embodiments.

A trench 2-607 adjacent to the active region and having a refractive index value n₁, n₂, n₃, and n₄However, it confines the optical mode of the laser to the lasing region 2-625 adjacent to the quantum wells and below the device central rib as shown in the drawing. The SCOWL can be designed to couple higher order transverse modes that may otherwise form and lase in the lasing region 2-625 to lossy higher order slab modes in adjacent regions. When properly designed, all higher order transverse modes from the lasing region 2-625 will be relatively lossy compared to the fundamental mode in that lasing region and will not lase. In some embodiments, the transverse optical mode of the SCOWL 2-600 may be a single transverse mode. The width of the optical mode may be between about 0.5 micrometers (0.5 microns) and about 6 micrometers (6 microns). The mode profile 2-622 taken in the x-direction may be shaped as shown in FIG. 2-6B according to some embodiments. In other embodiments, the SCOWL may generate multiple optical transverse modes to illuminate a region of interest. In some embodiments, the length of the active region (along the dimension into the paper) may be between about 20 micrometers (20 microns) and about 10 mm. The output power of the SCOWL can be increased by choosing a longer active region length. In some embodiments, the SCOWL can deliver an average output power of more than 300 mW.

Although a semiconductor laser (e.g., a SCOWL) and a pulser circuit can be combined to create a low-cost ultrafast pulsed laser suitable for many applications, the turn-off rate shown in FIG. 2-5D may not be suitable for some fluorescence lifetime analysis. In some cases, a more rapid turn-off may be required. For example, the inventors have found that some measurements based on fluorescence lifetime may require the tail of the pulse to decay to a level between about 25 dB and about 40 dB below the pulse peak within 250 ps after the pulse peak. In some cases, the pulse power may need to drop to this range within 100 ps after the pulse peak. In some embodiments, the pulse tail may need to drop to a level between about 40 dB and about 80 dB below the pulse peak within 250 ps after the pulse peak. In some embodiments, the pulse tail may need to drop to a level between about 80 dB and about 120 dB below the pulse peak within 250 ps after the pulse peak.

One approach to further suppress the emission tail of the pulse is to include a saturable absorber in the pulsed laser or high brightness LED system. According to some embodiments, a semiconductor saturable absorber 2-665 can be integrated on the same substrate as the semiconductor laser 2-600 or high brightness LED, as shown in FIG. 2-6C. The semiconductor laser can include a SCOWL structure including a quantum well region 2-620, according to some embodiments. The SCOWL can be driven by a pulse source 2-670, such as the pulser circuit 2-400 or other pulsing circuitry described above.

A saturable absorber 2-665 may be formed adjacent one end of the SCOWL. The saturable absorber 2-665 may include a region having a band gap tuned to absorb photons from the semiconductor laser. For example, the saturable absorber may include a single quantum well or multiple quantum wells with at least one energy band gap approximately equal to the intrinsic energy of the optical emission of the laser. In some embodiments, the saturable absorber may be formed by ion implantation into a region of the diode laser to electrically isolate the region within the diode laser cavity. A negative bias may be applied to this region to promote absorption rather than gain in the same laser diode structure. At high fluences from the laser 2-600, the valence band of the saturable absorber may be depleted of carriers and the conduction band may be filled, preventing further absorption by the saturable absorber. As a result, the saturable absorber whitens, reducing the amount of radiation absorbed from the laser. In this manner, the peak of the laser pulse may "punch through" the saturable absorber with less attenuation in intensity than the tail or wings of the pulse. Thus, the tail of the pulse may be further suppressed relative to the peak of the pulse.

According to some embodiments, a high reflector (not shown) can be formed or located at one end of the device. For example, a high reflector can be located at one end of the laser furthest from the saturable absorber to redirect the laser emission from the saturable absorber and increase the output power. According to some embodiments, an anti-reflective coating can be applied to one end of the saturable absorber and/or SCOWL to increase extraction from the device.

According to some embodiments, the saturable absorber can include a bias source 2-660. The bias source can be used to sweep carriers out of the active region after each pulse and improve the response of the saturable absorber. In some embodiments, the bias can be modulated (e.g., in pulse repetition rate) to make the saturable recovery time time dependent. This modulation can further improve the pulse characteristics. For example, the saturable absorber can suppress the pulse tail by absorbing otherwise more at low intensities if the recovery time of the saturable absorber is sufficient. Such differential absorption can also reduce the pulse length. The recovery time of the saturable absorber can be adjusted by applying or increasing a reverse bias to the saturable absorber.

III. System Timing and Synchronization Referring again to FIG. 1-1, regardless of the method and device used to generate short or ultrashort pulses, the system 1-100 can include circuitry configured to synchronize at least some electronic operations (e.g., data acquisition and signal processing) of the analysis system 1-160 with the repetition rate of the optical pulses from the light source. There are at least two methods for synchronizing the pulse repetition rate with the electronics on the analysis system 1-160. According to a first technique, a master clock can be used as a timing source to trigger both the generation of pulses in the pulsed light source and the instrument electronics. In a second technique, a timing signal can be derived from the pulsed light source and used to trigger the instrument electronics.

Figure 3-1 shows that the clock 3-110 has a synchronous frequency f_sync, to both a pulsed light source 1-110 (e.g., a gain-switched pulsed laser or a pulsed LED) and an analysis system 1-160, which may be configured to detect and process signals resulting from interactions between each excitation pulse 1-120 and biological, chemical, or other physical substances. By way of example only, each excitation pulse may excite one or more fluorescent molecules in the biological sample that are used to analyze a characteristic of the biological sample (e.g., cancerous or non-cancerous, viral or bacterial infection, blood glucose levels). For example, non-cancerous cells have an intrinsic fluorescence lifetime τ of a first value.₁while cancerous cells may exhibit a second value of lifetime τ that is different and distinguishable from the first lifetime value.₂As another example, the fluorescence lifetime detected from a sample of blood may have a lifetime and/or intensity value (relative to another stable marker) that depends on the blood glucose level. After each pulse or a series of pulses, the analysis system 1-160 may detect and process the fluorescent signal to determine characteristics of the sample. In some embodiments, the analysis system may generate an image of the area interrogated by the excitation pulses, including a two-dimensional or three-dimensional map of the area that indicates one or more characteristics of regions within the imaged area.

Regardless of the type of analysis being performed, the detection and processing electronics on the analysis system 1-160 may need to be carefully synchronized with the arrival of each optical excitation pulse. For example, when assessing fluorescence lifetimes, it is beneficial to know precisely the time of excitation of the sample so that the timing of the emission event can be precisely recorded.

The synchronization configuration shown in FIG. 3-1 may be suitable for systems in which optical pulses are generated by active methods (e.g., external control). Active pulse systems can include, but are not limited to, gain-switched lasers and pulsed LEDs. In such systems, a clock 3-110 can provide a digital clock signal that is used to trigger pulse generation (e.g., gain switching or current injection into an LED junction) in the pulsed light source 1-110. The same clock can also provide the same or a synchronized digital signal to the analysis system 1-160 so that electronic operations on the instrument can be synchronized to the pulse arrival times at the instrument.

The clock 3-110 may be any suitable clocking device. In some embodiments, the clock may include a crystal oscillator or a MEMS-based oscillator. In some implementations, the clock may include a transistor ring oscillator.

The frequency f of the clock signal provided by clock 3-110_syncf does not have to be at the same frequency as the pulse repetition rate R. The pulse repetition rate can be given by R=1/T, where T is the pulse separation interval. In FIG. 3-1, the light pulses 1-120 are shown to be spatially separated by a distance D. This separation distance corresponds to the time T between the arrival of the pulses at the analysis system 1-160 by the relationship T=D/c, where c is the speed of light. In practice, the time T between the pulses can be determined by a photodiode and an oscilloscope. According to some embodiments, T=f_sync/N, where N is an integer equal to or greater than 1. In some embodiments, T=Nf_syncwhere N is an integer equal to or greater than 1.

FIGURE 3-2 illustrates a system in which a timer 3-220 provides a synchronization signal to the analysis system 1-160. In some embodiments, the timer 3-220 can derive a synchronization signal from the pulsed light source 1-110, and the derived signal is used to provide the synchronization signal to the analysis system 1-160.

According to some embodiments, the timer 3-220 may receive an analog or digitized signal from a photodiode that detects the light pulses from the pulsed light source 1-110. The timer 3-220 may use any suitable method for forming or triggering a synchronization signal from the received analog or digitized signal. For example, the timer may use a Schmitt trigger or a comparator to form a train of digital pulses from the detected light pulses. In some embodiments, the timer 3-220 may further use a delay-locked loop or a phase-locked loop to synchronize a stable clock signal to the train of digital pulses generated from the detected light pulses. The train of digital pulses or the locked stable clock signal may be provided to the analysis system 1-160 to synchronize electronics on the instrument with the light pulses.

In some embodiments, as shown in FIG. 3-3, two or more pulsed light sources 1-110a, 1-110b may be required to provide light pulses at two or more different wavelengths to the analysis system 1-160. In such embodiments, it may be necessary to synchronize the pulse repetition rates of the light sources and the electronic operation on the analysis system 1-160. In some embodiments, when two pulsed light sources use an active method of generating pulses, the techniques described above in connection with FIG. 3-1 may be used. For example, a clock 3-110 may be provided with a synchronization frequency f_syncA clock or synchronization signal such as may be provided to both pulsed light sources 1-110a, 1-110b and the analysis system 1-160.

In some embodiments, it may be beneficial to interleave the pulses from the two pulsed light sources in time, as shown in Figures 3-4A and 3-4B. When the pulses are interleaved, a pulse 3-120a from a first source 1-110a occurs at a first time t₁At the first characteristic wavelength λ₁may excite one or more samples in the analysis system 1-160. Data representative of the interaction of the first pulse with the one or more samples may then be collected by the instrument.₂In the second source 1-110b, a pulse 3-120b has a second characteristic wavelength λ₂The first pulse may excite one or more samples in the analysis system 1-160. Data representative of the interaction of the second pulse with the one or more samples may then be collected by the instrument. By interleaving the pulses, the effects of the interaction of the pulse with the sample at one wavelength may be prevented from blending with the effects of the interaction of the pulse with the sample at a second wavelength. Additionally, properties associated with two or more fluorescent markers may be detected.

The pulses can be interleaved by timing and synchronization circuitry as shown in FIG. 3-4A. The method described in connection with FIG. 3-3 can be used to synchronize the pulse trains from the two pulsed light sources 1-110a, 1-110b and synchronize the electronics and operations on the analysis system 1-160 with the arrival of the pulses. To interleave the pulses, the pulses of one pulsed light source can be phase-locked or triggered out of phase with the pulses from the other pulsed light source. For example, the pulses of the first pulsed light source 1-110a can be phase-locked (using a phase-locked or delay-locked loop) or triggered 180 degrees out of phase with the pulses from the second pulsed light source 1-110b, although other phase or angle relationships may be used in some embodiments. In some implementations, a timing delay can be added to the trigger signal provided to one of the pulsed light sources. The timing delay can delay the trigger edge by approximately one-half the pulse separation interval T. According to some embodiments, a frequency doubling synchronization signal can be generated by a timer 3-220 and provided to the instrument 3-160 to synchronize the instrument electronics and operation with the arrival of interleaved pulses from the pulsed light source.

IV. TIME-DOMAIN APPLICATIONS OF PULSE LIGHT SOURCES The pulsed light sources described above are useful for a variety of time-domain applications. In some embodiments, the pulsed light sources can be used in systems configured to detect and/or characterize a state or property of a biological sample based on fluorescence lifetime, fluorescence wavelength, fluorescence intensity, or a combination thereof. The pulsed light sources can also be used in time-of-flight systems. Time-of-flight systems can include imaging and ranging systems that illuminate a target with a short or ultrashort light pulse and then detect backscattered radiation from the target to form a three-dimensional image of the target or determine the distance to the target.

In time-domain applications utilizing fluorescent emission, a pulsed light source operating at a first characteristic wavelength can excite one or more fluorescent molecules in a sample, and an analysis system can detect and analyze the fluorescent emission from the sample at one or more wavelengths different from the wavelength of the pulsed light source. According to some embodiments, one or more properties of the biological sample can be determined based on the analysis of the fluorescence lifetime from one or more fluorescent molecules present in the sample. In some embodiments, additional properties of the fluorescent emission (e.g., wavelength, intensity) may be analyzed to further assist in the determination of one or more properties of the biological sample. The system for determining the properties of the biological sample based on the fluorescence lifetime can be an imaging system or a non-imaging system. When configured as an imaging system, a pixel array can be used for fluorescence detection, and imaging optics can be placed between the sample and the pixel array to form an image of at least a portion of the sample on the pixel array. In some embodiments, the non-imaging system can detect the fluorescent emission from multiple samples in parallel using the pixel array.

An instrument 4-100 for determining a characteristic of a biological sample using a pulsed light source based at least in part on fluorescence lifetime analysis according to some embodiments is shown in FIG. 4-1. Such an instrument can include one or more pulsed light sources 4-120, a time-binning photodetector 4-150, an optical system 4-130 (which may be one or more lenses and may include one or more optical filters), and a transparent window 4-140 that can be pressed against a subject and onto which a biological sample can be placed. The one or more pulsed light sources and the optical system can be configured such that light pulses from the one or more light sources illuminate an area through the window 4-140. Fluorescence emissions excited by the optical excitation pulses can be collected by the optical system 4-130 and directed to a time-binning photodetector 4-150 that can discriminate the lifetime of one or more fluorescent molecules, as further described below. In some embodiments, the photodetector 4-150 can be non-imaging. In some embodiments, the photodetector 4-150 can comprise an array of pixels, each with time binning capability, for forming an image of the sample. The image data can include spatially resolved fluorescence lifetime information and conventional imaging information. The components of the instrument can be mounted in a casing 4-105, which can be small in size such that the instrument can be operated as a handheld device. The light source(s) 4-120 and the photodetector 4-150 may or may not be mounted on the same circuit board 4-110. In some embodiments, the instrument 4-100 can include a microprocessor or microcontroller and/or data communication hardware such that data can be transmitted to an external device (e.g., smartphone, laptop, PC) for processing and/or data storage.

A system configured to analyze a sample based on fluorescence lifetime can detect differences in fluorescence lifetime between different fluorescent molecules and/or the lifetime of the same fluorescent molecule in different environments that affect the fluorescence lifetime. By way of illustration, FIG. 4-2 plots two different fluorescence emission probability curves (A and B) that can represent, for example, fluorescence emission from two different fluorescent molecules or the same fluorescent molecule in different environments. With reference to curve A, the probability p of fluorescence emission from a first molecule after excitation by a short or ultrashort light pulse is_A(t) may decay over time as shown. In some cases, the reduction in the probability that a photon is emitted over time follows an exponential decay function

<math num="5"><img file="JP7631258B2_D0005.tif" /></math>where P_A0is the initial emission probability, and τ_Ais a time parameter associated with the first fluorescent molecule that characterizes the emission decay probability._Amay also be referred to as the "fluorescence lifetime," "luminescence lifetime," or "lifetime" of the first fluorescent molecule._AThe value of may be altered by the local environment of the fluorescent molecule. Other fluorescent molecules may have different emission characteristics than those shown in curve A. For example, another fluorescent molecule may have a decay profile different from a single exponential decay, and its lifetime may be characterized by a half-life value or some other metric.

The second fluorescent molecule has an exponential, but measurably different, lifetime τ_BIn some embodiments, the various fluorescent molecules may have lifetime or half-life values ranging from about 0.1 ns to about 20 ns. In the illustrated example, the lifetime of the second fluorescent molecule in curve B is shorter than that in curve A, and the probability of emission is higher immediately after excitation of the second molecule than in curve A.

The inventors have come to realize and appreciate that differences in fluorescence emission lifetimes can be used to distinguish between the presence or absence of different fluorescent molecules and/or to distinguish between different environments or conditions in a sample that affect the lifetime of one or more fluorescent molecules. In some cases, distinguishing fluorescent molecules based on lifetime (e.g., rather than emission wavelength) can simplify some aspects of the analytical system 1-160. As an example, when distinguishing fluorescent molecules based on lifetime, the number of wavelength discrimination optics (wavelength filters, dedicated detectors for each wavelength, dedicated pulsed light sources at different wavelengths, and/or diffractive optical elements) can be reduced or eliminated. In some cases, a single pulsed light source can be used to excite different fluorescent molecules that emit within the same wavelength region of the optical spectrum but have measurably different lifetimes. An analytical system that uses a single pulsed light source, rather than multiple light sources at different wavelengths to excite and distinguish different fluorescent molecules that emit within the same wavelength region, can be less complex to operate and maintain, can be more compact, and can be manufactured at a lower cost.

Although analytical systems based on fluorescence lifetime analysis can have certain advantages, by enabling additional detection techniques the amount of information obtained by the analytical system can be increased. For example, some analytical systems 1-160 may be further configured to determine one or more properties of the sample based on the fluorescence wavelength and/or fluorescence intensity.

Referring again to FIG. 4-2, according to some embodiments, different fluorescence lifetimes can be distinguished using a photodetector configured to time bin the fluorescence emission events following excitation of the fluorescent molecules. The time binning can be performed during a single charge accumulation cycle of the photodetector. The concept of determining the fluorescence lifetime by time binning of the emission events is shown graphically in FIG. 4-3.₁Or t₁At a time just before , a fluorescent molecule or a collection of fluorescent molecules of the same type (e.g., the type corresponding to curve B in FIG. 4-2) is excited by a short or ultrashort light pulse. For the collection of molecules, the intensity of the emission may have a time profile as shown in FIG. 4-3.

On the other hand, for a single molecule or a small number of molecules, the emission of fluorescent photons occurs according to the statistics of curve B in Figure 4-2. The time-binning photodetector 4-150 can accumulate the emission events into distinct time bins (three are shown in Figure 4-3) that are measured relative to the excitation time of the fluorescent molecule(s). When a large number of emission events are summed, the resulting time bins can approximate the decay intensity curve shown in Figure 4-3, and the binned signal can be used to distinguish between different fluorescent molecules or different environments in which the fluorescent molecules are located.

Examples of time-binned photodetectors are described in International Application No. PCT/US2015/044360, which is incorporated herein by reference, and an embodiment of such a photodetector is shown in FIG. 4-4 for illustrative purposes. A single time-binned photodetector 4-400 can include a photon absorption/carrier generation region 4-402, a carrier transfer region 4-406, and a number of carrier storage bins 4-408a, 4-408b, 4-408c, all formed on a semiconductor substrate. The carrier transfer region can be connected to the multiple carrier storage bins by carrier transport channels 4-407. Although only three carrier storage bins are shown, there may be more bins. There may be a readout channel 4-410 connected to the carrier storage bins. The photon absorption/carrier generation region 4-402, carrier transfer region 4-406, carrier storage bins 4-408a, 4-408b, 4-408c, and readout channel 4-410 may be formed by locally doping a semiconductor and/or forming tailored insulating regions to provide the photodetection function and confine the carriers. The time binned photodetector 4-400 may also include a plurality of electrodes 4-420, 4-422, 4-432, 4-434, 4-436, 4-440 formed on the substrate configured to generate an electric field within the device for transporting carriers through the device.

In operation, fluorescent photons can be received at the photon absorption/carrier generation region 4-402 at different times to generate carriers. For example, at approximately time t₁At t, three fluorescent photons can generate three carrier electrons in the depletion region of the photon absorption/carrier generation region 4-402. An electric field in the device (due to doping and/or externally applied biases on electrodes 4-420 and 4-422 and optionally or alternatively 4-432, 4-434, 4-436) can transport the carriers to the carrier transport region 4-406. In the carrier transport region, the distance traveled is converted to time after excitation of the fluorescent molecule. At a later time t₅At a later time t , another fluorescent photon can be received in the photon absorption/carrier generation region 4-402 and generate an additional carrier. At this point, the first three carriers have migrated to a position in the carrier transfer region 4-406 adjacent to the second storage bin 4-408b.₇In the embodiment, an electrical bias can be applied between electrodes 4-432, 4-434, 4-436 and electrode 4-440 to transport carriers laterally from the carrier transfer region 4-406 to the storage bins. The first three carriers can then be transported and stored in the first bin 4-408a, and later generated carriers can be transported and stored in the third bin 4-408c. In some embodiments, the time interval corresponding to each storage bin is on the sub-nanosecond time scale, although in some embodiments (e.g., embodiments in which the fluorophores have longer decay times) longer time scales may be used.

The process of generating and time-binning carriers after an excitation event (e.g., an excitation pulse from a pulsed light source) may be performed once after a single excitation pulse, or may be repeated multiple times after multiple excitation pulses during a single charge accumulation cycle of the photodetector 4-400. After charge accumulation is complete, carriers can be read out of the storage bins via the readout channel 4-410. For example, a suitable bias sequence can be applied to at least the electrode 4-440 and a downstream electrode (not shown) to remove carriers from the storage bins 4-408a, 4-408b, 4-408c.

The aspect of signal acquisition is shown in more detail for multiple excitation pulses in Figures 4-5A and 4-5B. In Figure 4-5A, multiple excitation pulses are_e1, t_e2, t_e3, ... is applied to the sample. After each excitation pulse, one or more fluorescence emission events occur at time t_fn, whereby carriers accumulate in different carrier storage bins depending on when the emission event occurs. After multiple excitation events, the accumulated signal in each carrier storage bin can be read out to result in a signal series that can be represented as a histogram 4-510 (shown in FIG. 4-5B). The signal series can indicate the number of photons detected during each binned time interval after excitation of the fluorophore(s) in the sample, representing the fluorescence emission decay rate. The signal series or histogram can be used to distinguish between different fluorescent molecules or different environments in which the fluorescent molecules reside.

As an example of distinguishing different fluorescent molecules, a photodetector with three time bins, as shown in Figures 4-3B and 4-4, can generate three signal values (35, 9, 3.5) represented by the histogram of bin 1 to bin 3 in Figure 4-5B and corresponding to curve B in Figure 4-2. These binned signal values can have different relative and/or absolute values than the binned signal values recorded from different fluorescent molecules, such as those corresponding to curve A in Figure 4-2, which can generate binned values (18, 12, 8). By comparing the signal sequence of binned values to a calibration standard, it is possible to distinguish between two or more fluorescent molecules or environments that affect the fluorescence lifetime. It can be beneficial to be able to distinguish between multiple different fluorescent molecules and/or environments based on lifetime information using a pulsed light source operating at only a single characteristic wavelength.

According to some embodiments, an excitation bin (e.g., bin 0) may be included in at least one time-binning photodetector to record the signal level of the excitation pulse (e.g., to accumulate carriers generated immediately by the excitation pulse). The recorded signal level may be used to normalize the fluorescence signal level, which may be useful for distinguishing fluorescent molecules based on intensity.

In some embodiments, the signal values from storage bins 4-408 can be used to fit a luminescence decay curve (e.g., a single exponential decay) to determine the detected lifetime. In some embodiments, the binned signal values can be fitted to a multiple exponential decay, such as a bi-exponential or triple exponential. A Laguerre decomposition process can be used to analyze the multiple exponential decay. In some embodiments, the signal values can be treated as vectors or locations and mapped into an M-dimensional space, and cluster analysis can be used to determine the detected lifetime. Once the lifetime is determined, the type of fluorescent molecule or characteristics of the environment in which the fluorescent molecule is located can be identified.

Although the examples described in connection with Figures 4-3 and 4-4 show three time bins, the time binning photodetector may have fewer or more time bins. For example, the number of time bins may be 2, 3, 4, 5, 6, 7, 8 or more. In some cases, there may be 16, 32, 64 or more time bins. According to some embodiments, the number of time bins in the photodetector may be reconfigurable. For example, one or more adjacent bins may be combined during readout.

Although the discussion of Figures 4-3 concerns detecting emission from a single type of fluorescent molecule at a time, in some cases a sample may contain two or more different fluorescent molecules with different lifetimes. When multiple different fluorescent molecules contribute to a temporal emission profile, an average fluorescence lifetime can be used to represent the collection. In some embodiments, the analysis system 1-160 can be configured to discriminate between combinations of fluorescent molecules. For example, a first combination of fluorescent molecules may exhibit a different average lifetime than a second combination of fluorescent molecules.

According to some embodiments, time-binned photodetectors may be used in an imaging array and imaging optics may be included between the time-binned photodetector array and the sample. For example, each imaging pixel of the imaging array may include a time-binned photodetector 4-400. The imaging optics may form an image of a region of the sample on the photodetector array. Each pixel in the photodetector array may record a time-binned signal value that is analyzed to determine the fluorescence lifetime of the portion of the imaged region corresponding to the pixel. Such an imaging array may thus provide spatially resolved fluorescence lifetime imaging information to distinguish different regions in an image having different fluorescence lifetime characteristics. In some embodiments, a conventional image of the same region may be obtained using the same time-binned photodetectors, for example, by summing all the bins of each pixel or by constructing an image from the excitation pulse bin (bin 0). The variation in fluorescence lifetime may be displayed as an overlaid color-coded map on a conventional gray scale or color image. In some cases, lifetime mapping may enable a surgeon to identify abnormal or diseased regions of tissue (e.g., cancerous or precancerous).

The inventors have recognized and come to appreciate that a pulsed light source and a time-binned photodetector for detecting fluorescence lifetimes can be combined into a low-cost, portable, point-of-care (POC) device that may have applications in clinical or home environments. Such devices may be imaging or non-imaging and may utilize fluorescence lifetime analysis to determine one or more characteristics of a biological sample (e.g., human tissue). In some cases, the device 4-100 for determining characteristics of a biological sample may be used in the field of biological material analysis (e.g., analysis of potentially harmful materials). Some aspects of the POC device and sample analysis using fluorescence lifetimes are described below.

The present inventors have come to recognize and appreciate that some endogenous biomolecules fluoresce with a characteristic lifetime that can be analyzed to determine the state of a patient or the state of a tissue or organ of a patient. Thus, some native biomolecules can serve as endogenous fluorescent molecules of an area of a patient to provide a label-free reporter of that area of a patient. Examples of endogenous fluorescent molecules can include, by way of example and not limitation, hemoglobin, collagen, nicotinamide adenine dinucleotide phosphate (NAD(P)H), retinol, riboflavin, cholecalciferol, folic acid, pyridoxine, tyrosine, dityrosine, glycoside adducts, idramin, lipofuscin, polyphenols, tryptophan, flavin, and melanin.

Intrinsically fluorescent molecules may differ in the wavelength of light they emit and their response to excitation energy. The excitation and fluorescence emission wavelengths of some exemplary intrinsically fluorescent molecules are shown in Table 1. Additional intrinsically fluorescent molecules and their inherent fluorescence wavelengths include retinol-500 nm, riboflavin-550 nm, cholecalciferol-380-460 nm, and pyridoxine-400 nm.

<tables><img file="JP7631258B2_D0006.tif" /></tables>Endogenous fluorescent molecules may also have different fluorescence lifetimes and/or fluorescence lifetimes that are influenced by the surrounding environment. Environmental factors that may affect the fluorescence lifetime of endogenous fluorescent molecules include changes in tissue structure, morphology, oxygenation, pH, vascularity, cell structure and/or cell metabolic state. In some embodiments, the fluorescence lifetime (or average binding lifetime) of healthy tissue may be different from that of unhealthy tissue. Analysis of the fluorescence lifetime detected from a patient's tissue illuminated by a short or ultrashort light pulse may enable clinicians to detect an earlier stage of a patient's disease than other evaluation techniques. For example, some types of skin cancer can be detected at an early stage using fluorescence lifetime analysis before the cancer becomes visible to the naked eye.

In some embodiments, the presence and/or relative concentration of certain biomolecules can be detected to determine the condition of the patient. For some biomolecules, the oxygenation state of the molecule can provide an indication of the patient's condition. The fluorescence lifetime of the molecule can change based on the oxygenation state of the molecule. Analysis of the detected fluorescence lifetimes can be used to determine the relative concentrations of the oxidized and reduced states of the biomolecule in the patient's tissue. The relative concentrations can be indicative of the patient's condition. In some cases, some biomolecules (e.g., NADH) can be bound to other molecules (e.g., proteins) in cells and can have an unbound or free solution state. The bound and unbound states can have different fluorescence lifetimes. Evaluation of the cell or tissue can include determining the relative concentrations of the molecule in free versus bound form based on the fluorescence lifetimes.

Certain biomolecules can provide indicators of various diseases and conditions, including cancer (e.g., melanoma), tumors, bacterial infections, viral infections, and diabetes. As an example, cancer cells and tissues can be distinguished from healthy cells and tissues by analyzing the fluorescence lifetime from certain biomolecules (e.g., NAD(P)H, riboflavin, flavin). Cancer tissues may have higher concentrations of one or more of these biomolecules than healthy tissues. As another example, diabetes in an individual can be assessed by detecting the fluorescence lifetime associated with biomolecules indicative of glucose concentration, such as hexokinase and glycogen adducts. As another example, general changes due to aging can be assessed by detecting collagen and lipofuscin concentrations based on fluorescence lifetime.

In some embodiments, exogenous fluorescent molecules can be incorporated into an area of tissue and used instead of or in addition to endogenous fluorescent molecules. In some cases, exogenous fluorescent markers can be included in a probe or provided as a marker to identify the presence of a target (e.g., a specific molecule, bacteria, or virus) in a sample. Examples of exogenous fluorescent molecules include fluorescent stains, organic dyes, fluorescent proteins, enzymes, and/or quantum dots. Such exogenous molecules can be conjugated to a probe or to a functional group (e.g., a molecule, ion, and/or ligand) that specifically binds to a specific target or component believed to be present in the sample. Attaching an exogenous fluorescent molecule to a probe can allow for target identification by detecting a fluorescence lifetime indicative of the exogenous fluorescent molecule. In some embodiments, the exogenous fluorescent molecule can be included in a composition (e.g., a gel or liquid) that can be easily administered to a patient (e.g., topical application to the skin, ingestion for gastrointestinal imaging).

As can be appreciated, compact POC imaging equipment can enable clinicians to non-invasively assess and/or diagnose a patient's condition. By imaging an acceptable area of tissue with an imaging device rather than extracting a biological sample from the patient, a patient assessment can be performed to reduce the time involved in obtaining a result, reduce the invasiveness of the procedure, reduce costs, and/or facilitate the clinician's ability to treat the patient without having to move the patient to a remote testing location or send a patient sample to a testing facility.

Another application of time-domain fluorescence lifetime imaging is in the field of microscopy. Fluorescence lifetime imaging microscopy (FLIM) can be performed by exciting a sample viewed by a microscope with a short or ultrashort light pulse and detecting the fluorescence emission from the sample by a time-binned photodetector array. The detected fluorescence emission can be analyzed at the pixel level to determine the lifetime of the corresponding imaged portion in the microscope's field of view, and the lifetime data can be mapped to the resulting image of the sample. Thus, based on the fluorescence lifetime, sample properties can be determined at the microscopic level.

The pulsed light source and the time-binned photodetector array can also be used for time-domain applications that do not involve fluorescence lifetime analysis. One such application includes time-of-flight (TOF) imaging. In TOF imaging, a light pulse can be used to illuminate a distant object. Imaging optics can be used to collect backscattered radiation from the pulse and form an image of the distant object on the time-binned photodetector array. At each pixel in the array, the time of arrival of the photon can be determined (e.g., determining when the peak of the backscattered pulse occurs). Because the time of arrival is proportional to the distance between the object and the photodetector array, a three-dimensional map of the object can be created that shows the surface topography of the imaged object.

V. CONFIGURATION Various configurations and embodiments of the apparatus and method can be implemented. Although several example configurations are described in this section, the invention is not limited to only the listed configurations and embodiments.

(1) A pulsed light source comprising: a semiconductor diode configured to emit light; and a drive circuit including a transistor coupled to a terminal of the semiconductor diode, the drive circuit configured to receive a unipolar pulse and to apply a bipolar electrical pulse to the semiconductor diode in response to receiving the unipolar pulse.

(2) The pulsed light source of configuration (1), wherein the bipolar electrical pulse includes a first pulse having a first magnitude and a first polarity followed by a second pulse of opposite polarity having a second magnitude different from the first magnitude.

(3) A pulsed light source according to (2), wherein the second magnitude is between 25% and 90% of the first magnitude.

(4) Any one of the pulsed light sources (1) to (3) further comprising a plurality of wire bonds connected to the terminals of the semiconductor diode.

(5) Any one of the pulsed light sources (1) to (4) further comprising a pulse generator coupled to the drive circuit and configured to form a unipolar pulse and output the unipolar pulse to the drive circuit.

(6) A pulsed light source (5) in which the pulse generator, driver circuit, and semiconductor diode are located on the same printed circuit board.

(7) A pulsed light source (5) in which the pulse generator, the driver circuit, and the semiconductor diode are located on the same substrate.

(8) A pulsed light source according to any one of (1) to (7), in which the pulse length of the unipolar pulse is between 50 ps and 500 ps.

(9) The pulse generator is any one of the pulse light sources (5) to (8) having a first logic gate that forms a unipolar pulse from two differential clock signals.

(10) The first logic gate is a pulsed light source (9) including an emitter-coupled logic gate.

(11) The pulsed light source of (9) or (10), wherein the pulse generator further comprises a fan-out gate configured to receive a single clock signal and output four clock signals to the first logic gate.

(12) The pulsed light source of any one of (9) to (11), wherein the pulse generator further comprises an adjustable delay element configured to vary the pulse length of the unipolar pulse in increments between 1 ps and 5 ps.

(13) A pulsed light source according to any one of (9) to (12), wherein the transistor has a conducting terminal connected between the cathode of the semiconductor diode and a reference potential, and has a gate terminal coupled to the first logic gate.

(14) The pulsed light source of (13), further comprising a capacitor connected between the gate terminal of the transistor and the output from the first logic gate.

(15) A pulsed light source according to any one of (1) to (14), wherein the transistor includes a high electron mobility field effect transistor.

(16) The pulsed light source of any one of (1) to (15), wherein the transistor is configured to switch up to 4 amperes through the semiconductor diode for a duration between 50 ps and 2 ns.

(17) Any one of the pulsed light sources of (9) to (13), further comprising a second logic gate connected in parallel with the first logic gate and configured to form a second unipolar pulse from the two differential clock signals, the output from the second logic gate being coupled to a gate terminal of the transistor.

(18) A pulsed light source according to any one of (1) to (17), in which the drain terminal of the transistor is directly connected to the cathode of the semiconductor diode.

(19) The pulsed light source of (18), further comprising a first capacitor and a resistor connected in parallel to the drain terminal.

(20) The pulsed light source of (18) or (19), further comprising a second capacitor connected between the anode of the semiconductor diode and the source terminal of the transistor.

(21) Any one of the pulsed light sources (5) to (20), wherein the pulse generator and driving circuit are configured to modulate the semiconductor diode with bipolar electrical pulses at a repetition rate between about 30 Hz and about 200 MHz.

(22) Any one of the pulsed light sources (1) to (21) in which optical pulses having a full width at half maximum duration between 50 ps and 500 ps are emitted from a semiconductor diode in response to application of a bipolar electric pulse.

(23) Any one of the pulsed light sources (1) to (21), wherein the optical pulse has a characteristic wavelength selected from the group consisting of 270 nm, 280 nm, 325 nm, 340 nm, 370 nm, 380 nm, 400 nm, 405 nm, 410 nm, 450 nm, 465 nm, 470 nm, 490 nm, 515 nm, 640 nm, 665 nm, 808 nm, and 980 nm.

(24) Any one of (1) to (23) pulsed light sources, in which the tail of the optical pulse remains at least 20 dB below the peak of the pulse 250 ps after the peak of the pulse.

(25) The pulse light source according to any one of (1) to (24), wherein the semiconductor diode includes a laser diode.

(26) A laser diode is a pulsed light source that contains multiple quantum wells.

(27) A pulsed light source according to any one of (1) to (26), wherein the semiconductor diode is a light-emitting diode.

(28) The pulsed light source according to any one of (1) to (27), wherein the semiconductor diode is a slab-coupled optical waveguide laser diode.

(29) The pulsed light source of any one of (1) to (28), further comprising a saturable absorber configured to receive optical pulses from the semiconductor diode.

(30) A pulsed light source according to any one of (1) to (29), wherein the saturable absorber is formed in the same substrate as the semiconductor diode.

(31) The pulsed light source of any one of (1)-(4), (15), (16), (18), and (22)-(30), wherein the driving circuit comprises a transmission line pulse generator.

(32) The pulse light source of (31) further comprising a transmission line formed in a U-shape.

(33) A pulsed light source according to claim (31) or (32), wherein the semiconductor diode is connected to a first end of the transmission line, and further comprising a termination impedance connected to a second end of the transmission line.

(34) The pulsed light source of claim (33), further comprising a shorting transistor configured to short the first end and the second end of the transmission line to a reference potential.

(35) Any one of the pulsed light sources (1) to (34), further comprising a photodetector array having a plurality of pixels, each configured to discriminate photon arrival times into at least two time bins during a single charge accumulation interval, and an optical system configured to form an image of an object illuminated by the pulsed light source on the photodetector array.

(36) The photodetector array is configured to generate a signal representative of the fluorescence lifetime of at least one fluorescent molecule located at the remote target.

(37) The pulsed light source of (35) or (36), further comprising signal processing electronics configured to receive signals representative of the fluorescence lifetime from the photodetector array and generate digital data of an electronic image of the object, the electronic image being indicative of at least one characteristic of the object based on the fluorescence lifetime.

(38) A method for generating light pulses comprising: receiving at least one clock signal; generating electrical pulses from the at least one clock signal; driving a gate terminal of a transistor with the electrical pulses, a current-carrying terminal of the transistor being connected to a semiconductor diode configured to emit light; and applying a bipolar current pulse to the semiconductor diode to generate a light pulse in response to activation of the transistor by the electrical pulse.

(39) The method of embodiment (38), wherein the electrical pulse is a unipolar pulse.

(40) The method of (38) or (39), further comprising adjusting the pulse duration, but not the pulse amplitude, of the unipolar pulse to control the amplitude of the optical pulse.

(41) Any one of the methods (38) to (40), wherein the optical pulse has a full width at half maximum duration between 50 ps and 2 ns.

(42) Any one of the methods (38) to (40), wherein the optical pulse has a full width at half maximum duration between 50 ps and 500 ps.

(43) Any one of the methods (38) to (42), wherein the light pulse has a characteristic wavelength selected from the group consisting of 270 nm, 280 nm, 325 nm, 340 nm, 370 nm, 380 nm, 400 nm, 405 nm, 410 nm, 450 nm, 465 nm, 470 nm, 490 nm, 515 nm, 640 nm, 665 nm, 808 nm, and 980 nm.

(44) Any one of the methods (38) to (43), further comprising repeating the receiving, generating, driving, and applying steps to generate a train of optical pulses at a repetition rate between 30 Hz and 200 MHz.

(45) Any one of the methods (38) to (44), wherein the bipolar current pulse includes a first pulse having a first amplitude and a second pulse having a second amplitude of opposite polarity and different magnitude than the first pulse.

(46) The method of any one of (38) to (45), wherein the semiconductor diode includes a laser diode or a light emitting diode.

(47) The method of any one of (38) to (46), further comprising the step of differentially attenuating a portion of the optical pulse using a saturable absorber.

(48) Any one of the methods (38) to (47), wherein the step of receiving at least one clock signal includes the step of receiving two differential clock signals at a logic gate coupled to gate terminals of the transistor.

(49) Any one of the methods (38) to (47), wherein the step of receiving at least one clock signal includes the step of receiving two differential clock signals at two logic gates coupled in parallel to the gate terminals of the transistor.

(50) Any one of the methods (38) to (49), wherein the step of generating an electrical pulse includes processing two differential clock signals using logic gates coupled to gate terminals of transistors to form an electrical pulse.

(51) The method of (50), further comprising the step of setting the length of the electrical pulse by a phase delay between two differential clock signals.

(52) Any one of the methods (38) to (51), wherein the step of generating an electrical pulse includes processing two differential clock signals using two logic gates coupled in parallel to gate terminals of a transistor to form an electrical pulse.

(53) The method of any one of (38) to (52), further comprising illuminating the sample with a light pulse from a semiconductor diode and detecting a fluorescence lifetime from the sample.

(54) The method of (53), further comprising the step of distinguishing between two different fluorescent molecules or at least two different fluorescence lifetimes having different decay rates associated with the environment in which the molecules are located, wherein the light pulses are at a single characteristic wavelength.

(55) The method of (53) or (54), further comprising determining at least one characteristic of the sample based on the detected fluorescence lifetime.

(56) The method of (55), further comprising generating an electronic image of an area of the sample and indicating at least one characteristic based on the fluorescence lifetime in the image.

(57) Any one of the methods of (38) to (52), further comprising the steps of illuminating the sample with a light pulse from a semiconductor diode and discriminating, using a single photodetector, the arrival times of photons backscattered from the sample into at least two time bins during a single charge accumulation interval of the single photodetector.

(58) The method of (57), further comprising generating an electronic three-dimensional image of the sample based on the differentiated times of arrival.

(59) A fluorescence lifetime analysis system comprising a semiconductor diode configured to emit light, a drive circuit configured to apply a bipolar current pulse to the semiconductor diode to generate a light pulse, an optical system configured to deliver the light pulse to a sample, and a photodetector configured to discriminate photon arrival times into at least two time bins during a single charge accumulation interval of the photodetector.

(60) The system of (59), further comprising a pulse generator configured to provide an electrical pulse to the current drive circuit, the current drive circuit configured to apply a bipolar pulse to the semiconductor diode in response to receiving the electrical pulse.

(61) The system of (60), wherein the electrical pulse is a unipolar pulse having a duration between 50 ps and 2 ns.

(62) The system of (60) or (61), wherein the current driver circuit comprises a transistor having a gate terminal coupled to an output from the pulse generator and having a conducting terminal connected between a terminal of the semiconductor diode and a reference potential.

(63) The system of (62), further comprising a first resistor and a first capacitor connected in parallel between the anode and cathode of the semiconductor diode, and a second resistor and a second capacitor connected in parallel between the gate terminal of the transistor and a reference potential.

(64) The system of any one of (59) to (63), wherein the semiconductor diode includes a laser diode or a light emitting diode.

(65) The system of any one of (59) to (63), further comprising a plurality of wire bonds connected to the terminals of the semiconductor diode.

(66) Any one of the systems (59) to (63), wherein the optical pulse has a full width at half maximum duration between 50 ps and 500 ps.

(67) Any one of the systems (59) to (63), wherein the optical pulse has a characteristic wavelength selected from the group consisting of 270 nm, 280 nm, 325 nm, 340 nm, 370 nm, 380 nm, 400 nm, 405 nm, 410 nm, 450 nm, 465 nm, 470 nm, 490 nm, 515 nm, 640 nm, 665 nm, 808 nm, and 980 nm.

(68) Any one of the systems (59) to (63), further comprising an array of photodetectors in which photodetectors are arranged, the array of photodetectors being configured to time bin fluorescence from the sample during a single charge accumulation interval of a light pulse.

(69) The system of (68), further comprising an imaging optical element positioned between the sample and the photodetector array, the imaging optical element configured to form an image of an area of the sample illuminated by the light pulse at the photodetector array.

(70) The system of (69), wherein the image formed at the photodetector array is an image of a small area of the sample.

(71) A pulsed light source comprising: a semiconductor diode configured to emit light; a first logic gate configured to form a first pulse at an output of the first logic gate; and a drive circuit coupled to the first logic gate, the drive circuit configured to receive the first pulse and apply a bipolar electrical pulse to the semiconductor diode to generate a light pulse in response to receiving the first pulse.

(72) A pulsed light source (71), wherein the first pulse is a unipolar pulse.

(73) The pulsed light source of (72), further comprising a fan-out gate and a delay element coupled to the first logic gate, the delay element delaying at least one output from the fan-out gate.

(74) The pulsed light source of (73), wherein the delay element is configured to vary the pulse length of the unipolar pulse in increments between 1 ps and 5 ps.

(75) The pulse light source of any one of (71) to (74), wherein the first logic gate is configured to form a first pulse from the two differential clock signals.

(76) The pulsed light source of any one of (71) to (75), wherein the bipolar electric pulse includes a first pulse having a first magnitude and a first polarity followed by a second pulse of opposite polarity having a second magnitude different from the first magnitude.

(77) A pulsed light source according to (76), wherein the second magnitude is between 25% and 90% of the first magnitude.

(78) The pulsed light source of any one of (71) to (77), further comprising a plurality of wire bonds connected to the terminals of the semiconductor diode.

(79) Any one of the pulsed light sources (75) to (78), further comprising a second logic gate configured to form a second pulse from two differential clock signals, the second logic gate being connected in parallel with the first logic gate, and an output of the second logic gate being coupled to a drive circuit.

(80) The pulsed light source according to any one of (71) to (79), further comprising a transistor in the drive circuit having a current-carrying terminal connected between the semiconductor diode and a reference potential.

(81) The optical pulses are a pulsed light source having a duration between 50 ps and 2 ns. (80)

(82) A pulsed light source comprising: a semiconductor diode configured to emit light; and a driver circuit including a transistor coupled to a terminal of the semiconductor diode, the driver circuit configured to receive a unipolar pulse and to apply a bipolar electrical pulse to the semiconductor diode in response to receiving the unipolar pulse, the transistor being connected in parallel with the semiconductor diode between a current source and a reference potential.

(83) A pulsed light source according to (82), further comprising a resistor and a capacitor connected in parallel between the semiconductor diode and a reference potential, and optionally having any one of features (2) to (4), (15), and (22) to (30), excluding feature (1).

(84) A pulsed light source of (82) or (83), wherein the transistor is normally conducting and configured to be pulsed off by a unipolar pulse.

(85) Any one of the pulsed light sources (82) to (84), further comprising a photodetector array having a plurality of pixels, each configured to discriminate photon arrival times into at least two time bins during a single charge accumulation interval, and an optical system configured to form an image of an object illuminated by the pulsed light source on the photodetector array.

(86) A pulsed light source comprising a semiconductor diode configured to emit light and a plurality of first circuit branches connected to a first terminal of the semiconductor diode, each circuit branch comprising a transistor having a current-carrying terminal connected between a reference potential and the first terminal of the semiconductor diode.

(87) A pulsed light source (86), wherein a first reference potential in a first circuit branch of the plurality of first circuit branches has a different value than a second reference potential in a second circuit branch of the plurality of first circuit branches, optionally having any one of features (4), (15), (16), and (22) through (30), except for feature (1).

(88) A pulsed light source of (86) or (87), wherein a first reference potential in a first circuit branch of the plurality of first circuit branches has a positive value and a second reference potential in a second circuit branch of the plurality of first circuit branches has a negative value.

(89) The pulsed light source of any one of (86) to (88), further comprising a resistor in each circuit branch connected between the conductive terminal of the transistor and a reference potential.

(90) The pulsed light source of any one of (86) to (89), further comprising a capacitor in each circuit branch connected between the conductive terminal of the transistor and ground potential.

(91) Any one of the pulsed light sources (86) to (90), further comprising a photodetector array having a plurality of pixels, each configured to discriminate photon arrival times into at least two time bins during a single charge accumulation interval, and an optical system configured to form an image of an object illuminated by the pulsed light source on the photodetector array.

(92) A pulsed light source comprising: a radio frequency amplifier providing a signal and an inverted signal; a logic gate configured to receive the signal and the phase-shifted inverted signal and to output a pulse and an inverted pulse; a combiner configured to combine the pulse and the inverted pulse to a common output; and a semiconductor diode coupled to the common output and configured to generate an optical pulse in response to receiving the pulse and the inverted pulse.

(93) A pulsed light source according to (92), further comprising a variable attenuator configured to attenuate the pulse or the inverted pulse, and optionally having any one of features (4) to (15), (16), and (22) to (30), excluding feature (1).

(94) The pulsed light source of (92) or (93), further comprising a delay element configured to delay in time the pulse or the inverted pulse.

(95) The pulsed light source of any one of (92) to (94), further comprising a DC block connected to the input of the radio frequency amplifier.

(96) Any one of the pulsed light sources (92) to (95), further comprising a photodetector array having a plurality of pixels, each configured to discriminate photon arrival times into at least two time bins during a single charge accumulation interval, and an optical system configured to form an image of an object illuminated by the pulsed light source on the photodetector array.

(97) A pulsed light source comprising: a radio frequency logic gate configured to receive a first signal and an inverted version of the first signal and to output a pulse and an inverted version of the pulse; and a semiconductor diode connected to the radio frequency logic gate, the semiconductor diode configured to receive the pulse at a first terminal of the semiconductor diode and the inverted version of the pulse at a second terminal of the semiconductor diode and to emit a light pulse.

(98) A pulsed light source according to (97), further comprising a first amplifier configured to receive a periodic signal and output a first signal and an inverted version of the first signal, and a phase adjuster configured to vary the phase of the first signal or the inverted version of the first signal, optionally having any one of features (4), (15), (16), and (22) to (30), except for feature (1).

(99) The pulsed light source of (97) or (98), further comprising a photodetector array having a plurality of pixels, each configured to discriminate photon arrival times into at least two time bins during a single charge accumulation interval, and an optical system configured to form an image of an object illuminated by the pulsed light source on the photodetector array. VI. Conclusion Thus, while certain aspects of certain embodiments of a pulsed laser have been described, it should be appreciated that various changes, modifications, and improvements will readily occur to those skilled in the art. Such changes, modifications, and improvements are intended to be part of this disclosure and are intended to be within the spirit and scope of the invention. While the present teachings have been described in connection with various embodiments and examples, it is not intended that the present teachings be limited to such embodiments or examples. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those skilled in the art.

While various inventive embodiments have been described and illustrated, those skilled in the art will readily envision various other means and/or structures for performing the functions and/or obtaining one or more of the results and/or advantages described, and each of such variations and/or modifications is deemed to be within the scope of the described inventive embodiments. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described are intended to be examples, and that the actual parameters, dimensions, materials, and/or configurations will depend on the particular application or applications in which the teachings of the present invention are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described. It is therefore to be understood that the above-described embodiments are presented by way of example only, and that within the scope of the appended claims and equivalents thereof, the inventive embodiments may be practiced otherwise than as specifically described and claimed. The inventive embodiments of the present disclosure may be directed to each individual feature, system, system upgrade, and/or method described. Additionally, any combination of two or more such features, systems, and/or methods is within the inventive scope of the present disclosure, provided that such features, systems, system upgrades, and/or methods are not mutually inconsistent.

Moreover, although several advantages of the present invention may be noted, it should be appreciated that not all embodiments of the present invention include all described advantages. Some embodiments may not implement any feature that is described as being advantageous. Accordingly, the foregoing description and drawings are for illustrative purposes only.

Numerical values and ranges may be described herein and in the claims as approximate or exact values or ranges. For example, in some instances, the terms "about," "approximately," and "substantially" may be used in reference to values. Such references are intended to encompass the referenced value as well as values plus and minus reasonable variations therein. For example, the phrase "between about 10 and about 20" is intended to mean "between exactly 10 and exactly 20" in some embodiments, and "between 10+δ1 and 20+δ2" in some embodiments. The amount of variation in values δ1, δ2 may be less than 5% of the value in some embodiments, less than 10% of the value in some embodiments, and even less than 20% of the value in some embodiments. In embodiments where a large range of values is given, such as a range including more than one order of magnitude, the amount of variation in values δ1, δ2 may be as high as 50%. For example, if the operable range extends from 2 to 200, "about 80" may encompass values between 40 and 120, and the range may be as large as between 1 and 300. Where an exact value is intended, the term "exactly" is used, such as, for example, "between exactly 2 and exactly 200."

All literature and similar materials cited in this application, including but not limited to patents, patent applications, articles, books, papers, and web pages, regardless of the form of such literature and similar materials, are expressly incorporated by reference in their entirety. In the event that one or more of the incorporated literature and similar materials differs or conflicts with this application, including but not limited to defined terms, term usage, techniques described, etc., this application controls.

The section headings used are for organizational purposes only and should not be construed as limiting the subject matter described in any way.

Also, the described techniques may be embodied as a method, at least one example of which is provided. Operations performed as part of a method may be ordered in any suitable manner. Thus, embodiments may be constructed in which operations are performed in an order different from that shown, and may include performing some operations simultaneously, even if shown as sequential operations in the example embodiments.

All definitions, and as used, should be understood to take precedence over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the terms defined.

The indefinite articles "a" and "an," as used in the specification and claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

The term "and/or," as used herein and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjointly present in some cases and disjointly present in other cases. Multiple elements listed with "and/or" should be construed in the same manner, i.e., as "one or more" of the elements so conjoined. Other elements, whether related or unrelated to those elements specifically identified, may optionally be present other than the elements specifically identified by the "and/or" clause. Thus, as a non-limiting example, when used with open-ended language such as "comprising," a reference to "A and/or B" may refer in one embodiment to only A (optionally including elements other than B), in another embodiment to only B (optionally including elements other than A), in yet another embodiment to both A and B (optionally including other elements), and so forth.

As used herein and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" should be interpreted as being inclusive, i.e., including at least one of a plurality of elements or a list of elements, but also including more than one, and optionally including additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of" or "exactly one of," or, when used in the claims, "consisting of," refer to the inclusion of exactly one element of a plurality of elements or a list of elements. In general, the term "or" as used should only be interpreted as indicating exclusive alternatives (i.e., "one or the other, but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, should have its ordinary meaning as used in the field of patent law.

As used herein and in the claims, the phrase "at least one" referring to a list of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of every element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows for elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified, may optionally be present. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B" or, equivalently, "at least one of A and/or B") can refer in one embodiment to at least one A with no B present, optionally including two or more As (and optionally including elements other than B), in another embodiment to at least one B with no A present, optionally including two or more Bs (and optionally including elements other than A), in yet another embodiment to at least one A, optionally including two or more As, and at least one B, optionally including two or more Bs (and optionally including other elements), etc.

In the claims and in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "including," "holding," "consisting of," and the like, are to be understood as open-ended, i.e., meaning including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively.

The claims should not be read as limited to the described order or elements unless so stated. It should be understood that various changes in form and detail can be made by those skilled in the art without departing from the spirit and scope of the appended claims. All embodiments that come within the spirit and scope of the following claims and equivalents thereto are claimed.