Scanning microscope for examining microscopic preparations and lighting device for same

The present invention relates to lighting methods and devices for objects.

Patent Document 1 discloses "an ultrashort pulse source capable of controlling multiple wavelength outputs. This ultrashort pulse source is particularly used for a multiphoton type microscope. This microscope system is" a constant wavelength. It has an ultrashort wavelength pulse laser that generates ultrashort wavelength photons and "at least one wavelength conversion channel."

Patent Document 2 discloses "a device for generating a wide band spectrum in the visible spectrum range and the infrared spectrum range. This device is based on a microstructure fiber to which the light of a pulse laser is coupled. The pump light is "diffused by a non-linear effect within the microstructured fibers. As the microstructured fibers," so-called photonic band gap materials or "photonic crystal fibers", "holey fibers", or "microstructured fibers" are used. The so-called "hollow fiber" is also known.

Another device for generating a wide band spectrum is "disclosed in Non-Patent Document 1. This device uses a conventional optical fiber with a fiber core, and" this optical fiber is at least partially tapered. This type of optical fiber is known as a so-called "tapered fiber".

From the PCT application of Patent Document 3, "an optical amplifier whose amplification degree can be adjusted according to a wavelength is known. Further, in this publication, a fiber light source based on this principle is disclosed.

The arc lamp is known as a wideband light source, and "it is used in many fields. As an example, here, Patent Document 4 (XENON PHOTOGRAPHY LIGHT) is mentioned, but in this publication, xenon for photographic lighting is mentioned. The arc lamp is disclosed.

In particular, microscopes, "endoscopes," flow cytometry, "chromatography," in lithography, "a universal illuminator with a high light illumination density is important for illuminating an object. In a scanning microscope. , "Samples are scanned by light rays. Therefore," lasers are often used as light sources. For example, the "multicolor fluorescence cofocal microscope system" described in Patent Document 5 "emits multiple laser beams. Devices with one laser are known. Most recently mixed gas lasers, "especially ArKr lasers are used. As samples," for example, biological tissue or sections prepared with a fluorescent dye are examined. Illumination light reflected by the sample is often detected in the material inspection area. Solid-state lasers, dye lasers, fiber lasers and optical parametric oscillator OPOs (with pump lasers located upstream) are also frequently used.

Microspot arrays, or so-called microplates, "in the field of genetic diagnostics," medical diagnostics, "biodiagnosis," have a large number of specially marked spots (favorably marked in a grid). Used for inspection. Patent Document 6 discloses "a microplate reader in which both the excitation wavelength and the detection wavelength can be adjusted.

The above-mentioned conventional lighting methods and lighting devices have some drawbacks. Known broadband luminaires "in most cases have a lower illumination density than laser-based luminaires," while "on the other hand, only discontinuous wavelength lines are provided to the user," their spectral state and width. "In most cases, it can be adjusted only slightly. Due to this limitation of the operating spectrum, known luminaires are not adaptable. In addition," laser-based luminaires and lighting methods are "laser light. Due to the high coherence of "the disadvantage is that defective interference phenomena, such as diffraction rings," Newton rings occur. To reduce such interference, "additional optical elements may be used. However, "the light power is reduced by the intrinsic absorption and scattering.

<p><patcit num="1"><text>German Federal Republic Patent Publication No. 19853669A1</text></patcit><patcit num="2"><text>U.S. Pat. No. 6097870</text></patcit><patcit num="3"><text>International publication number WO00 / 04613</text></patcit><patcit num="4"><text>U.S. Pat. No. 3,720,822</text></patcit><patcit num="5"><text>European Patent No. 049530</text></patcit><patcit num="6"><text>European Patent Publication No. 0841557A2</text></patcit></p>

<p><nplcit num="1"><text>Birks et al., "" Supercontinuum generation in tapered fibers ", Opt. Lett. Vol. 25," p. 1415 (2000).</text></nplcit></p>

<p> An object of the present invention is to provide an object lighting method and device that avoids or eliminates the above-mentioned drawbacks and problems.</p><p> In addition, "to provide the illumination method and apparatus that are universally usable," adaptable, "provide a broadband wavelength spectrum at high illumination densities," and avoid interference phenomena as much as possible. ..</p>

<p> In order to solve the above problems, the present invention provides an object lighting method.<u style="single">A step of injecting a laser beam into a microstructure optical element that spreads the spectrum, a step of shaping the spectrally diffused light into an illumination light beam, and a step of dividing the light beam into partial rays and directing the partial rays to an analyzer. And the step of supplying the electrical signal from the analyzer to the processing unit, where the spectral components of the partial light are displayed in the form of a graph on a monitor to which a computer is assigned and the spectrum through the processing unit. The step of adjusting the spectral component of the diffused light and the step of controlling the power variable device so that the illumination light is adjusted to the preselected spectral component of the spectrally diffused light, and the target of the illumination light. Steps to point things</u>It is characterized by containing.</p><p> In addition, in lighting equipment,<u style="single">Microstructure optics are provided upstream of the optical system that shapes the spectrally diffused light into illumination rays, the optical system is located upstream of the beam splitter, which the beam splitter splits into partial rays. Directing this to the analyzer, the processing unit receives an electrical signal from the analyzer, displays the spectral components in the form of a graph on the monitor, and adjusts the specific spectral components by transforming the graph on the monitor with the mouse. A means for this is provided, and a power variable device for changing the power of the spectrally diffused light based on the adjusted spectral component is provided.</u>It is characterized by that.</p><p> The advantages of the illumination methods and devices according to the invention are that they are "universally usable," easy to operate, "adaptable," and "illuminated with light in a wider wavelength range." , "Light has a very low coherence, so" a malfunctioning interference phenomenon is avoided.</p><p> Since the fine structure fiber is used, a wide-band continuous wavelength spectrum can be used. It should be noted that "microstructure fibers have already been described in Patent Document 2 and Non-Patent Document 1 by Birks et al., But the devices disclosed in these documents are particularly individual optical elements and their mutual positions. Since the adjustment is complicated, "the operation is troublesome," it is not adaptable, and "it is easy to break down again."</p><p> In particular, "an embodiment in which an optical system for shaping spectrally diffused light into one ray is provided on the downstream side of the" microstructure optical element "is advantageous. This optical system" houses the entire device. It is advantageous to place it inside the casing, just before the light exit hole or inside the light outlet hole. This optical system also provides "various divergent rays," collimated rays, "convergent rays. It is advantageous to have a variable magnification optical system (Variooptik) that produces.</p><p> As a laser, "all normal types of lasers can be used. In an advantageous configuration," the laser is a short pulse laser, "for example, emitting an optical pulse with a duration of 100 fs to 10 ps," mode-coupled. It is a solid-state laser. It is advantageous that the laser wavelength matches the "zero dispersion wavelength" of the fiber, and "or vice versa. On the surface, it is" zero dispersion. " The wavelength may "shift" over a particular wavelength range, but "this must be taken into account when pulling the fiber.</p><p> It is particularly advantageous for the luminaire to have a device that changes the power of the spectrally diffused light, in which case the power of the spectrally diffused light is applied with respect to at least one selectable wavelength. Alternatively, it is particularly advantageous to configure the illuminator so that it is variable or completely narrowed with respect to at least one selectable wavelength range.</p><p> It is advantageous to provide a device that varies the power of the spectrally diffused light. This optical power variable device is an acoustic or electro-optical element such as, for example, an acusto optical tunable filter AOTF. Similarly, dielectric filters or color filters arranged in a daisy chain may be used. For particular adaptability, the filter is attached to a revolver or slide frame that facilitates insertion of spectrally diffused light into the optical path.</p><p> A configuration in which at least one wavelength range is selected from the spectrally diffused light and the light in the selected wavelength range is directed toward the object is particularly advantageous. A device that achieves this, for example, is that the spectrally diffused light is spatially split and the spectral components are suppressed or completely narrowed down by an appropriate variable aperture or filter device, and then the remaining spectral components are again 1 Integrate into one ray. For spatial spectral division, for example, prisms or grids can be used.</p><p> The method according to the invention, in a particular embodiment, comprises adjusting the power of the spectrally diffused light. In other embodiments, a fabric perofilter is provided to change the power of the spectrally diffused light. LCD filters can also be used.</p><p> The illumination method according to the invention includes, in a particularly advantageous embodiment, an additional step of adjusting the spectral components of the spectrally diffused light. In a particularly advantageous embodiment, an operating element for adjusting the power of the spectrally diffused light and synthesizing the spectrum is provided directly on the casing. The operating element is an operating desk or personal computer. The adjustment data is sent in the form of an electrical signal to a lighting device or a device that changes the power of spectrally diffused light. Specifically, the adjustment is performed via a slider displayed on the display of a personal computer and operated by a mouse of the computer, for example.</p><p> According to the present invention, it has been clarified that the divergence of the light beam incident on the microstructure optical element significantly affects the spectral distribution of the specularly diffused light. In a particularly advantageous and adaptable configuration, the illuminator has focusing optics that focus the light of the laser to microstructured optics. It is particularly advantageous to configure the focusing optical system as a variable magnification optical system, for example, a zoom optical system.</p><p> Since the spectral distribution of specularly diffused light depends on the polarization and wavelength of the light incident on the microstructure optics, special embodiments are provided with devices for adjusting and controlling these parameters. For lasers that emit linearly polarized rays, a rotatably supported λ / 2 plate is used to rotate the plane of polarization. Using a Pockels cell, or Faraday rotator, which allows adjustment of any elliptical polarization, is somewhat costly, but adaptable. For wavelength adjustment, it is advantageous to provide a double refraction plate or a tiltable weights and measures meter in the laser.</p><p> In a particular embodiment, a device is provided that enables analysis of wavelength diffused light, especially with respect to spectral components and optical power. The analyzer is arranged so that a portion of the spectrally diffused light is split and supplied to the analyzer using, for example, a beam splitter. It is advantageous that the analyzer is a spectrometer. The illuminator has, for example, a prism or grid for dividing the spatial spectrum and a CCD element or multi-channel photomultiplier tube as a detector. In another modified embodiment, the analyzer has a multi-band detector. A semiconductor spectrometer can also be used.</p><p> To set the power of spectrally diffused light, the detector is configured to generate an electrical signal proportional to the optical power so that this electrical signal can be evaluated by an electronic system or computer.</p><p> In a particularly advantageous embodiment, a display device is provided for displaying the power of the spectrally diffused light and / or the spectral components of the spectrally diffused light. The display is mounted directly on the casing or operation desk. In other embodiments, a personal computer monitor is used to display the power or spectral components.</p><p> The method according to the invention, according to other configurations, comprises adjusting the polarization of the spectrally diffused light. For this reason, a rotatable arrangement polarizing filter, a λ / 2 plate or a Pockels cell or a Faraday rotator is provided.</p><p> In a very advantageous embodiment, the laser is a pulsed laser that advantageously emits an optical pulse with a pulse energy greater than 1nJ. In connection with this configuration, the method according to the invention includes an additional step of adjusting the pulse width of the spectrally diffused light. In addition, it would be advantageous if the method included other steps in adjusting the chapter of spectrally diffused light. These additional steps allow the pulse characteristics of the light directed at the object to be individually adapted to the object each time. A chapter is a series of light of different wavelengths in one pulse. For this reason, the device according to the invention advantageously has a prism or grid device. The prism or grid device is combined with an LCD striped grid in a particularly advantageous embodiment. Arrangement configurations that vary pulse time and chapter are conventionally known to those of skill in the art.</p><p> The illumination methods and devices according to the present invention are particularly suitable for illumination of micro-optical objects, particularly said illumination in microscopes, video microscopes, scanning microscopes or confocal scanning microscopes. In the case of using fluorescent light or relying on Felster transfer, it is particularly advantageous to accurately match the wavelength of the light directed at the object with the excitation wavelength of the fluorescent dye in the object.</p><p> The methods and devices according to the invention can also be advantageously used in endoscopes, flow cytometry and lithography. The microstructure optical element is composed of a large number of microoptical structural elements having at least two different optical densities in an advantageous embodiment of the scanning microscope. In a particularly advantageous embodiment, the optical element has a first region and a second region, the first region has a homogeneous structure, and within the second region is a microscopic structure consisting of microoptical structural elements. The structure is formed. It is also advantageous that the first region surrounds the second region. The micro-optical structural element is preferably a cannula, strip, honeycomb, tube piece or hollow space.</p><p> In other configurations, the microstructure optics consist of a glass or plastic material juxtaposed with each other and a hollow space. In a particularly advantageous modification embodiment, the microstructure optical element is made of a photonic band gap material and is configured as an optical fiber. In this case, it is advantageous to provide a light diode between the laser and the optical fiber that suppresses the back reflection of the laser beam at the end of the optical fiber.</p><p> According to a particularly advantageous and easily realized variant, a conventional optical fiber with a fiber core of about 9 μm is used as the microstructure optic, which has a taper along at least part of it. doing. This type of optical fiber is known as a so-called tapered fiber. Advantageously, the optical fiber should be 1 m long in total and 30 mm to 90 mm long with a tapered portion. The diameter of the optical fiber has been reduced to approximately 2 μm in the tapered region. Correspondingly, the diameter of the fiber core is in the nanometer range.</p>

<figref num="1">It is a flowchart of the method by this invention.</figref><figref num="2">It is a figure which shows the illuminating device by this invention which includes a power measuring instrument and a display device.</figref><figref num="3">It is a figure which shows an example which used the apparatus by this invention for a confocal scanning microscope.</figref><figref num="4">It is a figure which shows the microstructure optical element.</figref><figref num="5">It is a figure which shows the other embodiment of the microstructure optical element.</figref>

Next, an embodiment of the present invention will be described in detail with reference to the accompanying drawings. FIG. 1 is a flowchart of the method according to the present invention. In the first step 1, the light of the laser is incident on the microstructure optical element. Ultrastructure optics spread the spectrum of light. In this case, the light is guided to the microstructure optical element by, for example, a plurality of mirrors, and is advantageously focused on the microstructure optical element by the zoom lens. In the second step 3, the light emitted from the microstructure optical element is formed into an illumination ray, which is advantageously formed using a collimating optical system configured as a lens system. In the next step 5, the illumination ray is directed toward the object.

FIG. 2 shows the lighting device 7. The illuminating device 7 has a laser 9. The laser 9 is configured as a mode-coupling titanium sapphire laser 11 and emits light 13 (indicated by the dashed line) in the light pulse train. The duration of the optical pulse is approximately 100 fs when the number of repetitions is about 80 MHz. The light beam 13 is focused on the microstructure optical element 19 by the focusing optical system 15 (configured as the zoom optical system 17 and arranged so as to be movable along the propagation direction of the light beam). The microstructure optical element 19 is composed of an optical fiber 23 having a tapered portion 21. Laser light is spectrally diffused at microstructure optics 19 (spektral) verbreitern). All components are in a casing 25 with a light outlet hole 27, through which the illumination ray 29 leaves the casing 25 as a divergent ray. The spectrum of the spectrally diffused light 31 reaches a wavelength of approximately 300 nm to 1600 nm, and the optical power is sufficiently constant throughout the spectrum. The spectrum-diffused light 31 emitted from the optical fiber 31 is formed into a collimated illumination ray 29 by the optical system 33. The beam splitter-35 splits the illumination ray 29 into a partial ray 37, which is guided to the analyzer 39. The analyzer 39 has a prism 41 that branches a partial light ray 37 to form a luminous flux 43 that diverges and extends in the branch plane, and a photodiode cell 45 for photodetection. The photodiode cell 45 generates electrical signals proportional to the power of light in each spectral range, and these electrical signals are sent to the processing unit 47. The processing unit 47 is connected to the personal computer 49, and the monitor 51 of the personal computer 49 has two axes 55, whose spectral components are in the form of graph 53. It is displayed in the coordinate system with 57. Coordinate axis 55 represents the wavelength, and coordinate axis 57 represents the power of light. By clicking the graph 53 with the mouse 59 of the computer and moving the mouse 59 at the same time, the graph 61 shown by the broken line is obtained. This graph 61 can be deformed according to the movement of the mouse 59 of the computer. When a new click is made with the mouse 59, the power variable device 63 is controlled via the computer 49, and the preselected spectral components are displayed in the dashed graph 61. The power variable device 63 of the spectrally diffused light 31 is implemented as an AOTF (acousto optical tunable filter) 65 and is configured to control the wavelengths independently of each other and thus to adjust the spectral components of the spectrally diffused light 31. Has been done. Furthermore, the output of the laser 9 is also controlled via the computer 49. The user makes adjustments using the mouse 59 of the computer. A slider 67 is displayed on the monitor 51 and is used to adjust the total power of the spectrally diffused light 31.

FIG. 3 shows an example in which the apparatus according to the present invention is applied to a confocal scanning microscope 69. Illumination rays 29 coming from the illuminator 7 are reflected from the beam splitter-71 to the scan module 73. The scan module 73 includes a cardan-supported scan mirror 75 that allows light rays 29 to pass through the microscope optics 77 and is guided through or by the object 79. Let me. The illumination ray 29 is guided through the surface of the object 79 when the object 79 is non-transparent. If the object 79 is a biological object, or if it is transparent, the illumination ray 29 is also guided by the object 79. This means that the various focal planes of the object 79 are sequentially illuminated by the illumination rays 29 and thus scanned. In this case, the ex post facto composition yields a three-dimensional image of the object 79. At the imaging stage, the light rays 29 coming from the illuminating device 7 are shown by solid lines. The light 81 emitted from the object 79 passes through the microscope optical system 77, reaches the beam splitter-71 via the scan module 73, passes through this, and hits the detector 83 implemented as a photomultiplier tube. The light 81 emitted from the object 79 is indicated by a broken line. In the detector 83, a detection signal proportional to the power of the light 81 coming from the object 79 is generated and processed. The illumination pinhole 85 and the detection pinhole 87, which are usually provided in a confocal scanning microscope, are shown schematically for the sake of perfection, whereas the light rays are guided or shaped to make the figure easier to understand. The optical element to be used is not shown. This point is known to those skilled in the art.

FIG. 4 shows one embodiment of the microstructure optical element 19. The microstructure optical element 19 is made of a photonic band gap material and has a special honeycomb-like microstructure 89. This honeycomb structure is particularly suitable for generating wideband light. The diameter of the glass internal cannula 91 is approximately 1.9 μm. The internal cannula 91 is surrounded by a piece of glass 93. The glass strip 93 forms a honeycomb-shaped hollow space 95. These micro-optical structural elements work together to form a second region 97, which is surrounded by a first region 99, which is implemented as a glass coating.

FIG. 5 shows an embodiment of the microstructure optical element 19. In this embodiment, the microstructure optical element 19 is made of a conventional optical fiber 101, has an outer diameter of 125 μm, and includes a fiber core 103. The inner diameter of the fiber core 103 is 6 μm. The outer diameter of the optical fiber 101 is reduced to 1.8 μm in the region of the tapered portion 105 having a length of 300 mm. The diameter of the fiber core 103 in this region is only a fraction of a micrometer.

Although the present invention has been described above with respect to a specific embodiment, it goes without saying that various changes and modifications may be made without departing from the scope of protection of rights within the scope of claims of the present application.

7 Lighting equipment 9 laser 11 Titanium sapphire laser 13 Rays of light pulse train 15 Focusing optics 17 Zoom optics 19 Microstructure optics 21 Tapered part 23 optical fiber 25 casing 27 Light outlet hole 29 Illumination rays 31 Spectral diffused light 33 Optical system 35 Beam Splitter- 37 partial rays 39 Analyzer 41 prism 43 Luminous flux 45 photodiode cell 47 Processing unit 49 personal computer 51 monitor 53 Graph of spectral components 55 Coordinate axes 57 Coordinate axes 59 Computer mouse 63 Variable power device 65 AOTF (acousto optical tunable filter) 67 slider 69 Confocal scanning microscope 71 Beam Splitter- 73 Scan module 75 scan mirror 77 Microscope optics 79 Object 81 Light emitted from object 79 83 Detector 85 Lighting pinhole 87 Detection pinhole 89 Honeycomb microstructure 91 Cannula inside the glass 93 Glass strips 95 hollow space 97 Second area 99 First area 101 optical fiber 103 fiber core 105 Tapered part