Method and apparatus for confocal microscopy

The invention relates to a method for confocal microscopy, in which laser light from different spectral regions is coupled into a microscope beam path deflected in at least two coordinates and is directed temporally successively to the locations of a specimen, wherein the specimen is arranged in at least one plane by location and line by line The laser light is applied, and an image of the scanned plane is generated from the light reflected and / or emitted by the applied locations. The invention further relates to a laser scanning microscope for carrying out this method.

Whereas conventional optical microscopy is the only way to achieve the optical detection of an imaging plane, confocal microscopy offers the possibility to image and measure microstructures in the Z axis of the room as a special development of light microscopy. With the light microscope, it is not possible, for example, to obtain an impression of the spatial structure of the rough surface of a sample at high magnification, since only a small region of the specimen can be shown sharply, while depth details of the surface are determined by the high Scattered light component and the missing axial resolution are blurred.

In the confocal laser scanning microscope, however, the scattered light is largely eliminated and only the structures which are located in the focal plane of the objective are imaged. If the radiation is focused on different planes, three-dimensional images of a sample can be calculated from the scanning of these planes in the direction of the Z-axis.

For this purpose, a first perforated diaphragm is imaged in a point-shaped manner into the object plane, laser being used as the illumination source. The spot-shaped laser beam is moved in a grid-like manner by means of deflection mirrors in a location-by-line and line-by-line manner. Through the microscope objective, the light reflected and / or emitted by the sample is focused onto a second perforated diaphragm which is arranged conjugate to the first perforated diaphragm. The arrangement of these two apertured diaphragms has the result that only information from the focal plane passes to one or more detectors, which are arranged downstream of the second perforated diaphragm.

The scattered light, which is generated above and below the focus, is eliminated by the second pinhole aperture. The information obtained from a two-dimensional deflection from a plurality of superimposed imaging planes is stored and further processed to form images.

This principle of confocal laser scanning microscopy is described, for example, in FIG <nplcit id="ncit0001" npl-type="s"><text>Schroth: Confocal Laser Scanning Microscopy, a New Method of Investigation in Material Testing ", Journal of Material Testing, vol. 39 (1997), vol. 6, pages 264 ff</text></nplcit>.

In "<nplcit id="ncit0002" npl-type="s"><text>SCANNING MICROPHOTOLYSIS: A NEW PHOTOBLEACHING TECHNIQUE BASED ON FAST INTENSITY MODULATION OF A SCANNED LASER BEAM AND CONFOCAL IMAGING; Journal of Microscopy, Vol. 176 Pt 1, October 1994 pp. 22 - 33</text></nplcit>" and "<nplcit id="ncit0003" npl-type="s"><text>LINE-SCANNING MICROPHOTOLYSIS FOR DIFFRACTION-LIMITED MEASUREMENTS OF LATERAL DIFFUSION; Biophysical Journal, Vol. 71 September 1996, pages 1621-1632</text></nplcit>"Devices and methods for rastern microphotolysis are described. With the aid of a microscope setup, selected sample regions are subjected to a high-intensity laser radiation.

<nplcit id="ncit0004" npl-type="b"><text>FLUORESCENCE IMAGING SPECTROSCOPY AND MICROSCOPY; John Wiley &amp; Sons 1996, INC., Chapter 5, pages 125-156</text></nplcit> Refers to technical properties as well as possible applications of AOTF. In particular, the wavelength-dependent modulation of laser lines for use in a confocal laser scanning microscope is described there.

<patcit id="pcit0001" dnum="WO9418547A"><text>WO 94/18547</text></patcit> and <nplcit id="ncit0005" npl-type="s"><text>Carlsson et al. "Using intensity-modulated scanning beams in combination with lock-in detection for recording multiple-labeled fluorescent specimens in confocal laser microscope, SPIE Vol. 2184</text></nplcit>"Relate to devices and methods in which microscopic images with a plurality of wavelengths are recorded simultaneously. The different wavelengths with different frequencies are modulated in their intensity and separated by the aid of the lock-in technique.

Furthermore, from the "<nplcit id="ncit0006" npl-type="s"><text>Communications for Science and Technology ", Volume II, No. 1, pages 9-19, June 1995</text></nplcit> If the confocal microscope is used for fluorometric methods, conclusions can be drawn about changes in the concentration of ions and molecules. In this context, indicators are also important which, in addition to the intensity dependence, show a shift in the excitation or emission spectrum and thus enable a quantification of ion concentrations. Furthermore, the photobleaching method is proposed in this context in which a defined inhomogeneity is generated in order to be able to obtain object information such as fluidity and diffusion via the dynamics of the adjoining equilibrium. Which, in addition to the intensity dependence, exhibit a shift in the excitation or emission spectrum and thus enable a quantification of ion concentrations. Furthermore, the photobleaching method is proposed in this context in which a defined inhomogeneity is generated in order to be able to obtain object information such as fluidity and diffusion via the dynamics of the adjoining equilibrium. Which, in addition to the intensity dependence, show a shift in the excitation or emission spectrum and thus enable a quantification of ion concentrations. Furthermore, the photobleaching method is proposed in this context in which a defined inhomogeneity is generated in order to be able to obtain object information such as fluidity and diffusion via the dynamics of the adjoining equilibrium.

From the above publication, it is known to employ Ar-Kr lasers for fluorescence excitation in the visible spectral range with the lines 488 nm, 568 nm and 647 nm. These lines are combined in a laser beam and fed via optical fibers to the scanning device. For excitation in the UV range, an Ar laser of the wavelengths 351nm and 364nm is proposed. For this, too, the coupling into the scanning device takes place via optical fibers.

The methods and arrangements described here can be used to record 3D data sets, which permit, for example, a reliable assignment of spatial cell or tissue structures within a microarchitecture or the localization of several gene sites in the chromosomes in FISH experiments.

However, there is the disadvantage that the respective sample is subjected to the laser radiation generated in the laser module and coupled into the scanning device over the entire scanning area. Thus, the entire scanning range is subjected to a relatively high radiation load, which leads, in particular, to the investigation of living organisms to undesirable effects and inadequate results.

Furthermore, there is the disadvantage that, upon excitation of the sample with different wavelengths, for example the aforementioned laser lines, no clear detection and evaluation of the radiation emitted and / or reflected by a specific location of a sample is possible since the effect of the mutual pulsation of the individual spectral lines Occurs.

An object of the invention is to further develop a method for laser scanning microscopy of the type described above in such a way that both a lower beam loading of the sample and a more precise image evaluation are achieved.

According to the invention, this object is achieved by a method with the features of claim 1 as well as by a laser scanning microscope having the features of claim 13.

According to the invention, the method of the abovementioned type is further developed in that the laser light is altered in its spectral composition during the image recording and laser light of different spectral composition is applied in a scanning lane adjacent to one another,

A specific spectral composition of the laser light is assigned to each sample location with the laser light so that a change in the spectral composition of the laser light is specifically effected during the scanning of specific locations of the sample.

This is done either by intermittently coupling in single or multiple spectral components or by irradiating the light as a whole, or by temporarily adding individual or several spectral components into the microscope beam path while the deflection of the microscope beam path is continuously continued.

As a result, at least two juxtaposed locations of the sample are acted upon by light of different spectral characteristics and by laser radiation of varying intensity. By intermittently interrupting the coupling of the laser light during the deflection of the microscope beam path, it is possible to apply only selected sections of the image field with the laser radiation.

A preservation of the sample is achieved in that only the areas of a sample which are relevant for the image evaluation are subjected to laser radiation of higher intensity.

In a preferred embodiment variant of the method according to the invention, it is provided that the spectral composition and / or the intensity of the laser light is changed during the scanning of a plurality of juxtaposed locations, which form such a scanning line. The deflection over the locations of this line can take place several times in the same direction or also bidirectionally. According to the invention, it is, for example, intended to carry out the change in the spectral composition or the intensity always with respect to the juxtaposed places of this line during each scan across the locations of this line, irrespective of whether this is effected in the same direction or opposite Quality of the image evaluation is increased while the energy input into the sample remains limited.

The different spectral composition of the laser radiation coupled into the microscope beam path is achieved, for example, by the radiation provided by a plurality of line lasers having approximately wavelengths 633nm, 568nm, 543nm, 514nm, 488nm and 458nm as required or depending on the properties of the sample to be evaluated Single wavelength, with a selection of several individual wavelengths or with all available individual wavelengths. In addition to this radiation in the VIS region, wavelengths in the UV range, approximately 351 nm and 364 nm, can be provided for coupling.

According to the invention, the coupling of the laser radiation into the microscope beam path is polarization-maintaining via single-mode fibers. The setting of the laser lines provided in each case for the irradiation to a desired brightness is advantageously carried out with an acousto-optically tunable filter (AOTF), which may also be arranged downstream of an acousto-optical modulator (AOM). The adaptation of the respective laser wavelength to the microscope objective, respectively, placed in the beam path is effected by variable beam collimation for the UV as well as for the VIS area.

A further preferred embodiment of the method according to the invention consists in the fact that the light reflected and / or emitted by each individual site of the sample is evaluated with regard to its spectral properties and its intensity, the evaluation being carried out in synchronism with the exposure to the location and taking into account the spectral properties Composition and / or the intensity of the laser light with which this location has been applied. This makes it possible to evaluate the scanned portion of the sample with respect to the individual locations, which leads to a very high resolution and to the highest possible precision in the image evaluation.

It is also within the scope of the invention that the laser light reflected and / or emitted by each individual site is detected with a plurality of detection channels, the individual detection channels being designed to receive different spectral components. This provides very good conditions for the investigation of multifluorescence preparations, and identical optical sections can be generated over each detection channel in the simultaneous recording of multiple fluorescence preparations.

In this context, it is provided according to the invention that the spectral composition and / or the intensity of the laser light coupled into the microscope beam path correspond to the excitation radiation of a fluorescent dye contained in the sample or of the fluorescence dye applied to the sample and the individual detection channels are designed to receive the emission radiation emanating from the fluorescent dye . It is thus possible to generate laser light for excitation of different fluorescent dyes and to draw conclusions from the detection on the distribution of these fluorescent dyes on or in the sample.

A further very preferred embodiment of the invention consists in permanently evaluating the spectral composition and / or the intensity of the coupled laser light, and a mathematical linking of the evaluation results of the laser radiation directed to a specific location with the evaluation results of the reflected and / Or emitted light. As a result of this linking, the deflection position of the microscope beam path can be determined, for example, for two adjacent locations determined according to the coordinates x, y, z for which differences in the spectral properties of the light that are reflected from these locations can be detected beyond a predetermined threshold value And / or is emitted, Thus indicating the presence of an optical boundary layer between these two locations. These deflection positions are stored according to the invention and are based on the calculation of areas and / or volumes which are enclosed by optical boundary layers within the sample.

With the deflection positions thus obtained and stored, it is also possible to determine and prescribe control signals for the spectral composition and / or the intensity of the laser light for the application of these locations during a subsequent scanning cycle, with which an automatic optimization in the image evaluation taking into account the optical properties The sample or the fluorescent dye.

In particular, the process according to the invention is advantageously usable for the so-called photobleaching. During the scanning, a selected area of ​​a sample is initially subjected to a relatively high radiation intensity and a bleaching operation is thereby initiated. With the immediately following sampling cycles, the starting reactions are optically detected and evaluated, from which information can be obtained on the dynamic processes, such as diffusion and transport processes, which take place immediately after the bleaching process in the sample substance.

For this purpose, the sampling must be carried out with a very high time resolution, which is achieved according to the invention with the sufficiently fast switching between different intensities and different spectral compositions of the light striking individual locations of the sample in synchronism with the beam deflection.

The fast switching between different intensities and different spectral compositions of the laser radiation is carried out with an acousto-optically tunable filter (AOTF) which, in a sense, but significantly faster, takes over the function of different filters which are interchangeable in the beam path and also individual laser lines or, Can modulate arbitrary combinations of lines with high temporal dynamics in the intensity.

Functionality and application of the AOTF are, for example, described in detail in: <nplcit id="ncit0007" npl-type="s"><text>String, Kenneth, R .: "Wavelength Selection for Illuminaton in Fluorescence Microscopy", NIH, LKEM, Building 10 / 6N309, Bethexda, MD 20892, April 1993</text></nplcit>. Furthermore, concrete application examples for AOTF in the US patents<patcit id="pcit0002" dnum="US5444528A"><text>US 5,444,528</text></patcit>, <patcit id="pcit0003" dnum="US5377003A"><text>US 5,377,003</text></patcit> and <patcit id="pcit0004" dnum="US5216484A"><text>US 5,216,484</text></patcit> As well.

The time synchronism between the activation of the AOTF for modulating the laser radiation and the control of the scanning device for beam deflection is achieved by assigning specific control signals for the AOTF to the control signals output by the control device to the scanning device. Thus, the control of the San device and the control of the AOTF are always synchronous, ie control pulses for the AOTF are always added to the output of a control pulse for the scanning device.

This means, on the other hand, that a characteristic intensity and / or spectral composition of the light can be assigned to each deflection position and thus to each location of the sample.

For this purpose, the circuit arrangements for carrying out the method with regard to very short operating times of the control pulses from the output to the switching of the beam modulation by the AOTF are optimized. These are in the range of &lt;10 ms. A method variant consists in predetermining lead times for the switching of the intensity and spectral composition and / or for the deflection during the control of the AOTF or the scanning device, so that precisely the location provided also acts with the provided radiation intensity and spectral composition becomes.

The invention also relates to a laser scanning microscope for carrying out the aforementioned method steps, comprising a laser module for generating laser light with different selectable spectral components, with single-mode fibers for coupling the laser light into the microscope beam path, with a scanning which is at least two-dimensionally deflecting With a microscope objective, which focuses the laser light onto a sample, with a plurality of detectors for receiving different spectral components of the light reflected and / or emitted by the sample, and with an evaluation circuit, which is connected downstream of the outputs of the detectors.

According to the invention, a plurality of individually controllable individual and / or multi-wavelength lasers are provided in such a laser scanning microscope in the laser module. A beam combiner, an acoustically optically tunable filter (AOTF) and / or an acousto-optical modulator (AOM) are connected downstream of the laser module , The single-model fibers are arranged downstream of collimation optics whose distances to the respective fiber end are variable and which are coupled to controllable locations. Photomultipliers (PMT) are provided as detectors, one of which is assigned to a reflection or emission band and thus to a detection channel. In order to branch the radiation emerging from the sample into the individual detection channels, filters and / or color separators which are arranged on divider wheels and are interchangeable with one another by rotation of the divider wheels, each divider wheel also being coupled to a controllable positioning device. Furthermore, the control inputs of the laser module, the AOTF, the AOM, the scanning device as well as the positioning devices for the divider wheels and the collimation optics are connected to the outputs of the evaluation circuit.

According to the invention, the microscope beam path directed towards the sample is branched, and one of the branches is directed to an optoelectronic receiver whose output is also connected to the drive unit.

According to the invention, a mathematical linkage of the output signals of the optoelectronic receiver with the output signals of the PMT and with the deflection signals for the scanning device can take place in the evaluation circuit. Optimized control signals for the laser module, the AOTF, the AOM, the scanning device and the positioning device are provided at the output of the evaluation circuit.

The invention is to be explained in more detail below with reference to an exemplary embodiment. In the accompanying drawings<dl id="dl0001" compact="compact"><dt>FIG</dt><dd>The basic structure of a laser scanning microscope</dd><dt>FIG</dt><dd>The principle of deflecting the laser light over the individual locations of a sample</dd></dl>

In <figref idrefs="f0001">FIG</figref> A laser module 1 is shown which is equipped with the lasers 2, 3 and 4 for generating laser light in the visible region with the wavelengths 633 nm, 543 nm and 458 nm. The radiation emanating from these lasers is coupled via a plurality of beam combiner 5, an AOTF 6 and a fiber 7 into a scanning device 8 which is equipped with a unit 9 which is directed in the coordinates x and y. ,

In a second laser module 10, a UV laser is provided, the light of which is coupled into the scanning device 8 via an AOTF 11 and an optical fiber 12.

In both beam paths, collimation optics 13 are arranged downstream of the optical fibers 7 and 12, the distances of which to the respective fiber end can be varied and which for this purpose are coupled to a controllable location device (not shown).

The laser radiation from the beam deflecting device 9 is coupled through a scanning objective 14 into the beam path of the microscope 15, which is shown in simplified form, and is directed to a sample 16 here. The laser radiation passes through a tube lens 17, a beam splitter 18 and the microscope lens 19.

The light reflected and / or emitted by the respectively charged location of the sample passes through the microscope objective 19 back to the beam deflecting device 9, then passes through a beam splitter 20 and is directed with the aid of the imaging optical system 21 after diversion into a plurality of detection channels 22 onto photomultipliers 23, In each case one of the detection channels 22. For the purpose of branching into the individual detection channels 22, the light is directed from a deflection prism 24 to dichroic beamsplitters 25. In each detection channel 22, pinholes 26 and emission filters 27 which can be adjusted in the direction as well as perpendicular to the radiation direction and are variable in their diameters are provided.

The outputs of the photomultipliers 23 lead to the signal inputs of an evaluation circuit 28, which in turn is connected to a control device 29. The outputs of the control device 29 are connected to the signal inputs of the laser modules 1 and 10 as well as to signal inputs of the positioning devices for influencing the position of optical elements or assemblies, such as the position of the collimation optics 13, pinholes 26 and the like Shown).

By way of example, the laser radiation coupled into the scanning device 8 is branched by a beam splitter 30, one of the branches being directed to an optoelectronic receiver 31 which is arranged upstream of a line filter 32 which is arranged on filter wheels and is interchangeable by rotation of the filter wheels and neutral filters 33 which are interchangeable with one another are. The filter wheels on which the line filters 32 and the neutral filters 33 are arranged are coupled to control devices, the control inputs of which are connected to signal outputs of the control device 29 (not shown in the drawing).

When operating the laser scanning microscope, the optical axis 38 of the microscope beam path is passed through the scanning means 8 as shown in FIG <figref idrefs="f0002">FIG</figref> Symbolically, in the direction of the coordinate X, from place to place and in the direction of the coordinate Y, is guided in a raster-like manner from row to row over an object plane 34 to be scanned, in which the detail 35 to be evaluated of a sample lies.

According to the state of the art, laser light has thus far been coupled into the microscope beam path with constant spectral composition or intensity during scanning, which has the result that, particularly in the case of high-resolution structural investigations on extremely low-contrast objects, for example individual cells, organisms, organisms or parasites, High beam loading was required to obtain images with sufficient bright field or phase contrast.

In order to reduce the radiation load and to improve the quality of the image evaluation, it is provided according to the invention that during the scanning of a line and / or of the object plane 34 the coupling-in of one or more spectral components, if appropriate also of the entire spectrum, is intermittently interrupted or alternatively For this purpose, individual or several spectral components can additionally be coupled in time.

During the change in the spectral composition or the intensity of the laser light, the beam deflecting device 9 remains in continuous operation. In this way, it is achieved that, for example, the locations 36 and 37 are subjected to a different effect within a scanning line or within the sample to be scanned. This makes it possible to expose the locations 37, which are located within the detail 35 to be evaluated, for example a cell, to a lower radiation.

Conversely, an increase in the intensity and / or a change in the spectrum of the laser radiation is undertaken in the scanning of the locations 37, if this is desirable, for example in the application of the method according to the invention for the purpose of photobleaching, and it is important to select selected areas of the sample With a very high radiation intensity, in order to be able to follow immediately the dynamic processes which then occur.

With the method according to the invention and with the arrangement according to the invention, it is furthermore possible to receive the light reflected and / or emitted by each of the charged locations 36 and 37 in the individual detection channels 22, the individual detection channels 22 being used to receive different spectral components of the Respective location of outgoing light.

A special feature of the method according to the invention is that the detection and the evaluation of the light emanating from each applied location takes place synchronously with the application of the respective location. In this respect, the excitation wavelength and the emission wavelength can be assessed for each of the individual locations 36 and 37 of the sample, from which conclusions can be derived on the properties of the sample exactly at the location under consideration.

With the arrangement according to the invention, it is furthermore possible, by means of the signals emitted by the optoelectronic receiver 31, to permanently control the composition and the intensity of the laser light which is directed to the sample and to use these signals to compensate even the smallest intensity fluctuations via the control device 29.

The excitation radiation and emission radiation, which is related to one and the same location, are respectively evaluated with a calculation circuit integrated into the evaluation circuit 28. In this way, it is precisely ascertained whether a change in the emission wavelength or the intensity of the emitted radiation can be recorded during the deflection of the laser radiation from one location to the other, for example, from directly adjacent locations 36 and 37, the extent of which is above a predetermined threshold value Goes beyond If so, the presence of an optical boundary layer at the adjacent locations 36 and 37 can be inferred.

Since the data of the deflection positions in the control device 29 and / or the evaluation circuit 28 are also available for these locations 36, 37 as for each other sampled location of the sample, the course of such optical boundary layers can be determined using relevant deflection positions Based on these deflection positions, finally calculate the area or volume enclosed by the optical boundary layers.

For the sake of completeness, it should be pointed out that <figref idrefs="f0002">FIG</figref> Only refers to a scanning plane of the sample. It is, of course, possible to scan a plurality of planes of the sample by focusing the laser radiation on different coordinates in the z direction, ie, perpendicular to the displayed surface.