JP4646506B2 - Laser scanning microscope - Google Patents

Description

The present invention scans light from a light source and irradiates the sample with light generated by the sample (for example, fluorescence, reflected light, radiation derived from the sample generated according to the irradiation wavelength and physical properties of the material, etc.) The present invention relates to a laser scanning microscope having a spectroscopic optical device for spectroscopic observation and detection.

  In recent years, in the field of microscopy, it is hoped that various fluorescent lights can be detected simultaneously in multiple colors as the lineup of fluorescent reagents such as organic compounds and fluorescent proteins represented by GFP increases, and the labeling state changes. It is rare. In addition, in order to use spectral data for processing such as separation and analysis of fluorescent dyes, the entire spectral spectrum of multiple fluorescent wavelengths can be observed, and multiple regions of interest can be selected from the entire spectrum, and these can be quickly and simultaneously resolved at high resolution. A spectroscopic optical device with a high degree of freedom that can be observed in the field is expected.

  Conventionally, a laser scanning confocal microscope disclosed in Patent Document 1 is known as having such a spectroscopic optical device. This Patent Document 1 discloses a device using a light dispersion means and a multi-channel detector, and a prism or a diffraction grating is used as the light dispersion means, and is selected by a beam emitted from a monochromatic laser or a wavelength variable beam. A beam having one wavelength component is illuminated onto the sample as excitation light through the light dispersion means, and generated light emitted from the sample is spectrally detected again through the light dispersion means. Specifically, when the prism rotates, the excitation wavelength is guided to the illumination optical path so that the incident direction has the minimum deviation angle, and the light generated from the sample is dispersed through the prism positioned at this rotational position. To the detector. Moreover, this patent document 1 is also characterized by including a multi-channel detector as a detector.

  On the other hand, Patent Document 2 and Patent Document 3 are also known as other examples.

  Patent Document 2 is a combination of a Czerny-Turner type spectrometer using a diffraction grating and a multi-channel detector. Here, a light dispersion element that disperses radiant light generated in a sample into a spectrum, and detects dispersed light. And a means for electrically generating at least one sum signal from the multi-channel detector. Another feature is that a multi-channel detector is arranged in accordance with the spectral direction to detect spectral light and generate a combined signal of different spectral components.

Patent Document 3 corresponds to a modification of Patent Document 2, and similarly combines a Czerny-Turner type spectroscopic device and a multi-channel detector. Here, the light dispersion element, the detector, and the imaging element move or rotate accordingly, and the positional relationship between the dispersed spectrum and the detector can be relatively changed. Accordingly, the wavelength resolution is improved by providing a function as a wavelength scanner, shifting the position of the spectrum light with respect to the multi-channel detector and receiving the light, and processing the received light data. It is also described that the grating is switched to further improve the wavelength resolution.
Japanese Patent No. 3076715 US 2002/0020819 specification US 2002/0036775 Specification

  However, in Patent Document 1, the prism or diffraction grating used as the light dispersion means is rotated and positioned so that the excitation light and the light generated from the sample are guided to the sample and the detector, respectively. For this reason, it is impossible to simultaneously illuminate the sample with excitation light from a laser having two or more wavelength components and observe a plurality of fluorescence excited by the excitation light.

  Further, in Patent Document 2, since the dispersed spectrum light is fixed with respect to multi-channel detection, once the entire wavelength band is set, the band cannot be easily changed. Further, since the wavelength resolution of the detected spectrum is determined by the number of divisions of the multichannel detector regardless of the optical resolution, a higher wavelength resolution cannot be obtained. Furthermore, when a desired wavelength region is extracted and selected from spectral light, the desired number of channels is selected according to the wavelength resolution determined by this division number, and spectral light cannot be observed with high resolution in the region of interest. . Furthermore, since all the spectrum light dispersed by a single multi-channel detector is received, even if there is an intensity difference of several times between multiple fluorescent light quantities, appropriate sensitivity adjustments are made for each fluorescent light quantity. I can't do it. For this reason, it is difficult to observe fluorescence with low intensity with respect to bright fluorescence.

  Furthermore, since the high wavelength resolution obtained in Patent Document 3 uses pixel shift calculation processing by the wavelength scanner, time is generated by the wavelength scanner. In addition, switching of the grating is also described, but in order to obtain a high resolution, the grating has a large number of gratings, so that the diffraction efficiency is lowered and a light amount loss is unavoidable. Needless to say, the sensitivity adjustment has the same disadvantages as described above, and the light loss caused by the high dispersion grating adversely affects the fluorescence observation with low intensity.

The present invention has been made in view of the above circumstances, to provide a laser scanning microscope having a high spectral detector degree of freedom of the spectral region of interest with respect to the spectral region of the wide band observation light can accurately extract With the goal.

According to the first aspect of the present invention, in a laser scanning microscope that irradiates a sample with laser light as excitation light and observes the fluorescence emitted from the sample, a spectroscopic element that disperses the fluorescence into a spectrum, and a multi-spectrum that detects the spectrum. A channel detector, a spectrum projection optical system for projecting the spectrum onto the multi-channel detector, and disposed in front of the multi-channel detector to reflect the wavelength of the excitation light and be transparent to other wavelengths. A mask, driving means for rotating the spectroscopic element so as to move the position of the spectrum in the dispersion direction, and driving means for moving the multi-channel detector in parallel along the spectral dispersion direction. In order to reflect the wavelength of the excitation light even when the multi-channel detector moves or the spectroscopic element rotates. It is characterized in that in the dispersion direction of the spectrum is movable.

  According to the present invention, the first multi-channel spectroscopic detection system for detecting the first spectral region in the wide band and the second multi-channel for detecting the second spectral region in the wavelength range of interest included in the first spectral region. By having a spectroscopic detection system, it is possible to accurately extract a spectral region of a wavelength range of interest from a wide spectral range.

  Further, according to the present invention, a desired spectrum can be acquired with a relatively high degree of freedom by preparing wavelength separation means in accordance with the target fluorescence wavelength. Further, optimization of wavelength resolution and individual sensitivity adjustment can be appropriately performed for each spectrum of interest.

  Furthermore, since the first spectral region of the wide band is divided by the spectral region dividing means and the spectrum of each divided region can be detected, a wide spectral region or a spectral region of the wavelength range of interest can be arbitrarily selected and switched. Further, since these spectral regions can be determined by the spectral region dividing means, setting with a higher degree of freedom can be performed.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

(First embodiment)
FIG. 1 shows a schematic configuration of a spectroscopic optical device to which the first embodiment of the present invention is applied.

  In the figure, reference numeral 1 denotes an observation optical system including a pinhole for obtaining a confocal effect of a microscope, here, a laser scanning microscope for observing fluorescence generated in a sample. In addition, a light source part, a beam scanning part, a microscope part, a dichroic mirror that separates excitation light and fluorescence, which are other main parts of the laser scanning microscope, are omitted from the drawings.

  The fluorescence from the sample passing through the dichroic mirror of such a laser scanning microscope obtains a desired confocal effect by the observation optical system 1 and then is converted into substantially parallel light through a collimator lens (not shown) and emitted. Is done.

  An optical path switching unit 2 as an optical path switching unit is disposed in the observation light from the observation optical system 1, here, the optical path of fluorescence. The optical path switching unit 2 is for switching the optical path. Here, the hole (or the dummy glass for matching the optical axis) through which light passes and the reflection mirror are selectively positioned on the optical path. It has become. In this case, a turret, a slider, or the like is used for switching these optical paths.

  The reflection mirror 3 is disposed on the optical path that has passed through the hole while the hole of the optical path switching unit 2 is positioned on the optical path. A first multi-channel spectroscopic detection system 100 is disposed in the reflection optical path of the reflection mirror 3. The first multi-channel spectroscopic detection system 100 includes a diffraction grating 4 as a spectroscopic element, a spectral projection optical system 5 and a multi-channel detector 6. Then, the fluorescence reflected by the reflecting mirror 3 enters the diffraction grating 4, and the fluorescence spectrum dispersed by the diffraction grating 4 is condensed and projected onto the multichannel detector 6 by the spectrum projection optical system 5. It has become.

  In this case, the relative position of the multi-channel detector 6 and the fluorescence spectrum projected onto the multi-channel detector 6, in other words, the projection position onto the multi-channel detector 6 of the center wavelength dispersed by the diffraction grating 4 is moved. It is possible to receive an appropriate wavelength band. For this purpose, driving means (not shown) for rotating the diffraction grating 4 (not shown) and driving means for moving the multichannel detector 6 in parallel (shown arrow b) along the spectral direction. (Not shown). Although not shown, the multi-channel detector 6 is provided with sensitivity adjusting means.

  Thus, the diffraction grating 4 and the multichannel detector 6 constituting the first multichannel spectroscopic detection system 100 are arranged so as to cover a wide wavelength range, and which fluorescence wavelength band in the sample is detected. You can see at a glance. Further, the spectral region to be observed can be adjusted by rotating the diffraction grating 4 or translating the multi-channel detector 6 in accordance with the observation wavelength range.

  Next, in a state where the reflection mirror of the optical path switching unit 2 is located on the optical path, the second multi-channel spectroscopic detection system 200 is arranged in the shape of the reflection optical path of the reflection mirror. The second multi-channel spectroscopic detection system 200 includes a prism 7 as another spectroscopic element, a spectral projection optical system 8, and a multi-channel detector 9. Then, the fluorescence reflected by the reflection mirror of the optical path switching unit 2 enters the prism 7, and the fluorescence spectrum dispersed by the prism 7 is condensed and projected onto the multi-channel detector 9 by the spectrum projection optical system 8. It has become so.

  Also in this case, the prism 7 and the multi-channel detector 9 include a driving means (not shown) for rotating the prism 7 (not shown) and a multi-channel so that the relative position of the observed fluorescence spectrum can be varied. Driving means (not shown) for moving the channel detector 9 parallel to the spectral direction (arrow d in the figure) is provided.

  The spectral projection optical system 8 can change the focal length, the projection magnification, or the zoom ratio. Here, the zoom optical system is capable of changing the projection magnification of the spectrum onto the multi-channel detector 9. Although not shown here, the multi-channel detector 9 is provided with sensitivity adjusting means.

  The prism 7 and the multi-channel detector 9 constituting the second multi-channel spectral detection system 200 are introduced into the sample from the broadband spectral spectrum detected by the first multi-channel spectral detection system 100. The spectral region of the wavelength of interest can be observed according to the emission wavelength of the fluorescent reagent or fluorescent protein. There may be a plurality of spectral regions of the wavelength of interest. Here, for the sake of simplification, a case where one attention spectrum region is set is shown.

  In this case, the prism 7 is used as the spectroscopic element because it has dispersion as a characteristic of the glass material because the dispersion is different depending on the wavelength as compared with the diffraction grating 4, but the transmittance is excellent. It is because it has. That is, when only the selected wavelength region of interest of about 100 mm is observed, the difference in the amount of dispersion is so small that it can be ignored, so that bright observation can be performed with excellent transmittance. Further, by changing the zoom ratio of the spectral projection optical system 8, the projection magnification of the wavelength range of interest onto the multi-channel detector 9 changes, so that the wavelength range of light received in each detection channel can be changed. Thereby, the wavelength resolution observed in the attention area can be freely changed, and a desired wavelength resolution can be set in the area. This change in wavelength resolution can also be made variable step by step by switching glass prisms having different dispersion amounts. Of course, if the transmittance is not a problem, a plurality of diffraction gratings may be switched.

  Further, by providing sensitivity adjustment means for each of the multi-channel detectors 6 and 9, for example, when simultaneous observation of bright fluorescence and dark fluorescence is performed, the multi-channel detector 6 alone is set appropriately for dark fluorescence. However, it is also possible to perform appropriate sensitivity adjustment with the multi-channel detector 9 as a region where dark fluorescence is focused, and the spectrum can be examined in detail with sufficient brightness.

  Next, a method for cutting the excitation light on the multi-channel detector 6 will be described with reference to FIG. In this case, for example, when the excitation light from the argon laser 488 nm and the green He—Ne laser 543 nm excite the sample at the same time, the multi-channel detector 6 is informed of them according to the characteristics of the dichroic mirror provided in the laser scanning microscope. The beam is guided. Since these laser beams are stronger than fluorescence, they may be a factor that hinders appropriate fluorescence observation.

  As a method for solving this, for example, a coating that reflects the positions of 488 nm and 543 nm of the spectrum as shown in FIG. 2B is applied immediately before the multi-channel detector 6 shown in FIG. A mask M using a glass material that is transparent to other wavelengths is disposed. The mask M is mounted at a fixed position or driven in the same direction as the multi-channel detector 6 as required. In this way, as shown in FIG. 6C, the excitation light of 488 nm or 543 nm is always cut even if the wavelength range observed by the driving of the multichannel detector 6 or the rotation of the diffraction grating 4 is shifted. be able to.

  Therefore, according to such a configuration, for example, in the state where the first spectral region A is detected by the first multi-channel spectral detection system 100 as shown in FIG. It is divided into a plurality of parts (two in the illustrated example), and these divided regions can be detected as the second spectral region B by the second multi-channel spectroscopic detection system 200 as shown in FIG.

  In addition, as shown in FIG. 4A, in the state where the first spectral region A is detected by the first multi-channel spectral detection system 100, an arbitrary region A ′ selected in the first spectral region A is displayed. As shown in FIG. 4B, the second multi-channel spectroscopic detection system 200 can detect the second spectral region C.

  Thereby, observation of the whole spectrum area | region A and observation of the 2nd spectrum area | region B (C) of a detailed area | region can be switched, and a wavelength selectivity and the freedom degree as an apparatus increase. In addition, the first multi-channel spectroscopic detection system 100 and the second multi-channel spectroscopic detection system 200 are prepared individually for each spectral region A and B (C), so that the light intensity from the sample is adjusted. The sensitivity can be adjusted individually, and the sample can always be observed with appropriate settings.

  As described above, in the present invention, the first multi-channel spectroscopic detection system 100 that detects a wide band and the second multi-channel spectroscopic detection system 200 that detects a wavelength range of interest are combined to generate from a wide spectral range. The wavelength range to be noticed can be arbitrarily selected according to the fluorescence spectrum to be performed, and the wavelength resolution and the individual sensitivity adjustment can be appropriately optimized according to the fluorescence spectrum in the noticed range.

(Second Embodiment)
Next, a second embodiment of the present invention will be described.

  FIG. 5 shows a schematic configuration of a spectroscopic optical device to which the second embodiment of the present invention is applied.

  This case is also an example of a laser scanning confocal microscope, as described in the first embodiment, and the first path of the observation light from the sample that has passed through the observation optical system 11 including the pinhole is the first. Second and third multichannel spectroscopic detection systems 12, 13, and 14 are arranged.

  The first multi-channel spectroscopic detection system 12 corresponds to the first multi-channel spectroscopic detection system 100 described in the first embodiment, and a dichroic mirror 121 as a wavelength separation unit is provided on the optical path of the observation light. Has been placed. A diffraction grating 122 is arranged in the reflected light path of the dichroic mirror 121, and the fluorescence spectrum dispersed by the diffraction grating 122 is condensed and projected onto the multichannel detector 124 by the spectrum projection optical system 123. Yes.

  The second and third multichannel spectroscopic detection systems 13 and 14 correspond to the second multichannel spectroscopic detection system 200 described in the first embodiment. A dichroic mirror 131 is disposed on the transmitted light path of the dichroic mirror 121. A diffraction grating 132 is disposed in the reflected light path of the dichroic mirror 131, and the fluorescence spectrum dispersed by the diffraction grating 132 is condensed and projected onto the multichannel detector 134 by the spectrum projection optical system 133. Yes. In the third multi-channel spectroscopic detection system 14, the reflection mirror 141 is disposed on the transmission optical path of the dichroic mirror 131. A diffraction grating 142 is arranged on the reflection optical path of the reflection mirror 141, and the fluorescence spectrum dispersed by the diffraction grating 142 is condensed and projected onto the multichannel detector 144 by the spectrum projection optical system 143. Yes.

  Also in this case, the diffraction gratings 122, 132, 142 and the multi-channel detectors 124, 134, 144 of the multi-channel spectroscopic detection systems 12, 13, 14 are diffracted so that the relative positions of the observed fluorescence spectra can be varied. Driving means (not shown) for rotating the gratings 122, 132, and 142 separately (shown by an arrow e), and multi-channel detectors 124, 134, and 144 are respectively parallel along the spectral direction (shown by an arrow f). ) Is provided with a driving means (not shown). The spectral projection optical systems 123, 133, and 143 are zoom optical systems that can vary the projection magnification of the spectrum onto the multichannel detector. Further, although not shown here, the multi-channel detectors 124, 134, 144 are provided with sensitivity adjusting means.

  According to such a configuration, the first multi-channel spectral detection system 12 can detect a broadband spectral region, and the remaining multi-channel spectral detection systems 13 and 14 can detect the spectral region of the wavelength range of interest. .

  Further, the multi-channel spectroscopic detection systems 12, 13, and 14 can detect different wavelengths by the dichroic mirrors 121 and 131 for wavelength-separating fluorescence from the sample. If used in accordance with the fluorescence wavelength range for observing the wavelength separation characteristics, light in different wavelength ranges can be dispersed and detected. In this case, the dichroic mirrors 121 and 131 may be set to blue detection (400 to 500 nm), green detection (500 to 600 nm), red detection (600 to 700 nm), for example, depending on the wavelength region to be divided. Alternatively, it may be set according to the excitation wavelength to be excited or the fluorescence wavelength to be observed.

  In this way, according to the arrangement of the dichroic mirrors 121 and 131, the light of different wavelength regions can be spectrally detected and detected by the multichannel spectroscopic detection systems 12, 13, and 14. In this case, since the multichannel detectors 124, 134, and 144 each have individual sensitivity adjustment means, after correcting the sensitivity in order to eliminate individual differences in each of the multichannel spectral detection systems 12, 13, and 14, By matching the sensitivity and the projection magnification to the same conditions, the detected spectral data can be handled as observing a wide wavelength range. Further, if the sensitivity of the multichannel detectors 124, 134, and 144 is adjusted in accordance with the fluorescence intensity in each spectrum data, appropriate observation can be performed even for a dark sample.

  Further, in the multi-channel spectroscopic detection systems 12, 13, and 14, the diffraction gratings 122, 132, and 142 are rotated in accordance with the observation wavelength range, or the multi-channel detectors 124, 134, and 144 are translated in parallel. The wavelength band to be observed can be adjusted, and the wavelength resolution to be detected in each wavelength band can be changed by the zooming function of the spectrum projection optical systems 123, 133, and 143.

  Accordingly, by dividing the multi-channel spectroscopic detection systems 12, 13, and 14 by the dichroic mirrors 121 and 131 in this way, a wide spectral region and a spectral region of the wavelength range of interest can be arbitrarily selected according to the generated fluorescence spectrum. Can be selected and switched. This means that if the dichroic mirrors 121 and 131 are prepared in accordance with the target fluorescence wavelength, a desired fluorescence spectrum can be obtained with a relatively high degree of freedom. Further, in each of the multi-channel spectroscopic detection systems 12, 13, and 14, the wavelength resolution and the individual sensitivity adjustment can be appropriately optimized for the fluorescence spectrum in the target range.

(Third embodiment)
Next, a third embodiment of the present invention will be described.

  FIG. 6 shows a schematic configuration of a spectroscopic optical device to which the third embodiment of the present invention is applied.

  This case is also an example of a laser scanning confocal microscope as described in the first embodiment, and light dispersion is performed on the optical path of the observation light from the sample that has passed through the observation optical system 21 including the pinhole. A diffraction grating 22 is arranged as an element. Then, the fluorescence spectrum dispersed by the diffraction grating 22 is incident on the relay lens system 23 and returned to substantially parallel light again.

  On the optical path of the parallel light from the relay lens system 23, reflection mirrors 24 and 25 as spectral region dividing means are arranged side by side. These reflection mirrors 24 and 25 are for dividing a desired spectral region from the fluorescence spectrum dispersed by the diffraction grating 22.

  A first multi-channel spectral detection system 300 is disposed in the reflection optical path of the reflection mirror 24, and a second multi-channel spectral detection system 400 is disposed in the reflection optical path of the reflection mirror 25.

  Then, the spectral region divided by the reflection mirror 24 is condensed and projected onto the multi-channel detector 27 by the spectral projection optical system 26 constituting the first multi-channel spectral detection system 300. The divided spectral region is condensed and projected onto the multi-channel detector 29 by the spectral projection optical system 28 constituting the second multi-channel spectral detection system 400.

  In this case, the diffraction grating 22 is rotated (shown) so that the multi-channel detectors 27 and 29 and the relative positions of the fluorescence spectra projected onto the multi-channel detectors 27 and 29 can be moved to receive an appropriate wavelength band. The driving means (not shown) for moving the arrow g) is provided, and the multi-channel detectors 27 and 29 are also provided with driving means (not shown) for moving in parallel along the spectral direction (shown by the arrow h). It has been. Further, the reflecting mirrors 24 and 25 are also provided with driving means (not shown) for moving in the insertion direction (arrow i in the figure) with respect to the dispersed fluorescence spectrum so that the wavelength can be divided within a desired range. Yes. The movement direction at this time may be originally moved in a direction perpendicular to the fluorescence spectrum, but may be determined according to the arrangement of the multi-channel detectors 27 and 29 and the reflection mirrors 24 and 25. It is not limited to. Also. Although not shown here, the multi-channel detectors 27 and 29 are individually provided with sensitivity adjusting means. Further, as in the above-described embodiment, in order to make the wavelength resolution on the multi-channel detectors 27 and 29 variable, the spectrum projection optical systems 26 and 28 are zoom optical systems with variable projection magnification. Yes. In this case, a variable magnification optical system having a turret mechanism capable of changing the magnification in order to make the wavelength resolution variable may be used. In this case, the multichannel detectors 27 and 29 are arranged in front and rear according to the condensing position of the spectrum. You may provide a drive means (not shown) so that it can drive to (illustration arrow j).

  In this way, the fluorescence spectrum divided by the reflection mirrors 24 and 25 is received by the multi-channel detectors 27 and 29, respectively. At this time, since the division region of the fluorescence spectrum can be determined by the insertion position of the reflection mirrors 24 and 25, selection or division with a high degree of freedom can be performed. In addition, since each multi-channel detector 27 and 29 has a sensitivity adjustment means, the sensitivity and the projection magnification are adjusted after correcting the sensitivity in order to eliminate individual differences between the multi-channel detectors 27 and 29. By matching to the same conditions, the detected spectral data can be handled as observing a wide wavelength range. Furthermore, if the sensitivity of the multichannel detectors 27 and 29 is adjusted in accordance with the fluorescence intensity in each spectrum data, appropriate observation can be performed even for a dark sample. Furthermore, the detection system itself shown in the third embodiment is used as a detection system for the wavelength region of interest in the first embodiment (corresponding to the second multi-channel spectroscopic detection system 200 shown in FIG. 1). In this way, it is possible to construct a more advanced and detailed detection system by separating it from a broadband detection system (corresponding to the first multi-channel spectroscopic detection system 100 shown in FIG. 1). . In other words, if this third embodiment is applied to the first embodiment, the first embodiment can be further developed to detect a plurality of wavelength regions of interest. Again, the observation band can be adjusted by rotating the diffraction grating 22 and moving the multichannel detectors 27 and 29 parallel to the spectral direction (arrow h in the figure) in accordance with the desired observation wavelength range. In addition, the spectral projection optical systems 26 and 28 having a zoom mechanism can change the wavelength resolution to be detected in the multi-channel detectors 27 and 29 and the respective wavelength regions.

  Accordingly, in this way, the broadband spectral region dispersed by the diffraction grating 22 is divided through the reflection mirrors 24 and 25, and each is divided into the first multichannel spectroscopic detection system 300 and the second multichannel spectroscopic detection. Since detection is possible with the system 400, a wide spectral region or a spectral region of a wavelength range of interest can be arbitrarily selected and switched according to the fluorescence spectrum generated from the observation light. Further, since these spectral regions can be determined by the positions of the reflecting mirrors 24 and 25, setting with a higher degree of freedom can be performed. Furthermore, by using the detection system itself shown in the third embodiment as a detection system for the wavelength region of interest in the first embodiment described above, the number of detection channels can be increased, and more sophisticated and detailed information can be obtained. Observations can be made. Furthermore, in each of the multi-channel spectroscopic detection systems 300 and 400, it is possible to appropriately optimize the wavelength resolution and individual sensitivity adjustment for the fluorescence spectrum in the target range.

  In addition, this invention is not limited to the said embodiment, In the implementation stage, it can change variously in the range which does not change the summary. In the above description, the fluorescence observation is described. However, the present invention can be applied not only to the fluorescence observation but also to the radiated light generated in the sample according to the reflected light from the sample or the illumination light from the light source. The light dispersion element for obtaining the spectrum is not limited to a diffraction grating or a prism, and any light dispersing element that can obtain the same effect can be used. For example, an element widely used as a diffractive optical element such as an acousto-optic element or GRISM (an optical element combining a grating and a prism) can be assumed.

  Furthermore, the above embodiments include inventions at various stages, and various inventions can be extracted by appropriately combining a plurality of disclosed constituent elements. For example, even if some constituent requirements are deleted from all the constituent requirements shown in the embodiment, the problem described in the column of the problem to be solved by the invention can be solved, and is described in the column of the effect of the invention. If the above effect is obtained, a configuration from which this configuration requirement is deleted can be extracted as an invention.

The figure which shows schematic structure of the 1st Embodiment of this invention. The figure explaining the method of cutting excitation light on the multichannel detector of 1st Embodiment. The figure for demonstrating the effect | action of 1st Embodiment. The figure for demonstrating the other effect | action of 1st Embodiment. The figure which shows schematic structure of the 2nd Embodiment of this invention. The figure which shows schematic structure of the 3rd Embodiment of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 100 ... 1st multichannel spectral detection system 200 ... 2nd multichannel spectral detection system 1 ... Observation optical system, 2 ... Optical path switching part, 3 ... Reflection mirror 4 ... Diffraction grating, 5 ... Spectral projection optical system 6.9 DESCRIPTION OF SYMBOLS ... Multi-channel detector, 7 ... Prism 8 ... Spectral projection optical system, 11 ... Observation optical system 12 ... First multi-channel spectroscopic detection system, 121 ... Dichroic mirror 122 ... Diffraction grating, 123 ... Spectral projection optical system 124 ... Multi Channel detector, 13 ... second multi-channel spectroscopic detection system 131 ... dichroic mirror, 132 ... diffraction grating 133 ... spectral projection optical system, 134 ... multi-channel detector 14 ... third multi-channel spectroscopic detection system, 141 ... reflection Mirror 142 ... Diffraction grating, 143 ... Spectral projection optical system 144 ... Multi-channel detector, 300 ... First multi-channel Channel spectroscopic detection system 400 ... second multi-channel spectroscopic detection system, 21 ... observation optical system 22 ... diffraction grating, 23 ... relay lens system, 24.25 ... reflection mirror 26.28 ... spectral projection optical system, 27.29 ... Multi-channel detector

Claims (1)

In a laser scanning microscope that irradiates a sample with laser light as excitation light and observes fluorescence emitted from the sample,
A spectroscopic element for dispersing the fluorescence in a spectrum;
A multi-channel detector for detecting the spectrum;
A spectral projection optical system for projecting the spectrum onto the multi-channel detector;
A mask that is disposed immediately in front of the multi-channel detector to reflect the wavelength of the excitation light and is transparent to other wavelengths;
Driving means for rotating the spectroscopic element so as to move the position of the spectrum in a dispersion direction;
Driving means for translating the multi-channel detector along a spectral dispersion direction;
The laser scanning characterized in that the mask is movable in the dispersion direction of the spectrum so as to reflect the wavelength of the excitation light even when the multi-channel detector moves or the spectroscopic element rotates. Type microscope.

Images