JP5541978B2 - Laser scanning microscope - Google Patents

Description

  The present invention relates to a laser scanning microscope.

  2. Description of the Related Art Conventionally, a laser scanning microscope including a surface reflection type diffraction grating and a multichannel PMT composed of 32 cells arranged one-dimensionally is known (for example, see Patent Document 1). Further, a laser scanning microscope including a rotatable surface reflection type diffraction grating, a variable slit having a variable slit width, and a zero-dimensional detector is known (for example, see Patent Document 2).

JP 2003-185581 A JP 2006-010944 A

  The laser scanning microscope disclosed in Patent Document 1 measures fluorescence spectroscopic data for each pixel and performs arithmetic processing using a reference spectrum for each dye to separate a plurality of fluorescent lights with large crosstalk.

  However, when the wavelength resolution is increased, the amount of light detected by one cell is reduced, and the S / N is deteriorated. Further, if the wavelength width captured by one cell is widened, the wavelength becomes brighter but the wavelength resolution is lowered. Therefore, the wavelength range of about 10 nm width is detected by one cell, and there is a disadvantage that the wavelength resolution is poor. In addition, the multi-channel detector is expensive including the electric system, and the control system is complicated. Furthermore, the reflection type diffraction grating has a low diffraction efficiency, and has a diffraction efficiency of about 70% even at the most efficient wavelength.

  The laser scanning microscope disclosed in Patent Document 2 can arbitrarily set a wavelength width and a wavelength region to be captured at once by a rotatable surface reflection type diffraction grating and a variable slit. For example, by setting the slit width to a wavelength width of 10 nm and setting the wavelength feed of the rotation pitch of the diffraction grating to 2 nm, both brightness and wavelength resolution are realized.

  However, since correct data cannot be acquired unless the slit width of the variable slit and the rotation angle of the diffraction grating are accurately matched, there are inconveniences that adjustment is difficult and the apparatus becomes complicated. In addition, both the variable slit and the diffraction grating need to be controlled synchronously, and there is a disadvantage that the system becomes expensive. Furthermore, the reflection type diffraction grating has a wide wavelength range that can be taken in, but has a low diffraction efficiency, and has a diffraction efficiency of about 70% even at the most efficient wavelength.

  The present invention has been made in view of the above-described circumstances, and is bright and has a high wavelength resolution with a simple and inexpensive configuration without using a slit for cutting out a wavelength width or an expensive multichannel detector. An object of the present invention is to provide a laser scanning microscope capable of spectral detection.

In order to achieve the above object, the present invention provides the following means.
The present invention is directed to a laser light source that emits laser light, a scanning unit that scans laser light emitted from the laser light source, and a laser beam that is scanned by the scanning unit to irradiate the sample while light from the sample An objective lens that collects light from the specimen, a transmission-type Volume Phase Holographic (VPH) diffraction grating that disperses light from the specimen collected by the objective lens into a plurality of wavelength bands, and the VPH diffraction grating. A light detection unit that detects light; and an incident angle changing unit that changes an incident angle of the light from the specimen to the VPH diffraction grating. The incident angle changing unit includes a plurality of detection units that are detected by the light detection unit. the wavelength band light the Bragg reflection condition to satisfy respectively, the incident angle to the VPH grating of light from the specimen is varied for each detection wavelength band, before by the incident angle changing means Employing a laser scanning microscope for switching the incident angle of the light from the specimen in synchronization with scanning by the scanning unit.

According to the present invention, the laser beam emitted from the laser light source is scanned on the sample by the scanning unit, and the light from the sample is collected by the objective lens. The light from this sample is dispersed in a plurality of wavelength bands by a transmission type Volume Phase Holographic (VPH) diffraction grating, and detected by a light detection unit. At this time, the incident angle changing means changes the incident angle of the light from the sample to the VPH diffraction grating, thereby changing the dispersion state of the light from the sample. In this case, the incident angle of the light from the sample to the VPH diffraction grating is changed so that the Bragg reflection condition is satisfied for each of the light in the plurality of wavelength bands detected by the light detection unit. The Bragg reflection condition refers to a condition that satisfies the following expression. Under this condition, high diffraction efficiency can be obtained, and light with high intensity can be detected.
2 dsin Θ = nλ
Here, d is the period of the VPH diffraction grating, Θ is the incident angle of the light from the sample to the VPH diffraction grating, n is an integer, and λ is the wavelength.

Therefore, by changing the incident angle of the light from the sample to the VPH diffraction grating so as to satisfy the Bragg reflection condition for each wavelength band, the light from the sample with high intensity can be detected for each wavelength band.
As described above, according to the present invention, it is possible to perform bright and high spectral resolution spectral detection with a simple and inexpensive configuration without using a slit for cutting out the wavelength width or an expensive multichannel detector. it can.
Further, by switching the incident angle of light from the sample by the incident angle changing means in synchronization with the scanning by the scanning unit, the time difference for each detection wavelength can be extremely reduced, and a moving sample can be accurately captured. be able to.

In the above invention, the incident angle changing means may rotate the VPH diffraction grating about an axis orthogonal to the dispersion direction by the VPH diffraction grating.
By rotating the VPH diffraction grating around an axis orthogonal to the dispersion direction, the incident angle of light from the sample to the VPH diffraction grating changes. At this time, high diffraction efficiency can be obtained by rotating the VPH diffraction grating about the axis so as to satisfy the Bragg reflection condition for each of the light of the plurality of wavelength bands detected by the light detection unit, High intensity light can be detected.

In the above invention, the wavelength of the laser light is synchronized with the operation of the control unit for driving the incident angle changing unit and the operation of the incident angle changing unit according to the center wavelength of the wavelength band detected by the light detecting unit. It is good also as providing the wavelength selection means to select.
By doing in this way, according to the center wavelength of the wavelength band detected by the light detection unit, the light of the wavelength satisfying the Bragg reflection condition can be switched, and the wavelength of the laser light can be switched in synchronization with this switching. . This makes it possible to detect light from the sample for each wavelength dispersed by the VPH diffraction grating without using a wavelength selection slit, a multi-channel PMT, or a reflective diffraction grating, thereby easily making multiple stained images. Can be acquired. In addition, by changing the incident angle to the VPH diffraction grating so that the diffraction efficiency at the center wavelength of the wavelength band to be detected is maximized, the diffraction efficiency can be improved as compared with a conventional reflection diffraction grating. And a bright image can be acquired.

In the above invention, the detection wavelengths are switched in time series at equal intervals to acquire images (λ scan), and the spectral data for each pixel is acquired using the luminance change of the corresponding pixel in each of the images. Also good.
In this way, since the spectral data can be obtained in detail, it is possible to separate a dye having a large crosstalk.

In the above invention, the pitch of the center wavelength of the light detected by the light detection unit may be set to be narrower than the wavelength width when the diffraction efficiency of the VPH diffraction grating is 50%.
By doing in this way, the diffraction efficiency of a VPH diffraction grating can be improved and a bright image can be acquired.

In the above invention, the wavelength width when the diffraction efficiency of the VPH diffraction grating is 50% may be set to be narrower than the half width of the fluorescence spectrum having the narrowest bandwidth among the fluorescence spectra of the plurality of fluorescent dyes. .
The half-value width of the diffraction efficiency of the spectral characteristic at one rotation angle of the VPH diffraction grating is preferably narrower than the half-value width of the band of the fluorescent dye having the narrowest bandwidth among the fluorescent dyes, and particularly preferably half or less.

In the above invention, the wavelength pitch of the λ scan may be set to a half or less of the narrowest interval between the peak wavelengths of the plurality of fluorescent dyes.
The wavelength pitch of the λ scan is preferably less than half of the narrowest interval among the plurality of fluorescence spectra.

In the above invention, the incident angle of the light from the sample may be switched by the incident angle changing unit for each line in the main scanning direction of the scanning unit.
By doing so, it is possible to effectively synchronize the switching of the incident angle and the scanning by the scanning unit, and it is possible to accurately capture a moving sample.

In the above invention, the reciprocal of the diffraction efficiency when the relative angle of the light from the VPH diffraction grating and the sample incident on the VPH diffraction grating is matched to the Bragg reflection condition is detected in the detection data by the light detector for each wavelength It is good also as multiplying by.
By multiplying the detection data by the light detection unit for each wavelength by the reciprocal of the diffraction efficiency when the relative angle of the light from the VPH diffraction grating and the sample is matched to the Bragg reflection condition, the difference in diffraction efficiency for each wavelength is obtained. It can correct | amend and can acquire exact spectral data.

In the above invention, a condensing optical system for condensing light dispersed from the VPH diffraction grating on an effective light receiving surface of the light detection unit may be provided between the VPH diffraction grating and the light detection unit. .
In this way, the light dispersed from the VPH diffraction grating can be collected on the effective light receiving surface of the light detection unit, and the detection efficiency of light from the sample can be improved to obtain a bright image. .

In the above invention, the condensing optical system may be a cylindrical lens that condenses light from the specimen only in the dispersion direction.
By using a cylindrical lens as the condensing optical system, the light from the sample can be condensed only in the dispersion direction by the VPH diffraction grating, and can be efficiently condensed on the effective light receiving surface of the light detection unit.

In the above invention, a spectroscopic unit including the VPH diffraction grating, the incident angle changing unit, and the light detection unit, and a main scan unit including a detection system capable of selecting a wavelength to be detected from light from the sample; The spectroscopic unit and the main scan unit may be configured to be detachable.
By doing in this way, the light from a sample can be detected by either or both of the light detection unit and the detection system, and multicolor light can be detected at the same time. Further, by making the spectroscopic unit having the above-described configuration to be retrofitted to the main scan unit, a bright and inexpensive spectroscopic detection means can be easily added. In other words, the laser scanning microscope equipped with the main scan unit can be upgraded so that the light from the specimen can be detected efficiently for each fluorescence wavelength band and the multiple stained specimen with high wavelength resolution can be observed. be able to.

In the above invention, a second light detection unit that detects light (0th-order light) traveling straight through the VPH diffraction grating may be provided.
By doing so, both the mode in which broadband light is detected at once by the second light detection unit and the mode in which light in a relatively narrow wavelength band (for example, 10 to 20 nm) is detected by the VPH diffraction grating are performed. These modes can be switched by the incident angle changing means.

In the above invention, it is possible to provide re-input means for re-inputting light (0th-order light) traveling straight through the VPH diffraction grating to the light detection unit.
In this way, both the light diffracted by the VPH diffraction grating and the light traveling straight through the VPH diffraction grating can be detected by one photodetection unit, and one expensive photodetection unit (for example, PMT) is provided. Can be reduced. As the re-input means, for example, a mirror that reflects light traveling straight through the VPH diffraction grating toward the light receiving surface of the light detection unit may be used.

In the above invention, the light detector is a non-scan detector, and the non-scan detector detects light from the sample while changing the incident angle to the VPH diffraction grating by the incident angle changing means. A plurality of images may be generated, and pixel data between the plurality of images may be combined.
By doing so, spectral data for each pixel can be acquired by combining data of corresponding pixels in each image acquired by the non-descan detector while changing the incident angle to the VPH diffraction grating. it can.

In the above invention, the incident angle changing means is a reflection mirror that varies the angle of detection light incident on the VPH diffraction grating, and the reflection mirror and the light detection unit are substantially symmetrical with the VPH diffraction grating in between. It is good also as arrange | positioning in this position.
In this way, by rotating the mirror, it is possible to cope with wavelength switching that is faster than when the VPH diffraction grating is rotated. Further, the wavelength at which the diffraction efficiency is maximized is Bragg-reflected and gathered at one point on the light detection unit, so that the light receiving area of the light detection unit can be reduced.

  According to the present invention, there is an effect that spectral detection can be performed brightly and with high wavelength resolution with a simple and inexpensive configuration.

1 is a schematic configuration diagram of a laser scanning microscope according to a first embodiment of the present invention. It is the elements on larger scale of the wavelength selection apparatus periphery of FIG. It is sectional drawing of the diffraction grating of FIG. It is a figure explaining the light diffracted by the diffraction grating of FIG. 5 is a graph showing a VPH diffraction grating rotation angle and an absolute emission angle for each central wavelength in FIG. 4. It is a graph which shows the relationship between diffraction efficiency and a wavelength band. It is a graph which shows the spectral characteristics in the wavelength which satisfy | fills Bragg reflection conditions. It is a schematic block diagram of the laser scanning microscope which concerns on the 2nd Embodiment of this invention. It is a schematic block diagram of the laser scanning microscope which concerns on the 3rd Embodiment of this invention. FIG. 10 is a partially enlarged view around the wavelength selection device in FIG. 9. FIG. 11 is a partially enlarged view of a periphery of a wavelength selection device according to a modification of FIG. 10. It is a schematic block diagram of the laser scanning microscope which concerns on the 4th Embodiment of this invention. It is a schematic block diagram of the laser scanning microscope which concerns on the 5th Embodiment of this invention. It is a figure explaining the state at the time of incident light (420 nm) of an image right end Bragg-reflecting in the laser scanning microscope of FIG. It is a figure explaining the data acquisition area in the state of FIG. It is a figure explaining the state at the time of the Bragg reflection of the incident light (420 nm) of an image center in the laser scanning microscope of FIG. It is a figure explaining the data acquisition area in the state of FIG. It is a figure explaining the state at the time of the Bragg reflection of the incident light (420 nm) of the image left end in the laser scanning microscope of FIG. It is a figure explaining the data acquisition area in the state of FIG. FIG. 14 is a diagram illustrating a state when incident light (560 nm) at the left end of the image is Bragg-reflected in the laser scanning microscope of FIG. 13. It is a figure explaining the data acquisition area in the state of FIG. FIG. 14 is a diagram illustrating a state when incident light (700 nm) at the left end of the image is Bragg-reflected in the laser scanning microscope of FIG. 13. It is a figure explaining the data acquisition area in the state of FIG. It is a figure explaining the data acquisition area in each pixel in the laser scanning microscope of FIG.

[First Embodiment]
A laser scanning microscope 1 according to a first embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, a laser scanning microscope 1 according to the present embodiment includes a light source device 10 that emits laser light, a scan unit 20 that scans laser light from the light source device 10, an imaging lens 41, and A connection unit 40 having a mirror 42 and guiding the laser light from the scan unit 20, and an objective lens for irradiating the sample A with the laser light guided by the connection unit 40 and condensing the light from the sample A 45 and a control unit 50 for controlling them.

  The light source device 10 includes, for example, a laser light source 11 that emits laser light having a central wavelength of 560 nm, a laser light source 12 that emits laser light having a central wavelength of 488 nm, a laser light source 13 that emits laser light having a central wavelength of 405 nm, and the laser light sources, for example. A dichroic mirror 14 for synthesizing the optical path of the laser beam from the laser beam, and an acousto-optic element (wavelength selection means) 15 such as an AOTF (Acousto-optic tunable filter) for switching the wavelength of the emitted laser beam.

The light source device 10 and the scan unit 20 are connected by, for example, a visible single mode fiber 19, and the laser light from the light source device 10 is guided to the scan unit 20.
The scan unit 20 includes an irradiation optical system 25 that irradiates the sample A with the laser light from the light source device 10 and a detection optical system 30 that detects the light from the sample A.

  The irradiation optical system 25 reflects the laser light from the light source device 10, while the dichroic mirror 21 transmits the light from the specimen A, and the laser light reflected by the dichroic mirror 21 is two-dimensionally scanned on the specimen A. A galvano scanner (scanning unit) 22 and a pupil projection lens 23 that projects the pupil of the laser beam are provided.

  The detection optical system 30 includes a confocal lens 31 that condenses the light from the specimen A that has passed through the dichroic mirror 21 in the confocal pinhole 32, and a confocal pinhole that allows only light from the focal position of the specimen A to pass through. 32, a collimating lens 33 that collimates the light from the specimen A that has passed through the confocal pinhole 32, and laser light from the light source device 10 among the light from the specimen A that has been collimated by the collimating lens 33. A notch filter 34 that blocks unnecessary components such as a wavelength selector 35, a wavelength selector 35 that selects light from the sample A that has passed through the notch filter 34, from a plurality of wavelength bands, and a wavelength selector 35. And a light detection unit 36 that detects light in the wavelength band selected by (1).

  As shown in FIG. 2, the wavelength selection device 35 holds a transmission type diffraction grating 37 that disperses light from the specimen A that has passed through the notch filter 34 into a plurality of wavelength bands, and a diffraction grating 37. And a galvanometer (incident angle changing means) 38 that rotates 37 around an axis perpendicular to the direction of dispersion of light from the specimen A (the direction along the plane of the drawing in FIG. 2).

As shown in FIG. 3, the diffraction grating 37 is a transmission type VPH (Volume Phase Holographic) diffraction grating.
The transmission type VPH diffraction grating is a medium in which the refractive index of the medium is periodically changed, so that high diffraction efficiency is obtained at a wavelength satisfying the Bragg reflection condition, and almost no diffraction is performed at other wavelengths. it can.
For example, by rotating and adjusting the direction of the diffraction grating 37 with respect to the white light W, it is possible to efficiently extract light having a wavelength that meets the Bragg reflection conditions. Specifically, as shown in FIG. 3, when the diffraction grating 37 is at the position indicated by the symbol R, red wavelength light is emitted in the direction indicated by the arrow R, and the diffraction grating 37 is indicated by the symbol G. When the diffraction grating 37 is at the position indicated by the symbol B, the light having the blue wavelength is emitted in the direction indicated by the arrow B.
Note that the wavelength width of the light obtained as the first-order diffracted light (light emitted in the directions of arrows R, G, and B) varies depending on the thickness of the diffraction grating 37, and the thicker the thickness, the narrower the wavelength width. The thickness is selected so as to obtain a desired wavelength width.

The galvanometer 38 rotates the diffraction grating 37 around an axis orthogonal to the dispersion direction of the light from the specimen A based on an instruction from the control unit 50. Specifically, the galvanometer 38 is driven in accordance with the center wavelength of the wavelength band detected by the light detection unit 36 so that light in a plurality of wavelength bands detected by the light detection unit 36 satisfies the Bragg reflection condition. Further, the diffraction grating 37 is rotated around the axis. Here, the Bragg reflection condition means a condition satisfying the following expression, and high diffraction efficiency is obtained under this condition.
2 dsin Θ = nλ
In the above formula, d is the period of the diffraction grating 37, Θ is the incident angle of the light from the sample A to the diffraction grating 37, n is an integer, and λ is the wavelength. In the transmission type VPH diffraction grating, a high diffraction efficiency is obtained when n = 1.

  The light detection unit 36 is, for example, a PMT (Photomultiplier Tube). When a side-on type PMT is used as the light detection unit 36, since the sensitivity uniformity in the axial direction is relatively loose, the axial direction is arranged according to the dispersion direction.

  Here, the light receiving surface of the light detection unit 36 needs to have a size and an angle uniformity of the light receiving surface that can receive all dispersed wavelengths. However, if there is a problem with the size and angle uniformity of the light receiving surface, the size with respect to the light receiving surface can be adjusted by arranging a condensing optical system such as a cylindrical lens.

  Therefore, between the wavelength selector 35 and the light detector 36, as shown in FIG. 2, a cylindrical lens (collector) that collects light in the wavelength band selected by the wavelength selector 35 only in the dispersion direction. (Optical optical system) 39 is arranged. By the action of the cylindrical lens 39, light in a plurality of wavelength bands dispersed by the wavelength selection device 35 can be condensed on the light detection unit 36, and the light receiving surface of the light detection unit 36 can be reduced.

  The control unit 50 synchronously controls the XY galvano scanner 22, the acoustooptic device 15, the galvanometer 38, and the light detection unit 36. Specifically, the control unit 50 generates an image of the specimen A based on the scanning position of the XY galvano scanner 22 and the light intensity from the specimen A detected by the light detection unit 36. In addition, the control unit 50 switches the wavelength of the laser light by the acoustooptic device 15 according to the center wavelength of the wavelength band detected by the light detection unit 36 and drives the galvanometer 38 to change the incident angle to the diffraction grating 37. It is supposed to change.

The action of light from the specimen A detected by the light detection unit 36 when the galvanometer 38 is operated as described above will be described below using a specific example.
In FIG. 4, the symbol α is a rotation angle of the diffraction grating 37 (hereinafter referred to as “VPH diffraction grating rotation angle”), that is, an angle of a normal line of the diffraction grating 37 with respect to incident light (hereinafter referred to as “VPH diffraction grating normal line”). is there. A symbol β represents an emission angle of the first-order diffracted light with respect to the VPH diffraction grating normal line. Symbol γ is an absolute emission angle, that is, an emission angle of the first-order diffracted light with respect to incident light (0th-order light).

  FIG. 5 shows the relationship between the VPH diffraction grating rotation angle α and the absolute emission angle γ for each central wavelength. In FIG. 5, a VPH diffraction grating rotation angle α indicates a rotation angle for each wavelength satisfying the Bragg reflection condition.

  The thickness of the diffraction grating 37 determines the wavelength range that can be diffracted as the first-order diffracted light. That is, by adjusting the thickness of the refractive index distribution of the diffraction grating 37, it is possible to adjust the wavelength range having high diffraction efficiency with respect to the angle. Here, for example, by setting the grating interval of the diffraction grating 37 to 1200 lines / mm and the thickness of the refractive index distribution of the diffraction grating 37 to about 120 μm, the wavelength width is about 50% with a diffraction efficiency of 50% as shown in FIG. The wavelength width is about 20 nm with 10 nm, diffraction efficiency of about 0%. Note that the side rope has a low strength and can be ignored.

  That is, when the rotation angle of the diffraction grating 37 is set to 15.7 °, the diffraction efficiency shown by the solid line in FIG. 6 is obtained, and when it is set to 19.3 °, the diffraction efficiency shown by the dotted line in FIG. The diffraction efficiency indicated by the two-dot chain line in FIG. The characteristic of the diffraction efficiency with respect to each central wavelength is represented as a curve 51 in FIG.

The operation of the laser scanning microscope 1 having the above configuration will be described below.
Here, a case where fluorescence observation of the specimen A is performed by the light detection unit 36 using the laser scanning microscope 1 according to the present embodiment will be described.
First, the light source device 10 is operated, and laser light is incident on the scan unit 20. The laser light guided to the scan unit 20 is reflected by the dichroic mirror 21 and guided to the XY galvano scanner 22. The XY galvano scanner 22 scans the laser beam two-dimensionally on the specimen A. The laser beam thus scanned passes through the pupil projection lens 23 and the imaging lens 41, is deflected by the mirror 42, and is irradiated onto the sample A by the objective lens 45.

  On the focal plane of the objective lens 45 on the specimen A, the fluorescent substance in the specimen A is excited and fluorescence is generated. The generated fluorescence is collected by the objective lens 45 and guided to the XY galvano scanner 22 via the mirror 42, the imaging lens 41, and the pupil projection lens 23. The fluorescence that has passed through the XY galvano scanner 22 passes through the dichroic mirror 21, is condensed by the confocal lens 31, and only the fluorescence generated in the focal plane of the specimen A passes through the confocal pinhole 32. The fluorescence that has passed through the confocal pinhole 32 is converted into parallel light by the collimating lens 33 and is transmitted through the notch filter 34 to cut the wavelength of unnecessary excitation light. The fluorescence transmitted through the notch filter 34 is detected by the light detection unit 36 after the wavelength selection device 35 selects the fluorescence wavelength band that satisfies the Bragg reflection condition.

  Thus, by storing the intensity information of the fluorescence detected by the light detection unit 36 and the irradiation position of the laser beam by the XY galvano scanner 22 in association with each other, it is possible to construct a two-dimensional fluorescence image. .

In the image acquisition method described above, processing when acquiring the multiple stained image by switching the light detected by the light detection unit 36 will be described.
As a precondition, the specimen A triple-stained with the fluorescent dye DAPI (405 nm excitation / 450 nm detection), the fluorescent dye Alexa 488 (488 nm excitation / 530 nm detection), and the fluorescent dye Alexa 560 (560 nm excitation / 580 nm detection) is observed. In this case, the notch filter 34 having a characteristic of cutting light having wavelengths of 405 nm, 488 nm, and 560 nm is used.

First, the laser wavelength is set to 405 nm by the acoustooptic device 15.
In this case, by driving the galvanometer 38 so that the light having the center wavelength of 450 nm has the maximum diffraction efficiency, the VPH diffraction grating rotation angle α (15.7 °) is set, and the XY galvano scanner 22 is operated. An image is acquired (DAPI image).

Next, the laser wavelength is set to 488 nm by the acoustooptic device 15.
In this case, the galvanometer 38 is driven so that the light having the center wavelength of 530 nm has the maximum diffraction efficiency, thereby setting the VPH diffraction grating rotation angle α (18.5 °) and operating the XY galvano scanner 22. An image is acquired (Alexa 488 image).

Next, a laser wavelength of 560 nm is set by the acoustooptic device 15.
In this case, the galvanometer 38 is driven so that the light having the center wavelength of 580 nm has the maximum diffraction efficiency, thereby setting the VPH diffraction grating rotation angle α (20.4 °) and operating the XY galvano scanner 22. An image is acquired (Alexa 560 image).
Then, the three monochrome images acquired as described above are displayed with a pseudo color and displayed as a multi-channel image.

  As described above, according to the laser scanning microscope 1 according to the present embodiment, the galvanometer 38 is operated so as to satisfy the Bragg reflection condition in accordance with the detection wavelength band of each fluorescent dye, and the diffraction grating 37 is By rotating around the axis, the light from the specimen A can be efficiently detected for each fluorescence wavelength band. That is, according to the laser scanning microscope 1 according to the present embodiment, it is possible to multiplex light with high wavelength resolution with a simple and inexpensive configuration without using a slit for cutting out a wavelength width or an expensive multichannel detector. The stained specimen can be observed.

  Further, the diffraction grating 37 is a transmission type VPH diffraction grating and satisfies the Bragg reflection condition for each detection wavelength band. The amount of light to be detected can be increased, and a bright multiple stained image can be acquired.

[First Modification]
Below, the 1st modification of the laser scanning microscope 1 which concerns on this embodiment is demonstrated.
Processing in the case of performing λ scan by sequentially switching the wavelengths of light detected by the light detection unit 36 in this modification will be described below.
As a precondition, the specimen A double-stained with the fluorescent dye GFP and the fluorescent dye YFP is observed. In this case, the notch filter 34 having a characteristic of cutting light with a wavelength of 488 nm is used, and the dichroic mirror 21 is made of, for example, bare glass.

First, the laser wavelength is set to 488 nm by the acoustooptic device 15.
In this case, the galvanometer 38 is driven so that the light having the center wavelength of 500 nm has the maximum diffraction efficiency, thereby setting the VPH diffraction grating rotation angle α (17.5 °) and operating the XY galvano scanner 22. Get an image.

Next, the galvanometer 38 is driven so that the light with the center wavelength of 502 nm has the maximum diffraction efficiency, thereby setting the VPH diffraction grating rotation angle α and operating the XY galvano scanner 22 to acquire an image.
Hereinafter, the image acquisition is repeated by setting the VPH diffraction grating rotation angle α at which the diffraction efficiency is maximized while shifting the center wavelength of the detected light by 2 nm. These operations are performed by the control unit 50 while switching in time series.

  Based on the spectral data obtained for each pixel as described above, for example, by performing an unmixing process described in Japanese Patent Laid-Open No. 2003-185581, the fluorescent dye GFP and the fluorescent dye YFP are separated, and a multi-channel image is obtained. Display as.

  According to the laser scanning microscope 1 according to this modified example, in addition to the above-described effects, the diffraction grating 37 can be rotated to finely acquire spectroscopic data, so that even a dye having a large crosstalk can be separated. In particular, in the present modification, the diffraction grating 37 is rotated and the spectral data can be acquired in increments of 2 nm, so that a dye having a large crosstalk can be separated.

[Second Modification]
Below, the 2nd modification of the laser scanning microscope 1 which concerns on this embodiment is demonstrated.
In this modification, in the processing for acquiring the image, the rotation angle of the diffraction grating 37 by the galvanometer 38 is synchronized with the scanning by the XY galvano scanner 22, and X which is the main scanning of the XY galvano scanner 22 is performed. Do this for each line.
By doing in this way, the time difference for every detection wavelength can be made very small, and the moving sample A can be accurately captured.

[Third Modification]
Below, the 3rd modification of the laser scanning microscope 1 which concerns on this embodiment is demonstrated.
In the first embodiment described above, only one central wavelength is detected for each dye. However, in this modification, data is acquired at a plurality of central wavelengths in accordance with the spectral characteristics of the dye, and the same dye data is obtained. Add up.
By doing in this way, since the data of the same pigment | dye are added together, S / N in each image can be improved.

[Fourth Modification]
Below, the 4th modification of the laser scanning microscope 1 which concerns on this embodiment is demonstrated.
In this modification, the reciprocal of the diffraction efficiency (curve 51 in FIG. 6) when the relative angle of the light from the diffraction grating 37 and the sample A incident on the diffraction grating 37 is matched to the Bragg reflection condition is calculated for each wavelength. Multiply the detection data.
By doing so, the difference in diffraction efficiency for each wavelength can be corrected, and accurate spectral data can be acquired.

[Fifth Modification]
Below, the 5th modification of the laser scanning microscope 1 which concerns on the 1st modification of this embodiment is demonstrated.
In this modification, as shown in FIG. 7, spectral data are acquired by performing λ scan on three types of fluorescence (fluorescence spectra A, B, and C), and these fluorescences are separated by an unmixing process.

In this case, the half-value width of the diffraction efficiency of the spectral characteristic at one rotation angle of the diffraction grating 37 is smaller than the half-value width of the band of the fluorescent dye having the narrowest bandwidth in the spectral characteristic of each fluorescent dye, as shown below. Narrower is better.
Δλ _WS <Δλ _WB
here,
Δλ _WS : Wavelength width (half-value width) at the diffraction efficiency 50% point of the spectral characteristics of the diffraction grating 37
Δλ _WB : narrowest bandwidth, half width of spectral characteristic of fluorescence spectrum B

More preferably, the half value width of the diffraction efficiency of the spectral characteristic at one rotation angle of the diffraction grating 37 is half of the band of the fluorescent dye having the narrowest bandwidth in the spectral characteristic of each fluorescent dye, as shown below. Less than half of the price range is good.
Δλ _WS <Δλ _WB / 2

Further, as shown below, the wavelength pitch of λ scan is preferably less than half of the narrowest interval among the peak intervals of a plurality of fluorescence spectra.
Δλ _PS <Δλ _P / 2
here,
Δλ _PS : Wavelength pitch of λ scan by diffraction grating 37 Δλ _P : Peak wavelength interval of a fluorescent dye (between fluorescence spectra A and B) having the closest peak interval among a plurality of fluorescence spectra

Further, the wavelength pitch of the λ scan is preferably narrower than the half-value width of the diffraction efficiency of the spectral characteristic at one rotation angle of the diffraction grating 37 as shown below.
Δλ _WS > Δλ _PS

[Second Embodiment]
Next, a laser scanning microscope according to the second embodiment of the present invention will be described with reference to the drawings. The laser microscope apparatus 2 of the present embodiment is different from the laser microscope apparatus 1 of the first embodiment in that a detection unit is added to the optical path that guides light from the specimen. Hereinafter, regarding the laser microscope apparatus 2 of the present embodiment, description of points that are the same as those of the laser microscope apparatus 1 of the first embodiment will be omitted, and different points will be mainly described.

  As shown in FIG. 8, the laser scanning microscope 2 according to the present embodiment includes a light source device 10 that emits laser light, a scan unit (main scan unit) 60 that scans laser light from the light source device 10, and A connection unit 40 (not shown) that guides the laser light from the scan unit 60, and an objective lens 45 that irradiates the sample A with the laser light guided by the connection unit 40 and condenses the light from the specimen A ( (Not shown), a spectroscopic unit 70 that splits the light from the specimen A collected by the objective lens 45, and a control unit 50 that controls them.

  The scan unit 60 has a configuration similar to that of the scan unit 20 of the first embodiment (dichroic mirror 21, XY galvano scanner 22, pupil projection lens 23, confocal lens 31, confocal pinhole 32, and collimating lens 33). In addition, the dichroic mirrors 61 and 62 that branch part or all of the light from the specimen A that has been collimated by the collimator lens 33, and the laser light among the light from the specimen A that has been branched by the dichroic mirrors 61 and 62. Barrier filters 63 and 64 that block unnecessary components such as light detectors, and light detectors 65 and 66 that detect light from the specimen A that has passed through the barrier filters 63 and 64, respectively.

  As in the scan unit 20 of the first embodiment, the spectroscopic unit 70 is a notch that blocks unnecessary components such as laser light from the light source device 10 among the light from the specimen A that has been collimated by the collimator lens 33. A filter 34, a wavelength selection device 35 that disperses light from the specimen A that has passed through the notch filter 34 into a plurality of wavelength bands, and light detection that detects light in a wavelength band that satisfies the Bragg reflection condition by the wavelength selection device 35. Part 36.

  The dichroic mirrors 61 and 62 are inserted into and removed from the optical path of light from the specimen A by a drive mechanism (not shown) depending on the wavelength band to be detected. That is, when light from the sample A is detected by the light detection unit 65, the dichroic mirror 61 is disposed on the optical path of the light from the sample A. When the light detection unit 65 detects light from the sample A, the dichroic mirror 61 is removed from the optical path of the light from the sample A, and the dichroic mirror 62 is disposed on the optical path of the light from the sample A. . When the light from the specimen A is detected by the light detector 36, the dichroic mirrors 61 and 62 are removed from the optical path of the light from the specimen A.

  Alternatively, when light from the specimen A is detected by a plurality of light detection units, at least one of the dichroic mirrors 61 and 62 is disposed on the optical path of the light from the specimen A. For example, when both of the dichroic mirrors 61 and 62 are arranged on the optical path of the light from the sample A, the light reflected by the dichroic mirror 61 is detected by the light detection unit 65, and the dichroic mirror 62 is detected by the light detection unit 66. , And the light transmitted through the dichroic mirrors 61 and 62 is detected by the light detector 36. By doing in this way, three types of fluorescent dyes can be detected simultaneously.

  According to the laser scanning microscope 2 according to the present embodiment having the above-described configuration, by inserting / removing the dichroic mirrors 61 and 62 into / from the optical path of the light from the specimen A, any one of the light detection units 36, 65 and 66, Alternatively, light from the specimen A can be detected by a plurality of the light detection units 36, 65, and 66, and multicolor light can be detected at the same time. In addition, since such a spectroscopic unit 70 can be retrofitted to the scan unit 60 including the XY galvano scanner 22 and the light detection units 65 and 66, a bright and inexpensive spectroscopic detection means can be easily added. That is, the laser scanning microscope 2 provided with the scan unit 60 can efficiently detect the light from the specimen A for each fluorescence wavelength band, and observe the multiple stained specimen with high wavelength resolution. Can be upgraded.

  In the present embodiment, the dichroic mirrors 61 and 62 inserted into and removed from the optical path are used as means for branching a part of the light from the specimen A. Instead of this, for example, a mirror and a raw glass are used. It is good also as a mechanism which switches and arrange | positions on an optical path.

[Third Embodiment]
Next, a laser scanning microscope according to the third embodiment of the present invention will be described with reference to the drawings. The laser microscope apparatus 3 of the present embodiment is different from the laser microscope apparatus 1 of the first embodiment in that a detection unit that detects light (0th-order light) transmitted through the diffraction grating 37 is added. Hereinafter, with respect to the laser microscope apparatus 3 of the present embodiment, description of points that are common to the laser microscope apparatus 1 of the first embodiment will be omitted, and different points will be mainly described.

  As shown in FIG. 9, the laser scanning microscope 3 according to this embodiment includes a light source device 10 that emits laser light, a scan unit 20 that scans laser light from the light source device 10, and a scan unit 20. A connection unit 40 (not shown) for guiding laser light, an objective lens 45 (not shown) for irradiating the sample A with the laser light guided by the connection unit 40 and condensing the light from the sample A; And a control unit 50 for controlling them.

The scan unit 20 includes an irradiation optical system 25 that irradiates the sample A with the laser light from the light source device 10 and a detection optical system 30 that detects the light from the sample A.
The irradiation optical system 25 includes a dichroic mirror 21, an XY galvano scanner 22, and a pupil projection lens 23, as in the first embodiment.

  The detection optical system 30 has the same configuration as in the first embodiment (the confocal lens 31, the confocal pinhole 32, the collimating lens 33, the notch filter 34, the wavelength selection device 35, and the light detection unit 36). And a light detection unit 71 that detects light (0th order light) transmitted through the diffraction grating 37.

  As shown in FIG. 10, when the diffraction grating 37 is arranged perpendicular to the incident light, most wavelengths of light pass through as zero-order light. Therefore, according to the laser scanning microscope 3 according to the present embodiment, the light detection unit 71 is arranged in the emission direction of the zero-order light, so that the light detection unit 71 can detect light having a wide wavelength range at a time. Can do.

  Thus, both a mode for detecting broadband light at a time and a mode for detecting light in a relatively narrow wavelength band (for example, 10 to 20 nm) by the transmission type VPH diffraction grating 37 can be performed. The switching can be performed using a galvanometer 38 that rotationally drives the diffraction grating 37.

When observing 0th order light by the light detection unit 71, it is also effective to provide a broadband bandpass filter having a transmission band of 500 to 600 nm, for example, instead of the notch filter 34.
Further, video AF may be applied using the light detection unit 71 that detects the 0th-order light.
Further, the light detection unit 71 may detect DIC or the like with IR reflected light.

[Modification]
Below, the modification of the laser scanning microscope 3 which concerns on this embodiment is demonstrated.
In this modified example, as shown in FIG. 11, instead of the light detection unit 71, the reflection mirror 72 that reflects the 0th order light that passes through the diffraction grating 37 and the 0th order light that is reflected by the reflection mirror 72 A reflection mirror 73 that reflects toward the detection unit 36 is provided.

  According to the laser scanning microscope according to this modification, the 0th-order light transmitted through the diffraction grating 37 can be reflected by the reflection mirrors (re-input means) 72 and 73 and guided to the light detection unit 36. As a result, one expensive photodetection unit such as PMT can be reduced, and the manufacturing cost can be reduced.

  In the laser scanning microscope according to this modification, when only the primary light is detected, a shutter is provided between the diffraction grating 37 and the reflection mirror 72 so that the zero-order light does not enter the light detection unit 36. You should put in.

[Fourth Embodiment]
Next, a laser scanning microscope according to the fourth embodiment of the present invention will be described with reference to the drawings. The laser microscope apparatus 4 of the present embodiment is different from the laser microscope apparatus 1 of the first embodiment in that the incident angle to the diffraction grating 37 is changed instead of rotating the diffraction grating 37. Hereinafter, with respect to the laser microscope apparatus 4 of the present embodiment, description of points that are common to the laser microscope apparatus 1 of the first embodiment will be omitted, and different points will be mainly described.

  As shown in FIG. 12, the laser scanning microscope 4 according to this embodiment includes a light source device 10 that emits laser light, a scan unit 20 that scans laser light from the light source device 10, and a scan unit 20. A connection unit 40 (not shown) for guiding laser light, an objective lens 45 (not shown) for irradiating the sample A with the laser light guided by the connection unit 40 and condensing the light from the sample A; And a control unit 50 for controlling them.

The scan unit 20 includes an irradiation optical system 25 that irradiates the sample A with the laser light from the light source device 10 and a detection optical system 30 that detects the light from the sample A.
The irradiation optical system 25 includes a dichroic mirror 21, an XY galvano scanner 22, and a pupil projection lens 23, as in the first embodiment.

  The detection optical system 30 has the same configuration as the first embodiment (a confocal lens 31, a confocal pinhole 32, a collimator lens 33, a notch filter 34, a diffraction grating 37, and a light detection unit 36), and a notch filter. , And a galvanometer 76 that holds the reflection mirror 75 and rotates the reflection mirror 75 about an axis orthogonal to the incident light.

  Unlike the other embodiments, the laser scanning microscope 4 according to the present embodiment is different from the other embodiments in that the method of adjusting the incident angle at which the diffraction efficiency with respect to the center of the detection wavelength is maximized. The reflecting mirror 75 arranged in the previous stage is rotated by the galvanometer 76.

  Since the diffraction grating 37 has the highest diffraction efficiency at the Bragg-reflected wavelength, the light with the maximum diffraction efficiency can be detected by arranging the reflection mirror 75 and the light detection unit 36 symmetrically with respect to the diffraction grating 37. it can.

  According to the laser scanning microscope 4 according to the present embodiment, by rotating the reflection mirror 75, it is possible to cope with wavelength switching that is faster than when the diffraction grating 37 is rotated. Further, the wavelength at which the diffraction efficiency is maximized is Bragg-reflected and gathered at one point on the light detection unit 36, so that the light receiving area of the light detection unit 36 can be reduced.

[Fifth Embodiment]
Next, a laser scanning microscope according to the fifth embodiment of the present invention will be described with reference to the drawings. The laser microscope apparatus 5 of the present embodiment is different from the laser microscope apparatus 1 of the first embodiment in that it includes a non-descan detection unit 80 that non-scan detects light from the specimen. Hereinafter, regarding the laser microscope apparatus 5 of the present embodiment, description of points that are the same as those of the laser microscope apparatus 1 of the first embodiment will be omitted, and different points will be mainly described.

  As shown in FIG. 13, the laser scanning microscope 5 according to the present embodiment includes a light source device 10 that emits laser light, a scan unit 20 that scans laser light from the light source device 10, and a scan unit 20. A connection unit 40 that guides the laser light, an objective lens 45 that irradiates the sample A with the laser light guided by the connection unit 40 and condenses the light from the sample A, and non-decodes the light from the sample A. A non-descan detection unit 80 for detecting the scan and a control unit 50 for controlling them are provided.

The scan unit 20 includes an irradiation optical system 25 that irradiates the sample A with the laser light from the light source device 10 and a detection optical system 30 that detects the light from the sample A.
The irradiation optical system 25 includes a dichroic mirror 21, an XY galvano scanner 22, and a pupil projection lens 23, as in the first embodiment.

The detection optical system 30 includes a confocal lens 31, a confocal pinhole 32, a collimator lens 33, a notch filter 34, and a light detection unit 86.
The connection unit 40 includes an imaging lens 41 and a dichroic mirror 87 that reflects the laser light from the light source device 10 and transmits the light from the specimen A.

  The non-descanning detection unit 80 includes an IR cut filter 81 that cuts laser light and the like from the light source device 10 from light from the specimen A that has passed through the dichroic mirror 87, an imaging lens 82, a reflection mirror 83, and pupil projection. A wavelength selector 35 having a lens 84, a diffraction grating 37 and a galvanometer 38, a cylindrical lens 39, and a light detector 36 are provided.

Here, a detailed example of the detailed configuration of the laser scanning microscope 5 having the above configuration will be described.
The laser scanning microscope 5 according to the present embodiment non-scans the fluorescence generated by the multiphoton excitation without returning it to the XY galvano scanner 22, and spectrally detects the wavelength band of 420 to 700 nm using the diffraction grating 37. To do.

The laser light emitted from the light source device 10 is an IR pulse laser that causes multiphoton excitation in the specimen A, and can be tuned to a wavelength of 720 to 1000 nm.
A dichroic mirror 87 is disposed at the intersection of the optical axis of the XY galvano scanner 22 and the optical axis of the objective lens 45.

The dichroic mirror 87 has a characteristic of reflecting IR pulse laser light from the light source device 10 and transmitting visible light of 400 to 710 nm.
In the transmission optical path of the dichroic mirror 87, an IR cut filter 81, an imaging lens 82, a reflection mirror 83, a pupil projection lens 84, a wavelength selection device 35, a lens 39, and a light detection unit 36 are arranged.

The pupil of the objective lens 45 is projected onto the diffraction grating 37 by the imaging lens 82 and the pupil projection lens 84.
The light incident on the diffraction grating 37 is always converted into parallel light by the pupil projection lens 84, but the incident angle with respect to the optical axis changes according to the scanning position by the XY galvano scanner 22.

  Specifically, assuming that the number of image pixels by scanning is 512 * 512 with the X direction in the paper, the light beam corresponding to the left end (first pixel) of the image is 91a and the image center (256 pixels) as shown in FIG. The light beam corresponding to the eye) is represented by reference numeral 92a, and the light beam corresponding to the right end of the image (512th pixel) is represented by reference numeral 93a. That is, the incident angle of the light incident on the diffraction grating 37 increases as the scanning position by the XY galvano scanner 22 moves away from the optical axis center.

  In this case, the difference in the incident angle of the light corresponding to the left and right ends of the image in the X direction to the diffraction grating 37 is divided into the shortest wavelength (420 nm in this embodiment) and the longest wavelength (in this embodiment). (700 nm) to match the angle difference satisfying the Bragg reflection condition.

A method for observing the specimen A of the laser scanning microscope 5 having the above configuration will be described below.
First, as shown in FIG. 14, when the incident light 93a corresponding to the right end of the X image (512th pixel) is 420 nm, the VPH diffraction grating rotation angle (α1) is adjusted to the Bragg reflection angle, and an XY scan is performed. Get an image. At this time, as shown in FIG. 15, the data at the right end of the image (512th pixel) is stored as 420 nm data, and the data of 1 to 511 pixels are discarded.

  Thereafter, as shown in FIGS. 16, 18, 20, and 22, XY image acquisition is repeated while changing the rotation angle of the diffraction grating 37. Note that the variable pitch of the VPH diffraction grating rotation angle α engraved between the drawings may be determined according to the required wavelength resolution.

  FIG. 16 shows the VPH diffraction grating rotation angle (α2) that is Bragg-reflected when the image center (256th pixel) incident light 92a is 420 nm. In this case, as shown in FIG. 17, the data at the center of the image (256th pixel) is 420 nm, the data at the right end of the image (512th pixel) is stored as 560 nm data, and the data up to the 1st to 255th pixels is Discard.

  FIG. 18 shows the VPH diffraction grating rotation angle (α3) that is Bragg-reflected when the incident light 91a at the left end of the image (first pixel) is 420 nm. In this case, as shown in FIG. 19, data at the left end of the image (first pixel) is 420 nm, data at the center of the image (256th pixel) is 560 nm, and data at the right end of the image (512th pixel) is 700 nm. save. In this case, there is no data to discard.

  FIG. 20 shows the VPH diffraction grating rotation angle (α4) that is Bragg-reflected when the incident light at the left end of the image (first pixel) is 560 nm (intermediate between 420 nm and 700 nm). In this case, as shown in FIG. 21, data at the left end (first pixel) of the image is stored as 560 nm data, and data at the center of the image (256th pixel) is stored as 700 nm data, and 257 to 512 pixels are stored. Discard the data.

  FIG. 22 shows the VPH diffraction grating rotation angle (α5) that is Bragg-reflected when the incident light at the left end (first pixel) of the image is 700 nm, and the data at the left end (first pixel) of the image is stored as 700 nm data. In this case, the data of 2 to 512 pixels is discarded as shown in FIG.

FIG. 24 shows a diagram summarizing the operation of the observation method of the specimen A described above.
As indicated by an arrow 95 in FIG. 24, according to the laser scanning microscope 5 according to the present embodiment, spectral data of 420 nm to 700 nm for each pixel is obtained by collecting data of the same pixel in the X direction of each acquired image. You can get it.

  As mentioned above, although each embodiment of the present invention has been described in detail with reference to the drawings, the specific configuration is not limited to this embodiment, and includes design changes and the like without departing from the gist of the present invention. . For example, the present invention is not limited to those applied to the above-described embodiments and modifications, and may be applied to embodiments in which these embodiments and modifications are appropriately combined, and is particularly limited. is not.

A Sample 1, 2, 3, 4, 5 Laser scanning microscope 10 Light source device 15 Acousto-optic element (wavelength selection means)
20 Scan unit 22 XY galvano scanner (scanning unit)
25 Irradiation optical system 30 Detection optical system 35 Wavelength selection device 36 Light detection unit 37 Diffraction grating 38 Galvanometer (incident angle changing means)
39 Cylindrical lens (Condensing optical system)
40 connection unit 45 objective lens 50 control unit 60 scan unit (main scan unit)
70 Spectrometer unit

Claims (19)

A laser light source for emitting laser light;
A scanning unit that scans the laser light emitted from the laser light source;
An objective lens for condensing the light from the specimen while irradiating the specimen with laser light scanned by the scanning unit;
A transmission-type Volume Phase Holographic (VPH) diffraction grating that disperses light from the specimen collected by the objective lens into a plurality of wavelength bands;
A light detection unit for detecting light dispersed by the VPH diffraction grating;
An incident angle changing means for changing an incident angle of light from the specimen to the VPH diffraction grating,
The incident angle changing means detects the incident angles of light from the specimen to the VPH diffraction grating so that light in a plurality of wavelength bands detected by the light detection unit satisfies Bragg reflection conditions, respectively. Change for each wavelength band ,
A laser scanning microscope in which the incident angle changing unit switches the incident angle of light from the specimen in synchronization with scanning by the scanning unit .   2. The laser scanning microscope according to claim 1, wherein the incident angle changing unit rotates the VPH diffraction grating about an axis orthogonal to a dispersion direction by the VPH diffraction grating. The incident angle changing means is a reflection mirror that varies the angle of detection light incident on the VPH diffraction grating, and the reflection mirror and the light detection unit are arranged at substantially symmetrical positions with the VPH diffraction grating in between. The laser scanning microscope according to claim 1. A control unit that drives the incident angle changing unit according to the center wavelength of the wavelength band detected by the light detection unit,
The laser scanning microscope according to any one of claims 1 to 3 , further comprising: a wavelength selecting unit that selects a wavelength of the laser light in synchronization with an operation of the incident angle changing unit. Switch the detection wavelength in time series at equal intervals to acquire images (λ scan),
The laser scanning microscope according to any one of claims 1 to 3, wherein spectral data for each pixel is acquired using a luminance change of a corresponding pixel in each of the images. The laser scanning microscope according to claim 5 , wherein the pitch of the center wavelength of the light detected by the light detection unit is set to be narrower than the wavelength width when the diffraction efficiency of the VPH diffraction grating is 50%.   6. The laser scanning according to claim 5, wherein a wavelength width at a diffraction efficiency of 50% of the VPH diffraction grating is set to be narrower than a half-value width of a fluorescence spectrum having the narrowest bandwidth among fluorescence spectra of a plurality of fluorescent dyes. Type microscope. 6. The laser scanning microscope according to claim 5 , wherein the wavelength pitch of the [lambda] scan is set to a half or less of the narrowest interval among the plurality of fluorescent dyes. The laser scanning microscope according to any one of claims 1 to 8 , wherein an incident angle of light from the specimen is switched by the incident angle changing unit for each line in the main scanning direction of the scanning unit.   The detection data obtained by the light detection unit is multiplied for each wavelength by the reciprocal of the diffraction efficiency when the relative angle of light from the VPH diffraction grating and the sample incident on the VPH diffraction grating matches Bragg reflection conditions. Item 10. The laser scanning microscope according to any one of Items 1 to 9. 9. The condensing optical system for condensing light dispersed from the VPH diffraction grating on an effective light receiving surface of the light detection unit between the VPH diffraction grating and the light detection unit . The laser scanning microscope according to any one of the above.   The laser scanning microscope according to claim 11, wherein the condensing optical system is a cylindrical lens that condenses light from the specimen only in the dispersion direction. A spectroscopic unit including the VPH diffraction grating, the incident angle changing unit, and the light detection unit, and a main scan unit including a detection system capable of selecting a wavelength to be detected from light from the sample,
The laser scanning microscope according to any one of claims 1 to 8, wherein the spectroscopic unit and the main scan unit are configured to be detachable. The laser scanning microscope according to any one of claims 1 to 8, further comprising a second light detection unit that detects light (0th-order light) that travels straight through the VPH diffraction grating. 9. The laser scanning microscope according to claim 1, further comprising a re-input unit that re-inputs light (zero-order light) that travels straight through the VPH diffraction grating into the light detection unit. The light detection unit is a non-scan detector;
While changing the incident angle to the VPH diffraction grating by the incident angle changing means, the non-descan detector generates a plurality of images from the light from the sample and combines pixel data between the plurality of images. The laser scanning microscope according to claim 1. A laser light source for emitting laser light;
  A scanning unit that scans the laser light emitted from the laser light source;
  An objective lens for condensing the light from the specimen while irradiating the specimen with laser light scanned by the scanning unit;
  A transmission-type Volume Phase Holographic (VPH) diffraction grating that disperses light from the specimen collected by the objective lens into a plurality of wavelength bands;
  A light detection unit for detecting light dispersed by the VPH diffraction grating;
  An incident angle changing means for changing an incident angle of light from the specimen to the VPH diffraction grating,
  The incident angle changing means detects the incident angles of light from the specimen to the VPH diffraction grating so that light in a plurality of wavelength bands detected by the light detection unit satisfies Bragg reflection conditions, respectively. Change for each wavelength band,
  Laser that multiplies detection data by the light detection unit for each wavelength when the relative angle of light from the VPH diffraction grating and the sample incident on the VPH diffraction grating matches Bragg reflection conditions. Scanning microscope.   A laser light source for emitting laser light;
  A scanning unit that scans the laser light emitted from the laser light source;
  An objective lens for condensing the light from the specimen while irradiating the specimen with laser light scanned by the scanning unit;
  A transmission-type Volume Phase Holographic (VPH) diffraction grating that disperses light from the specimen collected by the objective lens into a plurality of wavelength bands;
  A light detection unit for detecting light dispersed by the VPH diffraction grating;
  An incident angle changing means for changing an incident angle of the light from the specimen to the VPH diffraction grating;
A spectroscopic unit including the VPH diffraction grating, the incident angle changing unit, and the light detection unit, and a main scan unit including a detection system capable of selecting a wavelength to be detected from light from the sample,
  The spectroscopic unit and the main scan unit are configured to be detachable,
  The incident angle changing means detects the incident angles of light from the specimen to the VPH diffraction grating so that light in a plurality of wavelength bands detected by the light detection unit satisfies Bragg reflection conditions, respectively. A laser scanning microscope that changes for each wavelength band.   A laser light source for emitting laser light;
  A scanning unit that scans the laser light emitted from the laser light source;
  An objective lens for condensing the light from the specimen while irradiating the specimen with laser light scanned by the scanning unit;
  A transmission-type Volume Phase Holographic (VPH) diffraction grating that disperses light from the specimen collected by the objective lens into a plurality of wavelength bands;
  A light detection unit for detecting light dispersed by the VPH diffraction grating;
  An incident angle changing means for changing an incident angle of light from the specimen to the VPH diffraction grating,
  The light detection unit is a non-scan detector;
  The incident angle changing means detects the incident angles of light from the specimen to the VPH diffraction grating so that light in a plurality of wavelength bands detected by the light detection unit satisfies Bragg reflection conditions, respectively. Change for each wavelength band,
  While changing the incident angle to the VPH diffraction grating by the incident angle changing means, the non-descan detector generates a plurality of images from the light from the sample and combines pixel data between the plurality of images. Laser scanning microscope.

Images