Scanning microscope device

A scanning microscope device includes a light source that emits laser light; an X-Y galvanometer mirror that scans the laser light on a sample; an objective lens that irradiates the sample with the scanned laser light and collects fluorescence generated at an irradiated position; a non-descan-detection excitation DM that is disposed between the X-Y galvanometer mirror and the objective lens and separates the laser light and the fluorescence from each other; a fiber that receives the separated fluorescence through an entrance end thereof and emits the fluorescence from an exit end thereof that is formed in a substantially linear shape; a diffraction grating that disperses the fluorescence emitted from the exit end of the fiber in a direction orthogonal to a longitudinal direction of the exit end; and a multi-anode PMT having plural cells arrayed in the dispersing direction of the dispersed fluorescence.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to scanning microscope devices.

This application is based on Japanese Patent Application No. 2009-123912, the content of which is incorporated herein by reference.

2. Description of Related Art

In the related art, a known microscope device has a spectroscope disposed in a light-detection optical path so as to perform spectrum detection of light released from a sample (for example, see Japanese Unexamined Patent Application, Publication No. 2003-185581). In Japanese Unexamined Patent Application, Publication No. 2003-185581, a laser scanning microscope (LSM) is configured to disperse light passing through a confocal pinhole in a descan optical path by using a diffraction grating, and to acquire spectral data by using a multi-anode photomultiplier tube (PMT) having 32 detectors (cells) disposed one-dimensionally at positions where the spectrum is generated. Furthermore, Japanese Unexamined Patent Application, Publication No. 2003-185581 also discusses disposing a diffraction grating and a multi-anode PMT in a non-descan optical path effective for multiphoton detection so as to perform spectrum detection of non-descan light in a similar manner to that of descan light.

However, when an image is formed in the non-descan optical path at a conjugate position with respect to a confocal pinhole in FIG. 6 of Japanese Unexamined Patent Application, Publication No. 2003-185581, the light moves in the confocal pinhole in a direction orthogonal to the optical axis simultaneously with the scanning process, which is a problem in that spectral data of only a single point near the optical-axis center of a sample surface can be acquired. Another problem is that, since scattered light is blocked by the confocal pinhole or a slit disposed in the light-detection optical path, the detection efficiency of fluorescence is significantly impaired. Moreover, when a pupil position of an objective lens is disposed at a conjugate position with respect to the aforementioned pinhole, the angle of incidence of light incident on the pinhole may change due to scanning, but the position thereof does not. However, since a pupil has a certain surface area, a large portion of projected incident light is blocked by the pinhole, resulting in a significant loss in the fluorescence.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a scanning microscope device that can achieve a high S/N ratio and can perform spectrum detection at high sensitivity and high speed.

A first aspect of the present invention provides a scanning microscope device that includes a light source that irradiates a sample with laser light; a scanner that scans the laser light from the light source on the sample; an objective lens that irradiates the sample with the laser light scanned by the scanner and collects fluorescence generated at an irradiated position of the laser light; a wavelength separator that is disposed between the scanner and the objective lens and separates the laser light and the fluorescence from each other; an fiber for epi-fluorescence that receives the fluorescence separated by the wavelength separator through an entrance end thereof, guides the fluorescence, and emits the fluorescence from an exit end thereof that is formed in a substantially linear shape; a dispersing element that disperses the fluorescence emitted from the exit end of the fiber for epi-fluorescence in a direction orthogonal to the longitudinal direction of the exit end; and a multi-anode photomultiplier tube having a plurality of detectors arrayed in the dispersing direction of the fluorescence dispersed by the dispersing element.

According to this aspect, when the sample is irradiated, via the objective lens, with the laser light emitted from the light source and scanned by the scanner, the fluorescence generated in the sample is collected by the objective lens and separated by the wavelength separator before being guided to the dispersing element by the fiber for epi-fluorescence. The fluorescence is then dispersed by the dispersing element and is detected by the plurality of detectors of the multi-anode photomultiplier tube.

In this scanning microscope device, the wavelength separator separates the fluorescence generated in the sample from the optical path of the laser light without returning the fluorescence to the scanner, and the fiber for epi-fluorescence guides the fluorescence to the multi-anode photomultiplier tube, thereby minimizing the loss of fluorescence in the optical path from the sample to the multi-anode photomultiplier tube.

In addition, since the exit end of the fiber for epi-fluorescence is formed in a linear shape extending in the dispersing direction of the dispersing element, that is, in a direction orthogonal to the arrayed direction of the detectors of the multi-anode photomultiplier tube, the fluorescence collected by the objective lens can be made incident on the detectors without loss. Thus, the dispersed fluorescence can be detected at once by each detector, thereby allowing for spectrum detection at a high S/N ratio, high sensitivity, and high speed.

In the first aspect, the scanning microscope device may further include a condenser lens that collects fluorescence generated in a transmission direction at the irradiated position of the laser light scanned on the sample by the scanner; a fiber for transmission fluorescence that receives the fluorescence collected by the condenser lens through an entrance end thereof, guides the fluorescence, and emits the fluorescence toward the dispersing element from an exit end thereof that is formed in a substantially linear shape extending in a direction orthogonal to the dispersing direction of the dispersing element; and a transmitted-fluorescence entrance section that causes the fluorescence emitted from the exit end of the fiber for transmission fluorescence to enter an optical path of the fluorescence emitted from the exit end of the fiber for epi-fluorescence so as to cause the fluorescence from the fiber for transmission fluorescence to be incident on the dispersing element in place of the fluorescence from the fiber for epi-fluorescence.

With this configuration, by actuating the transmitted-fluorescence entrance section, the fluorescence from the fiber for transmission fluorescence is made incident on the dispersing element in place of the fluorescence from the fiber for epi-fluorescence, whereby the multi-anode photomultiplier tube can detect the fluorescence generated in the sample in the transmission direction of the laser light. Consequently, spectrum detection of fluorescence generated in the direction in which it returns from the sample and spectrum detection of fluorescence generated in the direction in which it is transmitted through the sample can be performed in a switching manner.

A second aspect of the present invention provides a scanning microscope device that includes a light source that irradiates a sample with laser light; a scanner that scans the laser light from the light source on the sample; a condenser lens that collects fluorescence generated in a transmission direction at an irradiated position of the laser light scanned on the sample by the scanner; a fiber for transmission fluorescence that receives the fluorescence collected by the condenser lens through an entrance end thereof, guides the fluorescence, and emits the fluorescence from an exit end thereof that is formed in a substantially linear shape; a dispersing element that disperses the fluorescence emitted from the exit end of the fiber for transmission fluorescence in a direction orthogonal to the longitudinal direction of the exit end; and a multi-anode photomultiplier tube having a plurality of detectors arrayed in the dispersing direction of the fluorescence dispersed by the dispersing element.

According to this aspect, when the sample is irradiated with the laser light emitted from the light source and scanned by the scanner, the fluorescence generated in the sample in the transmission direction of the laser light is collected by the condenser lens and is guided to the dispersing element by the fiber for transmission fluorescence. Thus, the loss of fluorescence in the optical path from the sample to the multi-anode photomultiplier tube can be minimized, thereby allowing for spectrum detection at a high S/N ratio, high sensitivity, and high speed.

In the above aspect, the scanning microscope device may further include a fluorescence returner that is disposed in a replaceable manner with the wavelength separator and that returns the fluorescence collected by the objective lens to an optical path of the laser light; switching means that switches between the fluorescence returner and the wavelength separator; a confocal pinhole that is disposed at a conjugate position with respect to a focal position of the objective lens and that allows part of the fluorescence returned to the optical path of the laser light by the fluorescence returner, switched by the switching means, and transmitted through the scanner to pass therethrough; and a descanned fluorescence entrance section that causes the fluorescence passing through the confocal pinhole to enter the optical path of the fluorescence emitted from the exit end of the fiber for epi-fluorescence.

With this configuration, by disposing the fluorescence returner on the optical path in place of the wavelength separator by using the switching means, the fluorescence generated in the sample can be returned to the scanner by the fluorescence returner. Then, the fluorescence passes through the confocal pinhole and is made to enter the optical path from the fiber for epi-fluorescence by the descanned fluorescence entrance section. Thus, the fluorescence not to be returned to the scanner and the fluorescence to be returned to the scanner can be observed in a switching manner, whereby the configuration used for spectrum detection in the multiphoton excitation observation mode can be shared with the configuration used for spectrum detection in the single-photon excitation observation mode.

Furthermore, in the above aspect, the entrance end of the fiber for epi-fluorescence may be disposed at a conjugate position with respect to a pupil position of the objective lens, and may have a diameter and a maximum light-receivable angle that satisfy the following formulas:
ΦDr≧Φpo×βPL
αre≧θa
where ΦDrdenotes the diameter of the entrance end of the fiber for epi-fluorescence, Φpodenotes a pupil diameter of the objective lens, βPLdenotes the projection magnification from the pupil position of the objective lens to the entrance end of the fiber for epi-fluorescence, αredenotes the maximum light-receivable angle of the entrance end of the fiber for epi-fluorescence, and θa denotes a maximum angle of incidence at the entrance end of the fiber for epi-fluorescence, determined on the basis of a scan range of the scanner.

With the diameter and the maximum light-receivable angle of the entrance end of the fiber for epi-fluorescence satisfying the aforementioned formulas, the fluorescence from the entire scan range of the scanner can be made incident on the entrance end of the fiber for epi-fluorescence, thereby preventing optical loss in the fluorescence.

Furthermore, in the above aspect, the maximum light-receivable angle of the entrance end of the fiber for epi-fluorescence may further satisfy the following formula:
αre>θb
where αredenotes the maximum light-receivable angle of the entrance end of the fiber for epi-fluorescence, and θb denotes a maximum angle of incidence at the entrance end of the fiber for epi-fluorescence, determined on the basis of a capturable field of view of the objective lens.

Since there is a large amount of scattered light in a deep section of the sample (for example, about 500 μm from the surface of the sample), fluorescence is also generated from outside the scan range. With the maximum light-receivable angle of the entrance end of the fiber for epi-fluorescence satisfying the aforementioned formula, a greater amount of scattered fluorescence can be collected when observing a deep section.

Furthermore, in the above aspect, the exit end of the fiber for epi-fluorescence may have a widthwise dimension and a lengthwise dimension that satisfy the following formulas:
W×βPM<PW
Hr×βPM<Ph
αro÷βPM<θp
where W denotes the widthwise dimension of the exit end of the fiber for epi-fluorescence, βPMdenotes the magnification at which the exit end of the fiber for epi-fluorescence is projected onto the multi-anode photomultiplier tube, PWdenotes a widthwise dimension of each detector of the multi-anode photomultiplier tube in the arrayed direction thereof, Hrdenotes the lengthwise dimension of the exit end of the fiber for epi-fluorescence, Phdenotes a dimension of each detector of the multi-anode photomultiplier tube in a direction orthogonal to the arrayed direction, αrodenotes an emission angle of the fiber for epi-fluorescence, and θp denotes a permissible light-receiving angle of the multi-anode photomultiplier tube.

With the widthwise and lengthwise dimensions of the exit end of the fiber for epi-fluorescence satisfying the aforementioned formulas, the fluorescence emitted from the exit end of the fiber for epi-fluorescence can efficiently be made incident on the multi-anode photomultiplier tube without loss of wavelength resolution.

Furthermore, in the above aspect, the entrance end of the fiber for transmission fluorescence may be disposed at a conjugate position with respect to a pupil position of the condenser lens, and may have a diameter and a maximum light-receivable angle that satisfy the following formulas:
ΦDt>ΦPc×βcd
αte>θc
where ΦDtdenotes the diameter of the entrance end of the fiber for transmission fluorescence, ΦPcdenotes a pupil diameter of the condenser lens, βcddenotes the projection magnification from the pupil position of the condenser lens to the entrance end of the fiber for transmission fluorescence, αtedenotes the maximum light-receivable angle of the fiber for transmission fluorescence, and θc denotes a maximum angle of incidence at the fiber for transmission fluorescence, determined on the basis of a scan range of the scanner.

With the diameter and the maximum light-receivable angle of the entrance end of the fiber for transmission fluorescence satisfying the aforementioned formulas, the fluorescence from the entire scan range of the scanner can be made incident on the entrance end of the fiber for transmission fluorescence, thereby preventing optical loss in the fluorescence.

Furthermore, in the above aspect, the maximum light-receivable angle of the entrance end of the fiber for transmission fluorescence may further satisfy the following formula:
αte>θdt
where αtedenotes the maximum light-receivable angle of the fiber for transmission fluorescence, and θtdenotes a maximum angle of incidence at the fiber for transmission fluorescence, determined on the basis of a capturable field of view of the condenser lens.

With the maximum light-receivable angle of the entrance end of the fiber for transmission fluorescence satisfying the aforementioned formula, a greater amount of scattered fluorescence can be collected when observing a deep section.

Furthermore, in the above aspect, the exit end of the fiber for transmission fluorescence may have a widthwise dimension and a lengthwise dimension that satisfy the following formulas:
W×βPM<PW
Ht×βPM<Ph
αto÷βPM<θp
where W denotes the widthwise dimension of the exit end of the fiber for transmission fluorescence, βPMdenotes the magnification at which the exit end of the fiber for transmission fluorescence is projected onto the multi-anode photomultiplier tube, PWdenotes a widthwise dimension of each detector of the multi-anode photomultiplier tube in the arrayed direction thereof, Htdenotes the lengthwise dimension of the exit end of the fiber for epi-fluorescence, Phdenotes a dimension of each detector of the multi-anode photomultiplier tube in a direction orthogonal to the arrayed direction, αtodenotes an emission angle of the fiber for transmission fluorescence, and θp denotes a permissible light-receiving angle of the multi-anode photomultiplier tube.

With the widthwise and lengthwise dimensions of the exit end of the fiber for transmission fluorescence satisfying the aforementioned formulas, the fluorescence emitted from the exit end of the fiber for transmission fluorescence can efficiently be made incident on the multi-anode photomultiplier tube without loss of wavelength resolution.

Furthermore, in the above aspect, the scanning microscope device may further include a plurality of cylindrical lenses arrayed in the vicinity of light-receiving surfaces of the detectors of the multi-anode photomultiplier tube, in which the cylindrical lenses may be arrayed at a pitch that substantially matches a pitch at which the detectors are arrayed, and the cylindrical lenses may be disposed in correspondence with the respective detectors.

With this configuration, even when there are neutral zones (gaps) for forming electrodes between the detectors of the multi-anode photomultiplier tube, the cylindrical lenses can cause the fluorescence to be efficiently incident on the light-receiving surfaces of the respective detectors so as to prevent optical loss in the fluorescence caused by the gaps.

Furthermore, in the above aspect, a dimension of each cylindrical lens in a direction with no lens power may be greater than a dimension of an incidence range of the fluorescence to be incident on each detector.

With this configuration, optical loss in the fluorescence to be incident on the detectors of the multi-anode photomultiplier tube can be prevented.

Furthermore, in the above aspect, the scanning microscope device may further include an image processor that performs wavelength separation on the fluorescence detected by the detectors, and a monitor that displays an image of the fluorescence subjected to the wavelength separation performed by the image processor.

With this configuration, the image processor can separate multiple fluorochromes with large crossover and display them on the monitor.

Furthermore, in the above aspect, the scanning microscope device may further include a storage section that stores spectrum detection results of the sample at predetermined intervals of time.

With this configuration, temporal changes in the sample can be observed.

Furthermore, in the above aspect, each detector may include a photoelectric surface that performs photoelectric conversion on the fluorescence, and the multi-anode photomultiplier tube may include a cooling device that cools the photoelectric surfaces.

With this configuration, the cooling device can cool the photoelectric surfaces of the detectors so as to reduce noise in the multi-anode photomultiplier tube. Thus, the S/N ratio can be improved.

Furthermore, in the above aspect, a microscope section having the objective lens is separately provided from a detection unit, wherein the microscope section is optically connected to the detection unit via the fiber for epi-fluorescence, wherein each detector includes a photoelectric surface that performs photoelectric conversion on the fluorescence, wherein a cooling device that cools the photoelectric surfaces is equipped with the multi-anode photomultiplier tube, and wherein the detection unit including the multi-anode photomultiplier tube comprises a heat exhauster that externally releases heat generated when the photoelectric surfaces are cooled by the cooling device.

With this configuration, the heat exhauster can prevent a temperature increase in the multi-anode photomultiplier tube caused when the cooling device generates heat.

Furthermore, in the above aspect, a microscope section having the objective lens is separately provided from a detection unit, wherein the microscope section is optically connected to the detection unit via the fiber for epi-fluorescence and/or the fiber for transmission fluorescence, wherein each detector includes a photoelectric surface that performs photoelectric conversion on the fluorescence, wherein a cooling device that cools the photoelectric surfaces is equipped with the multi-anode photomultiplier tube, and wherein the detection unit including the multi-anode photomultiplier tube comprises a heat exhauster that externally releases heat generated when the photoelectric surfaces are cooled by the cooling device.

Furthermore, in the above aspect, a microscope section having the objective lens is separately provided from a detection unit, wherein the microscope section is optically connected to the detection unit via the fiber for transmission fluorescence, wherein each detector includes a photoelectric surface that performs photoelectric conversion on the fluorescence, wherein a cooling device that cools the photoelectric surfaces is equipped with the multi-anode photomultiplier tube, and wherein the detection unit including the multi-anode photomultiplier tube comprises a heat exhauster that externally releases heat generated when the photoelectric surfaces are cooled by the cooling device.

The present invention advantageously achieves the ability to perform spectrum detection at a high S/N ratio, high sensitivity, and high speed.

DETAILED DESCRIPTION OF THE INVENTION

First Embodiment

A scanning microscope device according to a first embodiment of the present invention will be described below with reference to the drawings.

As shown inFIG. 1, a scanning microscope device100according to this embodiment is a microscope device that allows for observation of a sample1by switching between a single-photon excitation observation mode and a multiphoton excitation observation mode. The sample1may be, for example, biological cells (multi-stained fluorescent sample) labeled with multiple fluorochromes with large crossover in fluorescence wavelengths, such as CFP (cyan fluorescent protein), GFP (green fluorescent protein), and YFP (yellow fluorescent protein).

The scanning microscope device100includes a single-photon-excitation light source (light source)2and a multiphoton-excitation light source (light source)3(simply referred to as “light sources2and3” hereinafter) that irradiate the sample1with laser light, a scan unit10and a scan-unit guiding projector tube50having an optical path for single-photon-excitation observation, an objective lens92that irradiates the sample1with the laser light emitted from the light sources2and3and collects fluorescence generated at the irradiated position of the sample1, and an epi-illumination-observation optical system90that constitutes an optical path for multiphoton-excitation observation. Reference numeral94denotes a microscope lens barrel for visual observation.

The single-photon-excitation light source2is, for example, an ArKr (argon-krypton) laser. The single-photon-excitation light source2is provided in a visible laser unit6. The visible laser unit6is provided with an AOTF (wavelength tunable filter)4that controls the transmission wavelength of the laser light emitted from the single-photon-excitation light source2. Reference numeral8denotes a visible-light single-mode fiber that guides the laser light from the visible laser unit6to the scan unit10.

The multiphoton-excitation light source3is, for example, an IR pulsed laser.

The scan unit10includes a scanner combining DM (dichroic mirror)12that guides the laser light emitted from the light sources2and3to the same optical path, an X-Y galvanometer mirror (scanner)14that reflects the laser light from the scanner combining DM12so as to scan the laser light on the sample1, and a pupil projection lens16that focuses the laser light reflected by the X-Y galvanometer mirror14.

The scan-unit guiding projector tube50includes an image forming lens52that collimates the laser light transmitted through the pupil projection lens16of the scan unit10, and a fluorescence returning mirror (fluorescence returner)54that reflects the laser light transmitted through the image forming lens52so as to cause the laser light to enter the objective lens92and that also reflects the fluorescence generated in the sample1so as to return the fluorescence to the scan unit10. Reference numeral93denotes a pupil position of the objective lens92.

In single-photon excitation observation, the fluorescence returning mirror54returns the fluorescence from the sample1to the X-Y galvanometer mirror14so as to perform descanning. The fluorescence returning mirror54is disposed in an insertable and removable manner in an optical path between the X-Y galvanometer mirror14and the objective lens92. The fluorescence returning mirror54can be replaced with a non-descan-detection excitation DM (wavelength separator)56by using switching means (not shown).

The non-descan-detection excitation DM56is used for detecting (descanning) the fluorescence from the sample1in multiphoton excitation observation without returning the fluorescence to the X-Y galvanometer mirror14. The non-descan-detection excitation DM56reflects the laser light from the image forming lens52so as to cause the laser light to enter the objective lens92, and transmits the fluorescence from the sample1so as to separate the laser light and the fluorescence from each other. The switching means is not limited in particular, and may be, for example, means for manually switching between the fluorescence returning mirror54and the non-descan-detection excitation DM56or may be an automatic switching device.

The scan unit10includes an excitation DM18that separates, from the laser light, the fluorescence that is generated in the sample1irradiated with the laser light and that is collected by the objective lens92before returning in the reverse direction along the optical path of the laser light via the fluorescence returning mirror54and the X-Y galvanometer mirror14, a confocal lens22that collects the fluorescence separated by the excitation DM18, and a confocal pinhole24that is disposed at a conjugate position with respect to a focal position of the objective lens92and that allows part of the fluorescence collected by the confocal lens22to pass therethrough.

Furthermore, the scan unit10also includes a first spectral DM26and a second spectral DM27that partially transmit and partially reflect the fluorescence passing through the confocal pinhole24, a one-channel photomultiplier tube (1CH_PMT)28that detects the intensity of the fluorescence reflected by the first spectral DM26, a two-channel photomultiplier tube (2CHPMT)29that detects the intensity of the fluorescence reflected by the second spectral DM27, and a spectrum detection unit30that performs spectrum detection of the fluorescence transmitted through the first spectral DM26and the second spectral DM27.

The spectrum detection unit30includes a diffraction grating32that disperses the fluorescence in one direction, a focusing lens34that focuses the fluorescence dispersed by the diffraction grating32, and a multi-anode photomultiplier tube (PMT)40having a plurality of cells42(detectors, seeFIG. 2) that detect the fluorescence focused by the focusing lens34.

The multi-anode PMT40is configured such that the cells42are arrayed one-dimensionally in the dispersing direction of the fluorescence dispersed in one direction by the diffraction grating32. The multi-anode PMT40may be, for example, a 32-channel multi-anode PMT (manufactured by Hamamatsu Photonics K. K.) having a one-dimensional array of 32 cells42.

As shown inFIG. 2, if there are gaps (neutral zones)44for forming electrodes between the cells42of the multi-anode PMT40, a cylindrical lens array46constituted of a plurality of cylindrical lenses45may be disposed in the vicinity of light-receiving surfaces of the cells42. In that case, as shown inFIG. 3, it is desirable that the cylindrical lenses45be arrayed at a pitch that substantially matches the array pitch of the cells42(see reference character P inFIG. 3) so as to be arranged in a one-to-one relationship with the respective cells42. InFIG. 2, reference character S denotes an image forming plane of spectral lines of the fluorescence dispersed by the diffraction grating32. It is desirable that an incidence plane of the cylindrical lenses45and the image forming plane of the spectral lines be substantially aligned with each other.

As shown inFIG. 3, regarding each of the cylindrical lenses45, it is desirable that the dimension thereof in a direction in which there is no lens power substantially match the dimension in a direction orthogonal to the arrayed direction of the cells42and be set greater than the dimension of an incidence range of fluorescence to be incident on each cell42(for example, a lengthwise dimension of an exit end74(seeFIG. 4C) of an epi-illumination fiber (fiber for epi-fluorescence)70projected onto the cell42). Consequently, the cylindrical lens45can make the fluorescence efficiently incident on the light-receiving surface of the cell42so as to prevent optical loss in the fluorescence caused by the gaps44. InFIG. 3, reference character T denotes a projected image of the exit end74of the epi-illumination fiber70.

The spectrum detection unit30is provided with a first switching mirror (descanned fluorescence entrance section)36that combines the fluorescence from the optical path for multiphoton excitation observation with the optical path of the fluorescence for single-photon excitation observation to be incident on the diffraction grating32(i.e., the optical path of the fluorescence from the second spectral DM27). The first switching mirror36causes the fluorescence emitted from the exit end74of the epi-illumination fiber70to enter the optical path of the fluorescence passing through the confocal pinhole24.

The first switching mirror36is disposed in an insertable and removable manner in the optical path between the second spectral DM27and the diffraction grating32, and is removed from the optical path by the switching means when performing single-photon excitation observation and is disposed in the optical path when performing multiphoton excitation observation.

The epi-illumination-observation optical system90includes an epi-illumination non-descan unit60that receives the fluorescence from the scan-unit guiding projector tube50having the non-descan-detection excitation DM56disposed therein in place of the fluorescence returning mirror54during multiphoton excitation observation, the epi-illumination fiber70that introduces the fluorescence from the epi-illumination non-descan unit60into the spectrum detection unit30of the scan unit10, and an epi-illumination-fiber guiding unit80.

The epi-illumination non-descan unit60includes a first projector lens62that receives the fluorescence transmitted through the non-descan-detection excitation DM56, a reflecting mirror64that reflects the fluorescence transmitted through the first projector lens62, an IR cut filter66that removes infrared light from the fluorescence reflected by the reflecting mirror64, and a second projector lens68that collects the fluorescence with the infrared light removed therefrom by the IR cut filter66and introduces the fluorescence into an end of the epi-illumination fiber70.

The pupil position93of the objective lens92and an entrance end (denoted by reference numeral72inFIG. 1) of the epi-illumination fiber70have an optically conjugate relationship achieved by the first projector lens62and the second projector lens68.

The first projector lens62and the reflecting mirror64are disposed in an insertable and removable manner in the optical path of the fluorescence. When the first projector lens62and the reflecting mirror64are removed from the optical path of the fluorescence, the fluorescence from the sample1enters the microscope lens barrel94so that visual observation can be performed using a transmission light source (not shown) or the like.

As shown inFIGS. 4A to 4C, the epi-illumination fiber70includes the entrance end72that receives the fluorescence collected by the second projector lens68of the epi-illumination non-descan unit60, and the exit end74that emits the guided fluorescence towards the epi-illumination-fiber guiding unit80, and is constituted of a fiber bundle formed by bundling multiple fibers together.

The entrance end72of the epi-illumination fiber70is disposed at a conjugate position with respect to the pupil position93of the objective lens92, and is formed in a substantially circular shape, as shown inFIG. 4A, by bundling multiple fibers together in a circle. The diameter and the maximum light-receivable angle of the entrance end72are set so as to satisfy the following formulas (1) and (2) (seeFIGS. 4A and 5):
ΦDr>Φpo×βPL(1)
αre>θa(2)
where ΦDrdenotes the diameter of the entrance end72of the epi-illumination fiber70, Φpodenotes the pupil diameter of the objective lens92, βPLdenotes the projection magnification from the pupil position of the objective lens92to the entrance end72of the epi-illumination fiber70, αredenotes the maximum light-receivable angle of the entrance end72of the epi-illumination fiber70, and θa denotes the maximum angle of incidence at the entrance end72of the epi-illumination fiber70, determined on the basis of a rotating-angle range of the X-Y galvanometer mirror14.

InFIG. 5, reference character S denotes an image height (i.e., height from the center of an image) of a scan range determined on the basis of the rotating-angle range of the X-Y galvanometer mirror14, reference character Fodenotes an image height determined on the basis of a capturable field of view of the objective lens92, and reference symbol θb denotes a maximum angle of incidence at the entrance end72of the epi-illumination fiber70, determined on the basis of the capturable field of view of the objective lens92.

As shown inFIG. 4C, the exit end74of the epi-illumination fiber70is formed by bundling fibers together in a linear form so as to be formed in a substantially linear shape extending in the dispersing direction of the diffraction grating32, that is, in a direction orthogonal to the arrayed direction of the cells42of the multi-anode PMT40. The widthwise and lengthwise dimensions of the exit end74are set so as to satisfy the following formulas (3), (4), and (5) (seeFIGS. 4C and 6):
W×βPM<PW(3)
Hr×βPM<Ph(4)
αro÷βPM<θp(5)
where W denotes the widthwise dimension of the exit end74of the epi-illumination fiber70(if the widthwise dimension of an entrance slit82is smaller than W, the widthwise dimension of the entrance slit82is defined as W), βPMdenotes the magnification at which the exit end74of the epi-illumination fiber70is projected onto each cell42of the multi-anode PMT40, PWdenotes the widthwise dimension of each cell42of the multi-anode PMT40in the arrayed direction thereof, Hrdenotes the lengthwise dimension of the exit end74of the epi-illumination fiber70, Phdenotes the dimension of each cell42of the multi-anode PMT40in the direction orthogonal to the arrayed direction thereof, αrodenotes an emission angle of the epi-illumination fiber70, and θp denotes a permissible light-receiving angle of the multi-anode PMT40(a sensitivity of about 80% is taken as a guide).

The epi-illumination-fiber guiding unit80is provided with the entrance slit82that shapes the fluorescence emitted from the exit end74of the epi-illumination fiber70. The entrance slit82is formed so as to extend in the same direction as the longitudinal direction of the exit end74of the epi-illumination fiber70. The epi-illumination-fiber guiding unit80includes a collimating lens84that substantially collimates the fluorescence shaped by the entrance slit82.

When performing multiphoton excitation observation, the first switching mirror36is disposed in the optical path between the second spectral DM27and the diffraction grating32so as to cause the fluorescence transmitted through the collimating lens84to enter the optical path for single-photon excitation observation to be incident on the diffraction grating32, whereby the fluorescence can be dispersed by the diffraction grating32.

The operation of the scanning microscope device100according to this embodiment having the above-described configuration will now be described.

When performing single-photon excitation observation, the fluorescence returning mirror54is disposed in the optical path of laser light within the scan-unit guiding projector tube50, and the first switching mirror36is set in the position where it is removed from the optical path in the spectrum detection unit30. The sample1is then disposed on a stage (not shown), and laser light is emitted from the single-photon-excitation light source2.

The laser light emitted from the single-photon-excitation light source2undergoes transmission-wavelength control by the AOTF4and is guided to the scan unit10by the visible-light single-mode fiber8. The laser light guided to the scan unit10is reflected by the scanner combining DM12and the excitation DM18and is scanned by the X-Y galvanometer mirror14. The laser light scanned by the X-Y galvanometer mirror14travels through the pupil projection lens16and the image forming lens52and is reflected by the fluorescence returning mirror54before the objective lens92irradiates the sample1with the laser light.

Fluorescence generated in the irradiated position of the sample1as a result of irradiation with the laser light is collected by the objective lens92and is reflected by the fluorescence returning mirror54so as to travel along the optical path of the laser light in the reverse direction. The fluorescence then travels through the image forming lens52and the pupil projection lens16so as to be incident on the excitation DM18via the X-Y galvanometer mirror14. The fluorescence incident on the excitation DM18is separated from the laser light and is subsequently collected by the confocal lens22before passing through the confocal pinhole24.

The first spectral DM26causes a portion of the fluorescence passing through the confocal pinhole24to be incident on the 1CH_PMT28where the intensity thereof is detected. Furthermore, the second spectral DM27causes a portion of the fluorescence transmitted through the first spectral DM26to be incident on the 2CH_PMT29where the intensity thereof is detected.

The fluorescence transmitted through the first spectral DM26and the second spectral DM27enters the spectrum detection unit30and is dispersed in one direction by the diffraction grating32. The dispersed fluorescence is collected by the focusing lens34before entering the plurality of cells42of the multi-anode PMT40. Thus, the dispersed fluorescence is detected in each cell42.

Alternatively, the fluorescence of all wavelengths can be guided to the spectrum detection unit30by removing the first spectral DM26and the second spectral DM27from the optical path.

Next, when performing multiphoton excitation observation, the switching means is used to dispose the non-descan-detection excitation DM56in the optical path of laser light within the scan-unit guiding projector tube50, and the first switching mirror36is set in the position where it is disposed in the optical path of fluorescence in the spectrum detection unit30. The sample1is then disposed on the stage, and laser light is emitted from the multiphoton-excitation light source3.

The laser light emitted from the multiphoton-excitation light source3is transmitted through the scanner combining DM12and is reflected by the excitation DM18before being scanned by the X-Y galvanometer mirror14. Then, the excitation light is transmitted through the pupil projection lens16and the image forming lens52and is reflected by the non-descan-detection excitation DM56before the objective lens92irradiates the sample1with the laser light.

Fluorescence generated in the sample1irradiated with the laser light is collected by the objective lens92and is subsequently transmitted through the non-descan-detection excitation DM56before entering the epi-illumination non-descan unit60. The fluorescence incident on the epi-illumination non-descan unit60is transmitted through the first projector lens62and is reflected by the reflecting mirror64before the IR cut filter66removes infrared light therefrom. The fluorescence with the infrared light removed therefrom is transmitted through the second projector lens68and is made incident on the entrance end72of the epi-illumination fiber70.

In this case, since the diameter and the maximum light-receivable angle of the entrance end72of the epi-illumination fiber70are set so as to satisfy formulas (1) and (2), the fluorescence from the entire scan range of the X-Y galvanometer mirror14can be made incident on the entrance end72, thereby preventing optical loss in the fluorescence.

The fluorescence incident on the epi-illumination fiber70is emitted from the exit end74so as to enter the entrance slit82of the epi-illumination-fiber guiding unit80. Then, the fluorescence is shaped by the entrance slit82and is subsequently transmitted through the collimating lens84where the fluorescence is substantially collimated before entering the spectrum detection unit30.

The widthwise and lengthwise dimensions of the exit end74of the epi-illumination fiber70are set so as to satisfy formulas (3), (4), and (5). Specifically, as shown in formula (3), since the widthwise dimension of the exit end74projected on the multi-anode PMT40is smaller than the widthwise dimension of each cell42of the multi-anode PMT40, sufficient wavelength resolution, determined on the basis of the dispersion by the diffraction grating32and the pitch of the cells42of the multi-anode PMT40, can be ensured. Furthermore, as shown in formula (4), when the exit end74of the epi-illumination fiber70is projected onto the multi-anode PMT40, the dimension thereof in the direction orthogonal to the dispersing direction of the diffraction grating32, that is, the longitudinal direction thereof, is smaller than the dimension of each cell42of the multi-anode PMT40in the direction orthogonal to the arrayed direction thereof, so that optical loss in the fluorescence to be incident on each cell42can be prevented. By satisfying formula (5), a reduction in the sensitivity caused by an increase in the numerical aperture for the fluorescence to be incident on each cell42can be prevented, and a reduction in the wavelength resolution can also be prevented. Therefore, the fluorescence emitted from the exit end74can be efficiently guided to the multi-anode PMT40without loss of wavelength resolution.

In the spectrum detection unit30, the fluorescence from the epi-illumination-fiber guiding unit80is reflected by the first switching mirror36so as to be made incident on the diffraction grating32along the same optical path as the optical path for single-photon excitation observation to be incident on the diffraction grating32. The fluorescence is then dispersed in one direction by the diffraction grating32and is focused by the focusing lens34before entering the plurality of cells42of the multi-anode PMT40.

In this case, since the exit end74of the epi-illumination fiber70is formed in a linear shape extending in the dispersing direction of the diffraction grating32, that is, the direction orthogonal to the arrayed direction of the cells42of the multi-anode PMT40, the fluorescence guided by the epi-illumination fiber70can be made to enter the cells42without loss. Furthermore, the dispersed fluorescence can be detected at once by the plurality of cells42instead of being detected wavelength-by-wavelength in a time-series fashion. Therefore, accurate observation can be performed even if the sample1is, for example, rapidly-moving biological cells labeled with multiple fluorochromes.

As described above, with the scanning microscope device100according to this embodiment, single-photon excitation observation and multiphoton excitation observation can be performed in a switching manner by using the scan unit10and the epi-illumination-observation optical system90. When performing multiphoton excitation observation, the non-descan-detection excitation DM56separates the fluorescence generated in the sample1from the laser light, and the epi-illumination fiber70introduces the fluorescence into the multi-anode PMT40without returning the fluorescence to the X-Y galvanometer mirror14, thereby minimizing the loss of fluorescence in the optical path from the sample1to the multi-anode PMT40. Furthermore, the multi-anode PMT40having the multiple cells42can detect the dispersed fluorescence at once so as to allow for immediate tracking of temporal changes in the biological cells. Thus, spectrum detection can be performed at a high S/N ratio, high sensitivity, and high speed.

In this embodiment, although the diameter and the maximum light-receivable angle of the entrance end72of the epi-illumination fiber70are set so as to satisfy formulas (1) and (2), for example, formula (1) may alternatively be ΦDr≧Φpo×βPL, and formula (2) may alternatively be αre≧θa.

Since there is a large amount of scattered light in a deep section of a sample (for example, about 500 μm from the surface of the sample1), fluorescence is also generated from outside the scan range. In light of this, the diameter and the maximum light-receivable angle of the entrance end72of the epi-illumination fiber70may be set so as to satisfy not only formulas (1) and (2), but also the following formula (6):
αre>θb(6)
where αredenotes the maximum light-receivable angle of the entrance end72of the epi-illumination fiber70, and θb denotes the maximum angle of incidence at the entrance end72of the epi-illumination fiber70, determined on the basis of the capturable field of view of the objective lens92.

In this manner, a greater amount of scattered fluorescence can be collected even when observing a deep section.

In this embodiment, although the scanning microscope device100is configured to perform single-photon excitation observation by using the fluorescence returning mirror54, the excitation DM18, the confocal pinhole24, and the like, the scanning microscope device100need not include, for example, the fluorescence returning mirror54, the excitation DM18, the confocal pinhole24, and the like and may be configured to perform only multiphoton excitation observation.

This embodiment can be modified as follows.

For example, instead of arranging the pupil position93of the objective lens92and the entrance end72of the epi-illumination fiber70in an optically conjugate relationship, the sample surface and the entrance end72of the epi-illumination fiber70may have an optically conjugate relationship achieved by the objective lens92and a third projector lens69, as shown inFIG. 7, such that the diameter and the maximum light-receivable angle of the entrance end72of the epi-illumination fiber70are set so as to satisfy the following formulas (7) and (8):
αob/βob<αre(7)
(Φ2×S)×βob<ΦDr(8)
where αobdenotes the aperture angle (half angle) of the objective lens92, βobdenotes the magnification for projecting the sample surface onto the entrance end72of the epi-illumination fiber70, αredenotes the maximum light-receivable angle (half angle) of the entrance end72of the epi-illumination fiber70, S denotes an image height of a scan range determined on the basis of the rotating-angle range of the X-Y galvanometer mirror14, Φ2×S denotes a scan range on the sample surface determined on the basis of the rotating-angle range of the X-Y galvanometer mirror14, and ΦDrdenotes the diameter of the entrance end72of the epi-illumination fiber70.

In this case, it is desirable that the epi-illumination fiber70be of a type in which the transmittance does not change depending on the incidence position of the fluorescence, instead of a fiber bundle formed by bundling fibers together.

Furthermore, in this modification, the diameter of the entrance end72of the epi-illumination fiber70may be set so as to satisfy the following formula (9):
(Φ2×F)×βob<ΦDr(9)
where F denotes an image height determined on the basis of the capturable field of view of the objective lens92, (Φ2×F) denotes the capturable range of the objective lens92, βobdenotes the magnification for projecting the sample surface onto the entrance end72of the epi-illumination fiber70, and ΦDrdenotes the diameter of the entrance end72of the epi-illumination fiber70.

In this manner, a greater amount of scattered fluorescence can be collected when observing a deep section.

Second Embodiment

Next, a scanning microscope device according to a second embodiment of the present invention will be described.

A scanning microscope device200according to this embodiment is a device for multiphoton excitation observation and differs from that in the first embodiment and the modification thereof in having a multiphoton excitation scan unit110and a spectrum detection unit30in place of the scan unit10; an epi-illumination-observation optical system90; and a transmission observation optical system190, as shown inFIG. 8.

Sections having the same configuration as those in the scanning microscope device100according to the first embodiment and the modification thereof will be given the same reference numerals, and descriptions of those sections will be omitted.

The multiphoton excitation scan unit110is constituted of an X-Y galvanometer mirror14and a pupil projection lens16.

A non-descan-detection excitation DM56is disposed in a scan-unit guiding projector tube50.

The transmission observation optical system190includes a condenser lens192that collects fluorescence generated in a transmission direction at an irradiated position of laser light scanned on the sample1by the X-Y galvanometer mirror14, a transmission non-descan unit160that receives the fluorescence collected by the condenser lens192, a transmission fiber (fiber for transmission fluorescence)170that guides the fluorescence from the transmission non-descan unit160to the spectrum detection unit30, and a transmission-fiber guiding unit180. Reference numeral193denotes a pupil position of the condenser lens192.

The transmission non-descan unit160is similar to the epi-illumination non-descan unit60in having a first projector lens162, a reflecting mirror164, an IR cut filter166, and a second projector lens168.

The pupil position193of the condenser lens192and an entrance end172of the transmission fiber170have an optically conjugate relationship achieved by the first projector lens162and the second projector lens168of the transmission non-descan unit160.

The transmission fiber170has a similar configuration to the epi-illumination fiber70in being disposed at a conjugate position with respect to the pupil position193of the condenser lens192. The diameter and the maximum light-receivable angle of the entrance end172of the transmission fiber170are set so as to satisfy the following formulas (10) and (11):
ΦDt>ΦPc×βcd(10)
αte>θc(11)
where ΦDtdenotes the diameter of the entrance end172of the transmission fiber170, ΦPcdenotes a pupil diameter of the condenser lens192, βcddenotes the projection magnification from the pupil position of the condenser lens192to the entrance end172of the transmission fiber170, αtedenotes the maximum light-receivable angle of the entrance end172of transmission fiber170, and θc denotes a maximum angle of incidence at the entrance end172of the transmission fiber170, determined on the basis of the rotating-angle range of the X-Y galvanometer mirror14.

The widthwise and lengthwise dimensions of an exit end174of the transmission fiber170are set so as to satisfy the following formulas (12), (13), and (14):
W×βPM<PW(12)
Ht×βPM<Ph(13)
αto÷βPM<θp(14)
where W denotes the widthwise dimension of the exit end174of the transmission fiber170(if the widthwise dimension of an entrance slit182is smaller than W, the widthwise dimension of the entrance slit182is defined as W), βPMdenotes the magnification at which the exit end174of the transmission fiber170is projected onto each cell42of a multi-anode PMT40, PWdenotes the widthwise dimension of each cell42of the multi-anode PMT40in the arrayed direction thereof, Htdenotes the lengthwise dimension of the exit end174of the transmission fiber170, Phdenotes the dimension of each cell42of the multi-anode PMT40in the direction orthogonal to the arrayed direction thereof, αtodenotes an emission angle of the transmission fiber170, and θp denotes a permissible light-receiving angle of the multi-anode PMT40(a sensitivity of about 80% is taken as a guide).

The transmission-fiber guiding unit180is configured to introduce the fluorescence guided by the transmission fiber170into the spectrum detection unit30and has a similar configuration to the epi-illumination-fiber guiding unit80.

The spectrum detection unit30includes a second switching mirror (transmitted-fluorescence entrance section)136that switches between the fluorescence from the epi-illumination-observation optical system90and the fluorescence from the transmission observation optical system190and introduces the fluorescence to the diffraction grating32. The second switching mirror136causes the fluorescence emitted from the exit end174of the transmission fiber170to enter the optical path of the fluorescence emitted from the exit end74of the epi-illumination fiber70so as to cause the fluorescence from the transmission fiber170to be incident on the diffraction grating32in place of the fluorescence from the epi-illumination fiber70.

By using switching means (not shown), the second switching mirror136is disposed on the optical path when performing spectrum detection of the fluorescence from the epi-illumination-observation optical system90, and is removed from the optical path when performing spectrum detection of the fluorescence from the transmission observation optical system190. By disposing the second switching mirror136in the optical path, the fluorescence emitted from the exit end74of the epi-illumination fiber70can be reflected and be made incident on the diffraction grating32. On the other hand, by removing the second switching mirror136from the optical path, the fluorescence from the exit end174of the transmission fiber170can be made incident on the diffraction grating32along the same optical path as the optical path from the epi-illumination fiber70.

The operation of the scanning microscope device200according to this embodiment having the above-described configuration will now be described.

When performing multiphoton excitation observation using the transmission observation optical system190, the second switching mirror136is set in the position where it is removed from the optical path of the spectrum detection unit30, the sample1is disposed on a stage, and laser light is emitted from the multiphoton-excitation light source3. The laser light emitted from the multiphoton-excitation light source3is scanned by the X-Y galvanometer mirror14, is transmitted through the pupil projection lens16and the image forming lens52, and is reflected by the non-descan-detection excitation DM56before the objective lens92irradiates the sample1with the laser light.

Fluorescence generated in the transmission direction at the irradiated position of the sample1as a result of irradiation with the laser light is collected by the condenser lens192and is subsequently transmitted through the first projector lens62and reflected by the reflecting mirror64before the IR cut filter66removes infrared light therefrom. The fluorescence with the infrared light removed therefrom is transmitted through the second projector lens68and is made incident on the entrance end172of the transmission fiber170.

In this case, since the diameter and the maximum light-receivable angle of the entrance end172of the transmission fiber170are set so as to satisfy formulas (10) and (11), the fluorescence from the entire scan range of the X-Y galvanometer mirror14can be made incident on the entrance end172, thereby preventing optical loss in the fluorescence.

The fluorescence entering the transmission fiber170is emitted from the exit end174and is substantially collimated via the entrance slit182and the collimating lens184of the transmission-fiber guiding unit180before entering the spectrum detection unit30.

In the spectrum detection unit30, the fluorescence from the transmission-fiber guiding unit180is made incident on the diffraction grating32by traveling along the same optical path as the optical path of the fluorescence from the epi-illumination fiber70. Then, the fluorescence is dispersed in one direction by the diffraction grating32and is focused by the focusing lens34before entering the plurality of cells42of the multi-anode PMT40. Consequently, the multi-anode PMT40can detect the fluorescence generated in the sample1in the transmission direction of the laser light.

As described above, with the scanning microscope device200according to this embodiment, by simply changing the position of the second switching mirror136, spectrum detection of fluorescence generated in the direction in which it returns from the sample1, performed by using the epi-illumination-observation optical system90, and spectrum detection of fluorescence generated in the direction in which it is transmitted through the sample1, performed by using the transmission observation optical system190, can be performed in a switching manner.

In this embodiment, although the diameter and the maximum light-receivable angle of the entrance end172of the transmission fiber170are set so as to satisfy formulas (10) and (11), the diameter and the maximum light-receivable angle thereof may additionally be set so as to satisfy the following formula (15):
αte>θdt(15)
where αtedenotes the maximum light-receivable angle of the entrance end172of the transmission fiber170, and θdtdenotes the maximum angle of incidence at the entrance end172of the transmission fiber170, determined on the basis of the capturable field of view of the condenser lens192.

In this manner, a greater amount of scattered fluorescence can be collected when observing a deep section.

Although the embodiments of the present invention have been described in detail above with reference to the drawings, specific configurations are not to be limited to those in the embodiments and may include design modifications within the scope of the invention. For example, the present invention is not limited to the above embodiments and the modifications thereof, and may be applied to an embodiment with an appropriate combination of these embodiments and modifications; the invention is not limited in particular.

For example, the scanning microscope device100according to the first embodiment that can perform single-photon excitation observation and multiphoton excitation observation in a switching manner may further include the transmission observation optical system190. In that case, as shown inFIG. 9, the second switching mirror136may be disposed in an insertable and removable manner in the optical path between the entrance slit82and the collimating lens84so that the fluorescence emitted from the exit end174of the transmission fiber170can be made to enter the optical path of the fluorescence emitted from the exit end74of the epi-illumination fiber70.

Furthermore, although fluorescence produced by multiphoton excitation is described as being detected using the epi-illumination-observation optical system90and the transmission observation optical system190in the above embodiments and the modifications, light generated by a nonlinear phenomenon, such as CARS light (coherent anti-Stokes Raman scattering light) or SHG light (second-harmonic-generation light), may be detected as an alternative. Since CARS light and SHG light are generally generated at the transmission side of the sample1, the light may be detected by using the transmission fiber170or the like constituting the transmission observation optical system190.

Furthermore, when observing a multi-stained fluorescent sample in each of the above embodiments, the scanning microscope device100or200may include an image processor that performs wavelength separation on multiple kinds of fluorescence on the basis of the spectrum of fluorescence detected by the cells42, and a monitor that displays an image of each kind of fluorescence having undergone the wavelength separation performed by the image processor. In this manner, the image processor can separate multiple fluorochromes with large crossover and display them on the monitor. Moreover, the scanning microscope device100or200may include a storage section that stores the spectrum detection results of the sample1at predetermined intervals of time. In this manner, temporal changes in the sample1can be observed.

Furthermore, in the above embodiments and the modifications thereof, for example, the multi-anode PMT40may include a Peltier device (cooling device) that cools photoelectric surfaces of the cells42. In this case, in the scanning microscope device200according to the second embodiment, for example, a cooling surface of a Peltier device248may be disposed so as to be joined to photoelectric surfaces243of all the cells42, as shown inFIG. 10or11. The Peltier device248can cool the photoelectric surfaces243to, for example, −5° C. so as to reduce noise in the multi-anode PMT40.

Furthermore, the spectrum detection unit30may include a forced air cooling fan (heat exhauster)237, as shown inFIG. 10, or a heat dissipating member238and a water-cooled tube239, as shown inFIG. 11, as a device that externally releases the heat generated when the Peltier device248cools the photoelectric surfaces243of the cells42. In this case, since a surface (heat dissipating surface) opposite the cooling surface of the Peltier device248generates heat due to heat exchange, the forced air cooling fan237may be disposed on the heat-dissipating-surface side of the Peltier device248, or the heat dissipating member238and the water-cooled tube239may be disposed on the heat-dissipating-surface side. By using the forced air cooling fan237or the water-cooled tube239to externally release the heat in the Peltier device248, a temperature increase in the multi-anode PMT40can be prevented.

In this case, since a microscope section constituted of the multiphoton excitation scan unit110, the scan-unit guiding projector tube50, the objective lens92, the epi-illumination non-descan unit60, the transmission non-descan unit160, and the like is optically connected to the spectrum detection unit30via the epi-illumination fiber70and the transmission fiber170, the microscope section can be prevented from being affected by vibrations occurring due to actuation of the forced air cooling fan237or by vibrations occurring due to pulsation of fluid flowing through the water-cooled tube239. Therefore, spectrum detection can be performed at a high S/N ratio and with high accuracy. Although this modification is described as being applied to the scanning microscope device200as an example, the modification can also be applied to the scanning microscope device100according to the first embodiment.