JP2013200329A - Microscope system - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a microscope system that can change a wavelength of light to be removed from signal light in accordance with a wavelength of excitation light.SOLUTION: A microscope system 1 comprises: a microscope 30 that allows the excitation light emitted from a light source system 10 to be scanned, allows an objective lens 32 to collect the excitation light on a specimen 33, and allows the objective lens 32 to collect fluorescence from the specimen 33; and a spectrometer 40 that includes a spectrum element 43 dispersing the fluorescence from the microscope 30 and a light reception part 45 receiving dispersion light dispersed by the spectrum element 43. The spectrometer 40 has a wavelength selection part 148 that suppresses an advancement of the excitation light toward the light reception part 45 and guides the fluorescence to the light reception part 45. The microscope system 1 includes a control part 50 that controls the wavelength selection part 148 to change a wavelength characteristic of the wavelength selection part 148 in accordance with the wavelength of the excitation light.

Description

  The present invention relates to a microscope system.

  In a microscope system having a scanning microscope and a spectroscopic unit (spectrometer) for spectrally detecting signal light (fluorescence) detected by the scanning microscope, the spectroscopic unit generally divides the signal light into a diffraction grating, etc. The light is separated by a light receiving element such as PMT (Photomultiplier Tube: photomultiplier tube) arranged in a line. In the spectroscopic unit used with the scanning microscope for detecting such signal light, the excitation light that excites the sample and generates signal light (fluorescence) is also mixed with the signal light. Therefore, it is necessary to cut the excitation light. . In order to cut off such excitation light, the conventional spectroscopic unit is provided with a strip-shaped slit for blocking light corresponding to the wavelength of the excitation light among the light (spectral light) dispersed by the spectroscopic element. (For example, see Patent Document 1).

US Pat. No. 7,675,617

  However, if the wavelength of the excitation light for exciting the sample is changed, the position where the light with the wavelength of the excitation light enters the receiver also changes, so the position of the strip slit must also be changed according to the wavelength. I must. Therefore, a strip-shaped slit has to be prepared for each wavelength of the excitation light, and there is a problem that the observation work of the specimen becomes complicated and the apparatus becomes complicated.

  The present invention has been made in view of such problems, and an object thereof is to provide a microscope system capable of changing the wavelength of light to be removed from signal light in accordance with the wavelength of excitation light.

  In order to solve the above problems, a microscope system according to the present invention scans excitation light emitted from a light source, condenses it on a specimen by an objective lens, and condenses fluorescence from the specimen by the objective lens. A microscope system comprising: a microscope; a spectroscopic element that splits fluorescence from the microscope; and a spectroscopic unit that includes a light receiver that receives spectroscopic light split by the spectroscopic element. The spectroscopic unit receives excitation light. A wavelength selection unit that suppresses the light from going to the detector and guides the fluorescence to the light receiver, and includes a control unit that controls the wavelength selection unit to change the wavelength characteristics of the wavelength selection unit according to the wavelength of the excitation light. It is characterized by that.

  In such a microscope system, the wavelength selection unit includes two or more optical elements having different wavelength characteristics, and a filter insertion / extraction unit that inserts / extracts each of these optical elements into / from the optical path of the spectroscopic unit. It is preferable that at least one of the optical elements is inserted into the optical path and the other optical elements are removed from the optical path by controlling the filter insertion / extraction unit.

  In such a microscope system, the wavelength selection unit includes an optical element having a predetermined wavelength characteristic, and includes an angle adjustment unit that changes the incident angle of the excitation light with respect to the optical element, and the control unit adjusts the angle. It is preferable to control the part.

  Further, such a microscope system has a storage unit that stores the incident angle of the angle adjusting unit controlled by the control unit in association with the wavelength of the excitation light, and has the incident angle read from the storage unit. Thus, it is preferable to control the angle adjustment unit by the control unit.

  In such a microscope system, the optical element is preferably a notch filter that reflects excitation light and transmits fluorescence.

  In such a microscope system, the wavelength selection unit includes two light transmission members arranged so that planes having a predetermined reflectance face each other at a predetermined interval, an interval adjustment unit that changes the interval, and a light transmission unit. It is preferable to have a filter unit including at least one of an angle adjustment unit that changes the incident angle of excitation light and fluorescence to the member, and to control at least one of the interval adjustment unit and the angle adjustment unit by the control unit.

  In addition, such a microscope system has a storage unit that stores the interval of the interval adjustment unit controlled by the control unit in association with the wavelength of the excitation light, so that the interval is read from the storage unit. It is preferable that the interval adjusting unit is controlled by the control unit.

  Further, such a microscope system has a storage unit that stores the incident angle of the angle adjusting unit controlled by the control unit in association with the wavelength of the excitation light, and has the incident angle read from the storage unit. Thus, it is preferable to control the angle adjustment unit by the control unit.

  In such a microscope system, it is preferable that the reflectance of the plane of the light transmitting member is 90% or more.

  In such a microscope system, at least two regions having different reflectivities are formed on each plane of the light transmitting member, and the two light transmitting members are moved in a plane perpendicular to the optical axis. It is preferable to have a position adjusting unit that changes the region where the excitation light is incident.

  In such a microscope system, the wavelength selection unit includes at least a first optical element that reflects light including at least fluorescence among incident light, and at least fluorescence among light reflected by the first optical element. It is preferable that at least one of the first optical element and the second optical element is a filter unit.

  In such a microscope system, it is preferable that the reflecting surfaces of the first optical element and the second optical element are arranged substantially in parallel.

  When the present invention is configured as described above, it is possible to provide a microscope system that can change the wavelength of light to be removed from signal light in accordance with the wavelength of excitation light.

It is a block diagram which shows an example of a structure of a microscope system. It is explanatory drawing which shows the structure of the spectroscopy unit which concerns on 1st Embodiment. It is a graph which shows the wavelength characteristic of a notch filter. It is explanatory drawing which shows the structure of the spectroscopy unit which concerns on 2nd Embodiment. It is explanatory drawing which shows the state of the light beam between two planes of the filter unit which comprises the wavelength selection part in 2nd Embodiment. It is explanatory drawing which shows the wavelength characteristic of the said filter unit, Comprising: (a) shows when a reflectance is 90%, (b) shows when a reflectance is 50%. It is a front view which shows the structure of the said filter unit. It is VII-VII sectional drawing of FIG. It is explanatory drawing which shows the structure of a detection part, Comprising: (a) shows the structure of the detection part which detects the wavelength characteristic of a wavelength selection part from the intensity ratio of transmitted light and reflected light, (b) Wavelength selection part by alignment microscope The structure of the detection part which detects the wavelength characteristic of is shown. It is a graph which shows the wavelength characteristic of a wavelength selection part. It is explanatory drawing which shows the structure of the detection part which detects the wavelength characteristic of a wavelength selection part with a length measurement interferometer.

  Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings. First, the configuration of a microscope system, which is an example of a scanning microscope with a spectral function, will be described with reference to FIG. The microscope system 1 includes a confocal microscope having a light source system 10, a confocal unit 20, and a microscope 30, and a spectroscopic unit 40 that is a spectroscopic unit that spectrally detects signal light (fluorescence) collected by the confocal microscope. And an information processing apparatus 50. In this microscope system 1, the confocal unit 20 and the spectroscopic unit 40 are optically connected by an optical fiber 28 via fiber couplers 29a and 29b.

  The light source system 10 includes a laser device 11, an optical fiber 13, and fiber couplers 12 and 14. The laser device 11 includes, for example, a laser diode, and emits excitation light (illumination light) having target wavelength characteristics. This excitation light is guided to the confocal unit 20 through the optical fiber 13. In the example of FIG. 1, light for exciting the specimen 33 to emit fluorescence is emitted as excitation light. The laser device 11 is configured to be able to select and emit excitation light having different wavelengths. Therefore, the observer can select the wavelength of light for exciting the specimen according to the specimen to be observed.

  The confocal unit 20 includes a collimating lens 21 that makes the excitation light from the light source system 10 a substantially parallel light beam, a dichroic mirror 22, a scanning unit 23, a scanner lens 24, a condenser lens 25, and a pinhole 26a. A pinhole plate 26 and a relay lens 27 are provided. The microscope 30 includes an objective lens 32 and a second objective lens 31, and a stage 34 on which the sample 33 is placed. The confocal unit 20 and the microscope 30 are combined to form a scanning confocal microscope. The dichroic mirror 22 is configured to reflect the laser light emitted from the light source system 10 toward the microscope 30 and transmit the fluorescence emitted from the specimen 33 excited by the laser light. Further, the image side focal point of the condenser lens 25 is disposed so as to substantially coincide with the pinhole 26 a of the pinhole plate 26.

  The spectroscopic unit 40 includes a collimating optical system 41 that makes signal light (fluorescence in the example of FIG. 1) incident from the optical fiber 28 via the fiber coupler 29b a substantially parallel light beam, and three types of spectroscopic elements having different spectroscopic characteristics. A certain diffraction grating 431, 432, 433 (collectively referred to as “diffraction grating 43”), a rotating table 42 that holds and rotates them, and a plurality of light receiving elements 45a are arranged in an array to constitute a line detector. A light receiver 45, a condensing optical system 44 that forms an image of the diffracted light (spectral light) emitted from the diffraction grating 43 (any of the diffraction gratings 431 to 433) on the light receiving surface of the light receiver 45, and the light receiver 45. A high voltage power supply 46 that supplies power for driving, and a detection circuit 47 that amplifies and digitizes the output of each light receiving element 45a of the light receiver 45 and outputs it as detection data. It has a. It should be noted that the preceding optical system (collimating optical system 41 or condensing optical system) is set so that the NA of the signal light (spectral light) incident on the light receiving element 45a is within the range of the effective NA or the optimum NA of the light receiving element 45a. 44) is formed.

  The information processing apparatus 50 includes a frame memory 51 that stores detection data output from the detection circuit 47 and a central processing unit (hereinafter referred to as “CPU”) that calculates target spectral characteristics based on the stored detection data. ) 52, a display device 53, an external storage device 54, an input device 55, and a scanner driving device 56. Here, although not shown, the CPU 52 includes a calculation unit and a main storage unit. For example, a liquid crystal display device is used as the display device 53. As the external storage device 54, for example, a hard disk device, an optical recording medium storage device, a semiconductor storage device, or the like is used. The input device 55 includes devices such as a keyboard and a mouse, for example.

  The external storage device 54 stores an operation program for the CPU 52 and various data. Specifically, it has a function as a storage means for storing spectral characteristics (spectral data) for each of a plurality of optical elements in association with the respective optical elements. Such a program and preliminarily given data are stored in the external storage device 54 by, for example, reading and installing from a storage medium such as a CD-ROM using a reading device (not shown).

  The CPU 52 loads a program stored in the external storage device 54 and performs various controls, arithmetic processing, and the like. The program stored in the external storage device 54 includes means for controlling the operation of the scanning unit 23 via the scanner driving device 56, and means for calculating spectral characteristics based on data obtained from the measurement target. The spectral characteristic information about the optical element to be used, which is stored by the storage unit based on the information for identifying the received optical element, is read out, and the spectral characteristic information of the optical system to be used is obtained. And a program for operating the CPU 52 by means for obtaining the spectral characteristics of the object using the spectral data output from the spectral unit 40 described above. In addition to the storage device, a program for operating the CPU 52 as storage means for storing the spectral characteristics of each of the plurality of optical elements in correspondence with each element is also included. Furthermore, a program for selecting and operating the wavelength of the excitation light emitted from the laser device 11 and a program for controlling the operation of the wavelength selection units 148 and 248 in the spectroscopic units 140 and 240 described later are also included.

  The spectroscopic unit 40 will be described in more detail. The spectroscopic unit 40 takes in the signal light through the optical fiber 28, splits it with any of the diffraction gratings 431 to 433, and receives the spectroscopic light at each light receiving element 45 a of the light receiver 45.

  As shown in FIG. 1, the optical fiber 28 is attached to the spectroscopic unit 40 with a fiber coupler 29b. The signal light from the optical fiber 28 is all collimated by the collimating optical system 41 and is incident on the diffraction grating 43 selected from the diffraction gratings 431 to 433. The signal light incident on the diffraction grating 43 is diffracted in the direction of the diffraction angle determined by the wavelength and is split. The split signal light is condensed by the condensing optical system 44, imaged on the light receiving surface of the light receiver 45, and incident on the light receiver 45. From the light receiver 45, an electrical signal corresponding to the amount of signal light incident on each light receiving element 45a is output and processed by the information processing device 50, whereby a spectral signal of wavelength versus intensity can be obtained.

  The diffraction gratings 431 to 433 differ in the number of engraved lines (the number of diffraction grating grooves per 1 mm). Further, these diffraction gratings 431 to 433 are installed on the rotary table 42 at different angular positions. Therefore, the wavelength band detected by the light receiver 45 can be changed by turning the rotary table 42 and changing the angle of incidence on the diffraction gratings 431 to 433. In addition, the wavelength resolution can be changed by switching from one diffraction grating to another among the diffraction gratings 431 to 433.

  The optical fiber 28 is a step index type made of a quartz core, and has a core diameter of 50 μm and an NA of 0.22. Light emitted from the optical fiber 28 via the fiber coupler 29 b is guided into the spectroscopic unit 40. In the present embodiment, the light is guided to the spectroscopic optical system via the collimating optical system 41. At this time, the microscope system 1 according to the present embodiment handles a weak light signal (fluorescence). For this reason, in order to obtain a high SN ratio, it is necessary to use the signal light beam introduced by the optical fiber 28 with high efficiency. Therefore, the role of the collimating optical system 41 is to take in the signal light beam (diverged light) introduced by the optical fiber 28 without loss and to guide it to the diffraction grating 43 which is a subsequent spectroscopic element.

  The light receiver 45 is composed of, for example, a 32-channel multi-anode line PMT (photomultiplier tube). This PMT has a structure in which the element size of one channel is about 0.8 mm × 7 mm, and 32 channels are arranged at a pitch P (= 1 mm) in the wavelength dispersion direction (the direction in which signal light is dispersed). ing.

  As the condensing optical system 44, a condensing lens can be used, and a condensing mirror can also be used. FIG. 1 shows a case where a condensing lens is used as the condensing optical system 44. In FIG. 2 and later described later, a case where a concave mirror is used as the condensing optical system 44 is shown. If the size of the signal light image (image of the core of the optical fiber) from the optical fiber 28 on the light receiving surface of the light receiver 45 is smaller than the size or pitch of the light receiving element 45a of the light receiver 45 in the wavelength dispersion direction, A high wavelength resolution corresponding to the number of elements of the light receiver 45 can be obtained. Furthermore, in consideration of the aberration of the optical system, it is desirable that the size of the signal light image is smaller than this. The condensing optical system 44 is configured such that an image of the light emitted from the diffraction grating 43 on the light receiver 45 is formed with a diameter smaller than the above-described pitch.

  Next, the operation of the microscope system 1 will be described. In this example, the specimen 33 to be observed is irradiated with laser light, and the fluorescence excited in the specimen 33 is captured by the microscope 30 and guided to the spectroscopic unit 40 to obtain spectroscopic data. The procedure of measurement or observation is roughly as follows.

  Laser light (excitation light, illumination light) emitted from the laser device 11 of the light source system 10 that is a light source is introduced into the optical fiber 13 through the fiber coupler 12. Further, the laser light passing through the optical fiber 13 enters the collimating lens 21 of the confocal unit 20 from the fiber coupler 14. The laser light is converted into substantially parallel light by the collimating lens 21, then reflected by the dichroic mirror 22 to the optical path on the microscope 30 side, and introduced into the scanning unit 23 and the scanner lens 24, which are composed of two orthogonally arranged galvanometers. And scanned two-dimensionally. The scanned laser light passes through the second objective lens 31, is condensed by the objective lens 32, and is condensed at one point on the sample 33. Note that the position on the specimen 33 scanned two-dimensionally by the scanning unit 23 is controlled by controlling the operation of the galvanometer in the scanning unit 23 by the CPU 52 via the scanner driving device 56 of the information processing apparatus 50. Then, the fluorescence (signal light) emitted from the specimen 33 excited by the laser light is converted into substantially parallel light by the objective lens 32, and follows the path opposite to the laser light (excitation light) to the dichroic mirror 22. Incident. The fluorescence that has entered the dichroic mirror 22 passes through the dichroic mirror 22 and is collected by the condenser lens 25 onto the pinhole 26 a of the pinhole plate 26.

  The light that has passed through the pinhole 26a is guided to the optical fiber 28 from the fiber coupler 29a through the relay lens 27. Through the relay lens 27, as shown in FIG. 1, the light passing through the pinhole 26a is condensed again as it is as a divergent light beam, so that at the opening end of the optical fiber 28, Apparently, even with a small aperture diameter, it becomes possible to enter effectively (with little loss).

  Here, since the condensing point formed in the pinhole 26a is an image of a light spot on the specimen 33, even if there is light emitted from another point on the specimen 33, the pinhole 26a. Then, an image is not formed and is blocked by the pinhole plate 26 and hardly reaches the fiber coupler 29a. Therefore, only the light that can pass through the pinhole 26 a can reach the fiber coupler 29 a via the relay lens 27. As a result, the scanning confocal microscope is a microscope capable of observing a specimen not only with high lateral resolution but also with high vertical resolution.

  The signal light (fluorescence) incident on the fiber coupler 29a passes through the optical fiber 28 and is introduced into the spectroscopic unit 40 via the fiber coupler 29b. The signal light introduced into the spectroscopic unit 40 becomes a substantially parallel light beam in the collimating optical system 41 and is introduced into any of the diffraction gratings 431 to 433. In the microscope system 1 according to the present embodiment, as described above, three types of these diffraction gratings 431 to 433 are prepared in order to make the wavelength resolution variable, and the rotation table 42 is rotated by being controlled by a pulse motor (not shown). Accordingly, any one of the diffraction gratings 43 is selected and used.

  The signal light diffracted by the diffraction grating 43 is collected by the condensing optical system 44 and is incident on the light receiver 45 at a spread angle corresponding to the wavelength resolution of the diffraction grating 43. The incident signal light is converted into an electric signal by the photoelectric effect of the light receiving element 45a. The converted electrical signal is amplified by the amplifier 47a of the detection circuit 47, converted into a digital signal by the A / D converter 47b, sent to the frame memory 51, and calculated and processed by the CPU 52 to the display device 53 as an image. Is displayed.

  In the spectroscopic unit 40 described above, the case where the diffraction grating 43 is used as a spectroscopic element for spectroscopically separating fluorescence included in the signal light has been described. The method is not limited. For example, at least one filter (long-pass filter or the like) that transmits light of a predetermined wavelength band and reflects light of the remaining wavelength is disposed on the optical path of the signal light, and the signal light is dispersed. It is also possible to detect the intensity of at least one of the light reflected by and transmitted light. At this time, a plurality of filters having different wavelength characteristics are arranged in series on the optical path of the signal light and dispersed to detect at least one intensity of the light reflected by each filter and the light transmitted through all the filters. It is also possible to configure. Further, when using a filter whose wavelength characteristic changes depending on the incident angle of incident light, the wavelength of transmitted and reflected light can be shifted by changing the angle of the filter with respect to the signal light.

  As described above, in the microscope system 1 according to the present embodiment, since the wavelength of the excitation light for exciting the specimen 33 can be selected, the wavelength of the excitation light incident on the spectroscopic unit 40 together with the signal light is also selected. Therefore, the wavelength of light to be removed from the signal light by the spectroscopic unit 40 also changes. Therefore, the microscope system 1 according to the present embodiment includes a wavelength selection unit configured to change the wavelength of light to be removed from the signal light according to the wavelength of excitation light emitted from the laser device 11 of the light source system 10. Is provided. Hereinafter, the structure of this wavelength selection part is demonstrated.

[First Embodiment]
First, the configuration of the spectroscopic unit 140 according to the first embodiment will be described with reference to FIG. The spectroscopic unit 140 according to the first embodiment is used in a configuration in which the spectroscopic unit 40 is replaced with the spectroscopic unit 140 in the microscope system 1 shown in FIG. Therefore, the detailed description of the configuration of the light source system 10, the confocal microscope having the confocal unit 20 and the microscope 30, and the information processing apparatus 50 is omitted by using the same reference numerals.

  The spectroscopic unit 140 according to the first embodiment includes a collimating optical system 41 that converts signal light (fluorescence) emitted from the fiber coupler 29b through the optical fiber 28 into a substantially parallel light beam, and the substantially parallel signal light. A wavelength selection unit 148 that removes light having a wavelength of excitation light to be generated, and diffraction gratings 431, 432, and 433 (diffraction grating 43), which are three spectral elements that are attached to the rotary table 42 and each have different spectral characteristics, A condensing optical system 44 composed of a concave mirror that condenses the light (spectral light) diffracted by any of the diffraction gratings 431 to 433, and the diffracted light (spectral light) condensed by the condensing optical system 44 And a light receiver 45 to detect.

  In the spectroscopic unit 140 according to the first embodiment, the wavelength selection unit 148 includes at least two filter units 149 arranged along the optical axis. Each of the filter units 149 inserts and removes the notch filter 149a on the optical path between the flat plate notch filter 149a, which is an optical element having different wavelength characteristics, and the collimating optical system 41 and the diffraction grating 43. A filter insertion / extraction section 149b, and a rotation stage 149c that changes the incident angle of the signal light (signal light made substantially parallel by the collimating optical system 141) to the notch filter 149a by rotating the notch filter 149a. Configured.

  Here, the notch filter 149a is a filter that reflects light of a predetermined wavelength and transmits light of other wavelengths. In the present embodiment, for example, four filter units 149 corresponding to 405 nm, 488 nm, 561 nm, and 640 nm, which are typical wavelengths used in the laser apparatus 11, are provided, and each notch filter 149a is any one. It is configured to reflect light having a wavelength of. Therefore, although not illustrated in FIG. 1, the information processing device 50 controls the wavelength of the excitation light emitted from the laser device 11, and the information processing device 50 is used as a control unit that controls the operation of the wavelength selection unit 148. The notch filter 149a corresponding to the wavelength of the excitation light emitted from the laser device 11 is inserted into the optical path by controlling the operation of the filter insertion / extraction portion 149b of each filter unit 149, and is not emitted from the laser device 11. By removing the notch filter 149a corresponding to the wavelength of the excitation light from the optical path, unnecessary excitation light is removed from the signal light.

  More preferably, the information processing device 50 controls the operation of the filter insertion / extraction section 149b of each filter unit 149 in synchronization with the switching control of the wavelength of the excitation light emitted from the laser device 11 by the information processing device 50. The notch filter 149a corresponding to the wavelength of the excitation light emitted from the laser device 11 is inserted on the optical path, and the notch filter 149a corresponding to the wavelength of the excitation light not emitted from the laser device 11 is removed from the optical path.

  Note that the notch filter 149a may be a multiband filter that reflects light in a plurality of wavelength bands. In addition, in FIG. 1, the configuration in which the four filter units 149 corresponding to the four wavelengths are provided has been described. However, the present invention is not limited to this configuration, and the laser device 11 is formed by any notch filter 149a. It is sufficient if the light having the wavelength of the excitation light emitted from the light can be reflected.

  Further, in the spectroscopic unit 140 according to the first embodiment, it is preferable that the laser stop band (wavelength band of reflected light) of the notch filter 149a is narrow because fluorescence used for observation is not removed. However, if the laser stopband is made too narrow, the excitation light passes through the wavelength selector 148 and enters the light receiver 45 when the wavelength of the emitted excitation light is shifted due to individual differences in the laser devices 11. End up. In general, the laser stop band (wavelength characteristics) of the notch filter 149a varies depending on the angle at which light enters the notch filter 149a. Specifically, when the substantially parallel signal light emitted from the collimating optical system 41 is perpendicularly incident on the notch filter 149a (incident angle = 0 degree) and the notch filter 149a is tilted, the laser stop band is reduced to the short wavelength side. Shift to. Therefore, the spectroscopic unit 140 is configured to change the incident angle of the signal light to the notch filter 149a by rotating the rotary stage 149c in accordance with the wavelength of the excitation light emitted from the laser device 11. More preferably, in synchronization with the switching control of the wavelength of the excitation light emitted from the laser device 11 by the information processing apparatus 50, the rotation stage 149c is rotated by the information processing apparatus 50 and the incident angle of the signal light with respect to the notch filter 149a. To change. In this case, when the incident angle of the signal light with respect to the notch filter 149a is 0 degree, it is preferable to design so that the laser stop band coincides with the upper limit on the long wavelength side of the variation of the excitation light. Thus, the rotary stage 149c functions as an angle adjustment unit that changes the angle of light incident on the notch filter 149a.

  The rotation stage 149c is intended to change the incident angle of the signal light with respect to the notch filter 149a, and therefore may be configured to incline the notch filter 149a in one direction with respect to the optical axis. Further, if the notch filter 149a can be tilted by 15 degrees, the laser stop band can be shifted by 3% or more, which is sufficient.

  When the spectroscopic unit 140 according to the first embodiment is configured as described above, only the light having the wavelength of the excitation light included in the signal light incident on the spectroscopic unit 140 can be removed, and an image with less noise is obtained. can do.

  By the way, as shown in FIG. 3, the above-described notch filter 149a does not have a constant spectral transmittance in the transmission band. Even when a filter having a high transmittance over the entire wavelength band is designed, ripples (fine irregularities with a high transmittance) exist as shown in FIG. Due to such spectral transmittance characteristics of the notch filter 149a, the amount of transmitted light is not constant. Therefore, it is difficult to quantitatively evaluate the signal light as in the spectroscopic unit 40 according to the present embodiment. Become.

  Therefore, in the microscope system 1 according to the present embodiment, the wavelength characteristic of the notch filter 149a (transmittance with respect to the wavelength of incident light as shown in FIG. 3) is previously provided for each filter unit 149 constituting the wavelength selection unit 148. ) Is measured and stored in the external storage device (storage unit) 54 of the information processing device 50. The wavelength characteristic of the notch filter 149a may be a design value instead of actually measuring. Then, the information processing apparatus 50 reads the wavelength characteristic (wavelength characteristic corresponding to the incident angle) of the notch filter 149a inserted on the optical path as described above from the external storage device 54, and the wavelength pair detected by the light receiver 45. A high-accuracy spectral signal can be obtained by correcting the intensity spectral signal with the wavelength characteristic of the notch filter 149a. As a correction method for the spectral signal, a correction coefficient for each wavelength may be stored in the external storage device 54, and the signal intensity obtained by the light receiver 45 may be multiplied by this correction coefficient, or the light receiver The gain may be changed according to the correction coefficient for each of the light receiving elements 45a constituting the 45. Further, when a plurality of notch filters 149a are arranged on the optical path, a product obtained by multiplying each correction coefficient may be used as the correction coefficient.

  Further, when the rotation stage 149c is rotated to change (shift) the laser stop band by changing the incident angle of the signal light to the notch filter 149a, the above-described ripple also causes a wavelength shift. The phase of this wavelength has the relationship of the formula (1) described later and varies depending on the wavelength. For this reason, the amount of shift of the ripple due to the rotation of the notch filter 149a also depends on the wavelength. Therefore, it is necessary to perform the above correction in consideration of this change.

  It should be noted that such correction of the spectral signal based on the wavelength characteristics of the filter needs to be performed in the same manner when a filter is used as the spectral element.

[Second Embodiment]
Next, the configuration of the spectroscopic unit 240 according to the second embodiment will be described with reference to FIGS. The spectroscopic unit 240 according to the second embodiment is also used in a configuration in which the spectroscopic unit 40 is replaced with the spectroscopic unit 240 in the microscope system 1 shown in FIG. Therefore, the detailed description of the configuration of the light source system 10, the confocal microscope having the confocal unit 20 and the microscope 30, and the information processing apparatus 50 is omitted by using the same reference numerals. In the second embodiment, the information processing apparatus 50 is used as a control unit that controls the operation of the wavelength selection unit 248.

  As shown in FIG. 4, the spectroscopic unit 240 according to the second embodiment includes a collimating optical system 41 that converts the signal light (fluorescence) emitted from the fiber coupler 29b through the optical fiber 28 into a substantially parallel light beam, Diffraction gratings 431, 432, and 433, which are attached to the rotary table 42 and three spectral elements each having different spectral characteristics, are attached to the wavelength selection unit 248 that removes the light of the excitation light included in the substantially parallel signal light. (Diffraction grating 43), Condensing optical system 44 composed of a concave mirror that condenses the light diffracted by any of diffraction gratings 431 to 433, and diffracted light collected by this condensing optical system 44 is detected. And a light receiver 45.

  In the spectroscopic unit 240 according to the second embodiment, the wavelength selection unit 248 includes two filter units 249F and 249R (collectively referred to as “filter unit 249”). Each of the filter units 249 includes plate-like light transmitting members 249a and 249b having two planes S1 and S2 arranged in parallel at a predetermined interval, and these light beams in a direction in which the planes S1 and S2 extend. A parallel stage 250a that translates the transmission members 249a and 249b and a rotary stage 250b that rotates the parallel stage 250a are configured. The filter unit 249 is configured to transmit light of a predetermined wavelength and reflect light of the remaining wavelength by the two planes S1 and S2 described above. A reflective film is formed on the opposing planes S1 and S2 of the light transmitting members 249a and 249b, and has at least two regions having different reflectances along the direction moved by the parallel stage 250a. Configured.

  Here, as shown in FIG. 5, a light beam incident on a space (hereinafter referred to as “interference space 249c”) sandwiched between the planes S1 and S2 facing the light transmitting members 249a and 249b described above is divided into two planes. While part of the light is transmitted through S1 and S2 and the rest is reflected, reflection is repeated in the interference space 249c, and multiple interference occurs between the planes S1 and S2. At this time, as shown in FIG. 5, regarding the light beam incident from one plane (for example, plane S1 in FIG. 5), the incident angle of this incident light beam in the interference space 249c is φ ′, and the incident position of the light beam The distance between the planes S1 and S2 (hereinafter referred to as “surface distance”) is d, and the refractive index of the medium of the interference space 249c is n ′. In the configuration shown in FIG. 5, the interference space 249 c is filled with air, and the refractive index n ′ is approximately 1. Further, when the refractive index of the medium of the light transmitting members 249a and 249b is n and the incident angle of the light flux in the light transmitting members 249a and 249b is φ, nsinφ = n′sinφ ′ is established according to Snell's law.

Then, assuming that the energy reflectance of each of the planes S1 and S2 is R ₀ , the energy transmittance Tfp due to the multiple interference between the two planes S1 and S2 arranged close to each other as described above is expressed by the equation (1) δ. Is expressed as in the following equation (2).

As is clear from these equations (1) and (2), the wavelength characteristics of the wavelength of the light transmitted through the filter unit 249 having the above-described configuration and the wavelength of the light reflected by the filter unit 249 are the plane S1 and the plane. The reflectance R _{0 of} S2 is determined by the incident angle φ ′ of the light beam in the interference space 32c sandwiched between these planes S1 and S2 and the surface interval d between the planes S1 and S2.

First, the reflectance R ₀ of the planes S1 and S2 will be described. For example, the interference space 249c is filled with air (n ′ = 1), the surface interval d is 286.4 nm, the energy reflectance R ₀ of the planes S1 and S2 is 99%, and the incident angle φ of the light flux in the interference space 249c. FIG. 6A shows the wavelength characteristics of the filter unit 249 when ′ is 45 °. FIG. 6 shows the wavelength characteristics of the transmitted light. In the filter unit 249 having such a configuration, light having a wavelength near 405 nm is transmitted, and light having other wavelengths is reflected. At this time, since the wavelength band of the transmitted light is very narrow, for example, by using the light of 405 nm as the excitation light and the light of other wavelengths as the signal light, only the light of the wavelength of the excitation light can be reliably obtained from the signal light. Can be removed.

On the other hand, FIG. 6B shows the wavelength characteristics of the filter unit 249 when the reflectance R ₀ of the planes S1 and S2 is 50% and other conditions are the same as those of the above-described configuration. The filter unit 249 having such a configuration does not change the peak wavelength of the transmitted light as in the case of FIG. 6A described above, but since the band is widened, light having a wavelength in the vicinity of the peak wavelength is also transmitted. Therefore, when the wavelength of the excitation light and the wavelength of the signal light are close to each other, the signal light is cut. Therefore, it is desirable that the reflectance R ₀ of the planes S1 and S2 is 90% or more.

  As described above, in the spectroscopic unit 240 according to the second embodiment, the wavelength selection unit 248 uses the light transmitting members 249a and 249b of the filter unit 249 and the reflectivity of the planes S1 and S2 by the parallel stage 250a. It can be moved in a changing direction. Therefore, desired wavelength characteristics can be obtained by moving the light transmitting members 249a and 249b by the parallel stage 250a and changing the reflectance at the position where the signal light is incident. For example, when the wavelengths of the excitation light and the signal light are close, the wavelength band of the transmitted light is narrowed by increasing the reflectance to prevent loss of the signal light, and the wavelengths of the excitation light and the signal light are separated. In this case, the excitation light can be reliably cut by reducing the reflectance and widening the wavelength band of the transmitted light. Here, the reflectivities of the planes S1 and S2 may be changed continuously in the moving direction by the parallel stage 250a, or may be changed stepwise. Furthermore, if films having different reflectances are formed in a circular shape, the reflectance at the position where the signal light is incident can be changed by rotating the light transmitting members 249a and 249b. As described above, the parallel stage 250a has a function of a position adjusting unit that changes the position where the signal light enters the light transmitting members 249a and 249b (regions having different reflectivities).

  Further, as apparent from the above formulas (1) and (2), the wavelength characteristic of the filter unit 249 can be changed even if the incident angle φ ′ of the light beam in the interference space 249c of the planes S1 and S2 is changed. Can do. When changing the incident angle φ ′ of the light beam, the light transmitting members 249a and 249b are rotated by the rotating stage 250b of the filter unit 249. That is, the rotary stage 250b functions as an angle adjusting unit that changes the incident angle of the signal light with respect to the planes S1 and S2 of the light transmitting members 249a and 249b. The operations of the parallel stage 250a and the rotary stage 250b are controlled in synchronism with the control of the wavelength of the excitation light emitted from the laser device 11 by the information processing device 50, whereby the reflectivity of the planes S1 and S2 is controlled. Further, by adjusting the wavelength characteristics by changing the incident angle, the light having the wavelength of the excitation light emitted from the laser device 11 can be separated from the signal light.

  Further, as apparent from the above formulas (1) and (2), the wavelength characteristics of the filter unit 249 can be changed even if the plane distance d between the planes S1 and S2 is changed. A method for changing d will be described. As shown in FIGS. 7 and 8, the filter unit 249 constituting the wavelength selection unit 248 of the spectroscopic unit 240 according to the second embodiment is a flat light transmitting member on which the above-described planes S1 and S2 are formed. 249a and 249b, and these light transmission members 249a and 249b are arranged so that the planes S1 and S2 face each other. The light transmitting members 249a and 249b are configured to form an interference space 249c by sandwiching spacers 249d at three positions on the peripheral edge. The filter unit 249 is provided with a holding member 249e so as to surround the light transmitting members 249a and 249b and the spacer 249d. The holding member 249e has an annular shape in which an opening 249j is formed when viewed from a direction orthogonal to the planes S1 and S2 so that light can be transmitted and reflected on the planes S1 and S2. Have. Further, as shown in FIG. 8, the cross section has a U-shape, and the peripheral portions of the light transmitting members 249a and 249b and the spacer 249d are arranged in a space surrounded by the holding member 249e. Then, by using three fixing members 249f attached to one surface side of the holding member 249e so as to be aligned with each of the three spacers 249d, the light transmitting members 249a and 249b and the spacer 249d are held by the holding member 249e. It is comprised so that it may fix by pressing on the other surface of this and pinching these. Therefore, this filter unit 249 can adjust the amount by which the spacer 249d is compressed by adjusting the pressing force by the fixing member 249f, and can accurately adjust the surface interval d between the two light transmission members 249a and 249b. It becomes. As described above, the spacer 249d, the holding member 249e, and the fixing member 249f constitute an interval adjusting unit 249g that adjusts the surface interval d of the filter unit 249.

  In the above-described interval adjusting unit 249g, the case where the interval d is adjusted by the amount of compression of the spacer 249d by the fixing member 249f has been described, but instead of these, an element using a piezoelectric effect such as a piazo element The surface distance d may be adjusted, or the temperature of the spacer 249d may be changed by a heater or a Peltier element to expand and contract the spacer 249d to adjust the surface distance d. good.

  Further, in the case of a configuration in which the surface interval d between the planes S1 and S2 of the filter unit 249 constituting the wavelength selection unit 248 is controlled by an electrical signal like a piazo element, the surface interval can be controlled by the information processing device 50. . For example, the information processing device 50 determines the wavelength of the fluorescence (signal light) generated in the sample from the property of the sample and the wavelength of the excitation light (illumination light) emitted from the laser device 11, and the above equation (1). And the expression (2), the filter unit 249 has a desired wavelength characteristic (transmits the excitation light and reflects the signal light) by calculating a surface interval d, and the filter unit so that the surface interval d is obtained. 249 can be configured to control the interval adjustment unit 249g.

Further, the excitation light radiated from the laser device 11 of the light source system 10 and the wavelength of the fluorescence generated by this excitation light are input by the input device 55 and set for the information processing device 50. Based on (1) and equation (2), at least one of the surface spacing d, the incident angle φ ′, and the energy reflectivity R ₀ is controlled to interlock with the wavelength of the laser device 11 and the wavelength selector 248. It is also possible to configure so as to control the wavelength characteristics of (filter unit 249 (249F, 249R)). Alternatively, the type of the fluorescent dye of the specimen 33 and at least one of the excitation light and the fluorescence wavelength corresponding to this kind are stored in the external storage device 54, and the type of the fluorescent dye input from the input device 55 Based on the wavelengths of the excitation light and the fluorescence, at least one of the surface distance d, the incident angle φ ′, and the energy reflectance R ₀ is calculated by the equations (1) and (2) to calculate the wavelength selector 248 (filter unit 249). (249F, 249R)) may be adjusted (that is, the wavelengths of the excitation light and the fluorescence are determined from information set in the external storage device 54 in accordance with the fluorescent dye input from the input device 55). It is also possible to directly input the wavelength of excitation light and fluorescence used for observation, or to input the wavelength of either excitation light or fluorescence and determine the other wavelength therefrom. good) .

  Note that the above-described surface distance d and incident angle φ ′ may control the filter unit 249 (249F, 249R) of the wavelength selection unit 248 according to values calculated from the above formulas (1) and (2). (For example, the surface spacing of the light transmitting members 249a and 249b is controlled so as to have a desired surface spacing d), a predetermined reference position, or a correction value (a reference position or The filter unit 249 (249F, 249R) of the wavelength selection unit 248 may be controlled by obtaining the difference from the current position.

  Further, from the above formulas (1) and (2), the filter unit 249 can be made to have a multi-peak that transmits two or more wavelengths by increasing the surface interval d.

  According to the spectroscopic unit 240 configured as described above, the signal light that has become a substantially parallel light beam by the collimating optical system 41 enters the first filter unit 249F, and is included in the signal light by the first filter unit 249F. Light of a predetermined wavelength is transmitted, and light of other wavelengths is reflected and enters the second filter unit 249R. The second filter unit 249R transmits light of a predetermined wavelength included in the signal light, reflects light of other wavelengths, and is diffracted by any one of the diffraction gratings 431 to 433 to collect light. The light is condensed at 44 and detected by the light receiver 45. Therefore, by adjusting the wavelength characteristics of the first and second filter units 249F and 249R of the wavelength selection unit 238 by the method described above, the wavelength selection unit 238 allows the light having the wavelength of the excitation light included in the signal light to be adjusted. Since it can be removed by transmission, an image with less noise can be acquired.

  At this time, by combining the two filter units 249F and 249R as described above, the incident angles of the signal light to the respective filter units are made the same, that is, the first and second filter units 249F and 249R. When the planes S1 and S2 are substantially parallel, signal light is emitted in the same direction from the same position of the wavelength selection unit 238 regardless of the incident angle of the signal light. Therefore, the position of the optical system after the diffraction grating 43 can be fixed. Note that one of the two filter units 249F and 249R can be configured by a mirror. In that case, a rotating stage is necessary for the mirror, but there is no need to provide a parallel stage.

In the above description, at least one of the surface distance d, the incident angle φ ′, and the energy reflectance R ₀ of the filter unit 249 (249F, 249R) is controlled so that the wavelength selection unit 248 has a desired wavelength characteristic. However, each of the first and second filter units 249F and 249R may be provided with a detection unit 260 that detects the wavelength characteristics. In this case, not only the wavelength characteristic of the filter unit 249 (249R, 249F) of the wavelength selection unit 248 is confirmed, but also the wavelength characteristic detected by the detection unit 260 is fed back to obtain the surface interval d, the incident angle φ ′, or the energy reflection. By adjusting the value of the rate R ₀ , it is possible to control so that the wavelength characteristics of the entire wavelength selection unit 248 become a desired value. As a method of detecting the wavelength characteristics of the filter unit 249 (249F, 249R) by the detection unit 260, there is a method of detecting by detecting the distance between the planes S1, S2 of the light transmitting members 249a, 249b. Will be explained.

  First, the configuration of the detection unit 260 that detects the wavelength characteristics of the filter unit 249 from the intensity ratio of transmitted light and reflected light will be described with reference to FIG. The detection unit 260 has a first light source 261 and a second light source 262 that emit light having different wavelengths, and the light travels substantially in parallel and enters the light transmitting member 249a at a predetermined incident angle. A first transmission sensor 263 that detects light emitted from the first light source 261 and transmitted through the light transmission members 249a and 249b, and planes S1 and S2 of the light transmission member 249b emitted from the first light source 261. A first reflection sensor 264 that detects light reflected by multiple interference and transmitted through the light transmission member 249a, and a second transmission that detects light emitted from the second light source 262 and transmitted through the light transmission members 249a and 249b. A second reflection sensor that detects light radiated from the sensor 265 and the second light source 262 and reflected by the multiple interference of the planes S1 and S2 of the light transmission member 249b and transmitted through the light transmission member 249a. And 266, and a. Here, the wavelength of light emitted from the first light source 261 is set to 400 nm, the wavelength of light emitted from the second light source 262 is set to 550 nm, and emitted from the first and second light sources 261 and 262. The incident light incident on the light transmitting member 249a is 15 °, the energy reflectance of the planes S1 and S2 of the filter unit 249 is 80%, the distance d is 300 nm, and the wavelength characteristics shown in FIG. Explain that it is.

  As described above, the wavelength characteristic of the filter unit 249 is determined by the incident angle of the light beam in the interference space between the plane S1 and the plane S2, the interval between the plane S1 and the plane S2, and the energy reflectance. That is, if these values are appropriate values, the intensity of light detected by the first transmission sensor 263 and the first reflection sensor 264 corresponding to the first light source 261 in the detection unit 260 is the first value. Since the wavelength of the light emitted from one light source 261 is 400 nm, the transmittance of the filter unit 249 is 95% and the reflectance is 5% as shown in FIG. Become. Similarly, the intensity of light detected by the second transmission sensor 265 and the second reflection sensor 266 corresponding to the second light source 262 is that the wavelength of the light emitted from the second light source 262 is 550 nm. Accordingly, as shown in FIG. 10, the transmittance of the filter unit 249 is 50% and the reflectance is 50%. As shown in FIG. 9A, the incident angle of the light emitted from the first and second light sources 261 and 262 is set to the incident angle of the signal light in the spectroscopic unit 240, and the sensors 263, 264 and 265 are set. , 266, the wavelength characteristics of the filter unit 249 can be set to a desired value by adjusting the distance between the light transmitting members 249a, 249b so that the intensity of the light detected at 266 is the above-described ratio.

  Next, the configuration of the detection unit 260 ′ that detects the wavelength characteristics of the filter unit 249 using an alignment microscope will be described with reference to FIG. In order to accurately measure the positions of the two light transmission members 249a and 249b constituting the filter unit 249, the detection unit 260 ′ includes the first alignment microscope 261 ′ and the third alignment in the light transmission member 249a. A microscope 263 ′ is arranged, and a second alignment microscope 262 ′ and a fourth alignment microscope 264 ′ are arranged on the light transmission member 249 b. Thereby, the position of each plane S1, S2 which forms each light transmission member 249a, 249b, or the position of the alignment mark (not shown) formed in the side part of light transmission member 249a, 249b is measured accurately. Thus, the wavelength characteristic of the filter unit 249 is detected, that is, the distance between the light transmitting member 249a and the light transmitting member 249b is set to a desired value. In order to set the interval between the two light transmission members 249a and 249b (planes S1 and S2) more in parallel, three sets of the light transmission members 249a and 249b are spaced apart by approximately 120 degrees. It is good to arrange an alignment microscope. In order to simplify the system, an alignment microscope may be arranged at least one of the light transmission member 249a and the light transmission member 249b.

  Finally, the configuration of a detection unit 260 ″ that detects the wavelength characteristic of the filter unit 249 using a length measurement interferometer will be described with reference to FIG. 11. This detection unit 260 ″ includes two light transmitting members that constitute the filter unit 249. In order to accurately measure the respective positions of 249a and 249b, reflecting surfaces R1 and R2 are provided on the side surfaces in the same direction to constitute a length measuring interferometer. Specifically, the light beam emitted from the interferometer head 261 ″ is separated into a P wave and an S wave by the polarization beam splitter 262 ″, and the P wave transmitted through the polarization beam splitter 262 ″ is reflected on the reflection surface of the light transmission member 249b. The S wave reflected by the polarization beam splitter 262 ″ is incident on R2 to the reflection surface R1 of the light transmission member 249a by the first mirror 263 ″. Here, the two optical paths separated by the polarization beam splitter 262 ″ In either case, a first λ / 4 plate 264 ″ and a second λ / 4 plate 265 ″ are disposed. Therefore, the polarization direction is changed by 90 degrees when the light is reflected by the respective reflecting surfaces R1 and R2 of the light transmitting members 249a and 249b and again enters the polarizing beam splitter 262 ″. That is, the light transmitting member 249a. The light reflected by the reflecting surface R1 becomes a P wave, is reflected by the first mirror 263 ″, passes through the polarization beam splitter 262 ″, and the light reflected by the reflecting surface R2 of the light transmitting member 249b becomes an S wave. By being reflected by the polarization beam splitter 262 ″, these lights are combined and incident on the second mirror 266 ″, reflected by the second mirror 266 ″ and incident on the receiver 267 ″. The interval between the light transmitting members 249a and 249b is accurately controlled based on the output signal output from 267 ″, that is, the wavelength characteristic of the filter unit 249. Rukoto can.

  In the detection unit 260 ″ shown in FIG. 10, only one interferometer is arranged. More ideally, a plurality of interferometers are arranged and controlled so that the light transmission members 249a and 249b are parallel to each other. The degree can be kept good, which is preferable.

  In the spectroscopic units 140 and 240 according to the first and second embodiments described above, the wavelength selection units 148 and 248 are arranged before entering the diffraction grating 43, but the present invention is limited to this configuration. The optical fiber 28 may be disposed anywhere on the optical path from the emission end (fiber coupler 29b) to the light receiver 45.

1 Microscope system 29b Fiber coupler (exit end)
DESCRIPTION OF SYMBOLS 10 Light source system (light source) 30 Microscope 32 Objective lens 33 Sample 40 Spectroscopic unit (spectral part) 41 Collimate optical system 43 (431-433) Diffraction grating (spectral element) 44 Condensing optical system 45 Light receiver 50 Information processing apparatus (control) 54) External storage device (storage unit)
140 Spectroscopic unit 148 Wavelength selection unit 149 Filter unit 149a Notch filter (optical element) 149b Filter insertion / extraction unit 149c Rotation stage (angle adjustment unit) 240 Spectroscopy unit 248 Wavelength selection unit 249 (249F, 249R) Filter unit 249a, 249b Light transmission member (Optical element) 249g Interval adjustment unit 250a Parallel stage (position adjustment unit) 250b Rotation stage (angle adjustment unit)

Claims (12)

A microscope that scans the excitation light emitted from the light source and collects it on the sample by the objective lens, and collects the fluorescence from the sample on the objective lens,
A spectroscopic element that splits the fluorescence from the microscope, and a spectroscopic unit that receives the spectroscopic light split by the spectroscopic element;
A microscope system comprising:
The spectroscopic unit includes a wavelength selection unit that suppresses the excitation light from being directed to the light receiver and guides the fluorescence to the light receiver.
A microscope system comprising: a control unit that controls the wavelength selection unit to change a wavelength characteristic of the wavelength selection unit according to the wavelength of the excitation light. The wavelength selector is
Two or more optical elements having different wavelength characteristics;
A filter insertion / extraction section for inserting / extracting each of the optical elements into / from the optical path of the spectroscopic section,
The control unit controls the filter insertion / extraction unit to insert at least one of the optical elements on the optical path and to remove the other optical elements from the optical path. The microscope system according to 1. The wavelength selection unit includes an optical element having a predetermined wavelength characteristic, and includes an angle adjustment unit that changes an incident angle of the excitation light with respect to the optical element,
The microscope system according to claim 1, wherein the control unit controls the angle adjustment unit. A storage unit that stores the incident angle of the angle adjustment unit controlled by the control unit in association with the wavelength of the excitation light;
The microscope system according to claim 3, wherein the angle adjustment unit is controlled by the control unit so that the incident angle is read from the storage unit.   The microscope system according to any one of claims 2 to 4, wherein the optical element is a notch filter that reflects the excitation light and transmits the fluorescence. The wavelength selection unit includes two light transmission members arranged so that planes having a predetermined reflectance face each other at a predetermined interval, an interval adjustment unit that changes the interval, and the excitation light for the light transmission member, A filter unit including at least one of angle adjustment units that change the incident angle of the fluorescence;
The microscope system according to claim 1, wherein at least one of the interval adjustment unit and the angle adjustment unit is controlled by the control unit. A storage unit that stores the interval of the interval adjustment unit controlled by the control unit in association with the wavelength of the excitation light;
The microscope system according to claim 6, wherein the interval adjustment unit is controlled by the control unit so as to be the interval read from the storage unit. A storage unit that stores the incident angle of the angle adjustment unit controlled by the control unit in association with the wavelength of the excitation light;
The microscope system according to claim 6, wherein the angle adjustment unit is controlled by the control unit so that the incident angle is read from the storage unit.   The microscope system according to any one of claims 6 to 8, wherein a reflectance of the plane of the light transmitting member is 90% or more. Each of the planes of the light transmission member is formed with at least two or more regions having different reflectivities,
The two light transmissive members include a position adjusting unit that moves in a plane orthogonal to the optical axis to change the region where the excitation light is incident. 10. The microscope system described in 1. The wavelength selection unit includes a first optical element that reflects at least the fluorescence-containing light among incident light; and
A second optical element that reflects at least the light containing the fluorescent light among the light reflected by the first optical element;
The microscope system according to claim 6, wherein at least one of the first optical element and the second optical element is the filter unit.   The microscope system according to claim 11, wherein reflecting surfaces of the first optical element and the second optical element are arranged substantially in parallel.

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