Microscope

Abstract

To provide a microscope capable of achieving multiband detection, in other words multicolor simultaneous detection.SOLUTION: There is provided a microscope comprising an illumination optical system for irradiating a sample with excitation light, a detector for detecting fluorescence emitted from the sample, and an observation optical system for guiding fluorescence to the detector. The observation optical system includes a first optical filter whose reflecting and permeating waveform characteristics change in accordance with a position at which light enters, a second optical filter which is located in the optical path of light reflected by the first optical filter and whose boundary wavelength of permeation changes with respect to the position along a first direction, and which passes light of a longer wavelength through than a first boundary wavelength at a position where reflected light enters, and a third optical filter which is located in the optical path of light reflected by the first optical filter and whose boundary wavelength of permeation changes with respect to the position along the first direction, and which passes light of a shorter wavelength through than a second boundary wavelength at the position where reflected light enters. The first boundary wavelength is shorter than the second boundary wavelength.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a microscope. [Background technology]

[0002] There is a fluorescence microscope equipped with a bandpass filter whose wavelength characteristics are adjustable (see, for example, Patent Document 1). [Prior art document] [Patent documents] [Patent Document 1] JP 2000-056228 A Summary of the Invention

[0003] In a first aspect of the present invention, the microscope may have an illumination optical system that irradiates excitation light onto a specimen. The microscope may have a detector that detects fluorescence emitted from the specimen. The microscope may have an observation optical system that guides the fluorescence to the detector. The observation optical system may have a first optical filter whose reflection and transmission wavelength characteristics vary depending on the position at which light is incident, a second optical filter that is arranged in the optical path of light reflected by the first optical filter, whose transmission boundary wavelength changes with respect to the position along the first direction, and transmits light with a wavelength longer than the first boundary wavelength at the position at which the reflected light is incident, and a third optical filter that is arranged in the optical path of light reflected by the first optical filter, whose transmission boundary wavelength changes with respect to the position along the first direction, and transmits light with a wavelength shorter than the second boundary wavelength at the position at which the reflected light is incident. The first boundary wavelength may be shorter than the second boundary wavelength.

[0004] The first optical filter may have different wavelength characteristics depending on the position along the first direction. The first optical filter may be arranged inclined at less than 45 degrees with respect to the incident light in a plane intersecting the first direction. The second optical filter and the third optical filter may be movable along the first direction. The first direction may be the direction of gravity. Either one of the second optical filter and the third optical filter may be arranged inclined in a plane intersecting the first direction with respect to the other. The observation optical system may further include a concave mirror that collects the light reflected by the first optical filter between the second optical filter and the third optical filter. The observation optical system may further include a concave mirror that converts the light reflected by the first optical filter into a parallel light beam and causes the parallel light beam to be incident on the second optical filter and the third optical filter.

[0005] The observation optical system may include a reflecting element that receives the light transmitted through the first optical filter and reflects at least a portion of the light, a fourth optical filter that is disposed in the optical path of the light reflected by the reflecting element, has a boundary wavelength that changes with respect to the position along the first direction, and transmits light having a wavelength longer than the third boundary wavelength at the position where the reflected light is incident, and a fifth optical filter that is disposed in the optical path of the light reflected by the reflecting element, has a boundary wavelength that changes with respect to the position along the first direction, and transmits light having a wavelength longer than the fourth boundary wavelength at the position where the reflected light is incident. The third boundary wavelength may be shorter than the fourth boundary wavelength.

[0006] The reflecting element may be a sixth optical filter whose wavelength characteristics of reflection and transmission change depending on the position where the light is incident, or a total reflection mirror. The first optical filter, the second optical filter, and the third optical filter may be stored in the first unit, and the sixth optical filter or the reflecting element, the fourth optical filter, and the fifth optical filter may be stored in the second unit. The first unit and the second unit may be configured to be removable. The optical detector may include a first detector that receives a part of the light reflected by the first optical filter and that has passed through the second optical filter and the third optical filter, and may include a second detector that receives a part of the light that has passed through the first optical filter. The light receiving surface of the first detector and the light receiving surface of the second detector may face the same direction.

[0007] The above summary of the invention does not list all of the features of the present invention. Also, subcombinations of these features may also be inventions. [Brief description of the drawings]

[0008] [Figure 1] 1 is a schematic diagram showing the structure of a microscope 101 for observing a specimen 210. FIG. [Diagram 2] An example of the observation system downstream 140 and the detection unit 160 is shown diagrammatically. [Diagram 3] FIG. 2 is a schematic diagram showing the function of the LVF 254 as an example. [Figure 4] 1 is a schematic diagram for explaining a wavelength range that is transmitted through a wavelength selection unit 151 and can be detected by a detector 161. FIG. [Diagram 5] The detectable ranges of the four wavelength selection units 151, 152, 153, and 154 are shown diagrammatically. [Figure 6] Schematically shows how the detectable range is changed in the wavelength selection unit 151. [Figure 7] The light source 110 is shown diagrammatically. [Figure 8] 4 is an example of a flowchart showing an observation procedure of the microscope 101. [Figure 9]13 is a flowchart showing details of step S12 for creating and editing an imaging channel. [Figure 10] An example of the setting screen 300 in FIG. 9 is shown. [Figure 11] 13 shows an example of a setting screen 310 in step S14 for setting an LVF etc. for an imaging channel. [Figure 12] 13 is a flowchart showing details of step S16 for setting a combination of an imaging channel and a real channel. [Figure 13] An example of the setting screen 320 in FIG. 12 is shown. [Figure 14] 13 is an example of a setting screen 330 for setting the laser intensity in step S18 and the detector sensitivity in step S18. [Figure 15] 13 is a flowchart showing details of step S24, which is an internal control for acquiring an image. [Figure 16] 1 is a flow diagram for acquiring a fluorescence spectral distribution using the microscope 101. [Figure 17] A corresponding setting screen 450 is shown in FIG. [Figure 18] 18 is a schematic diagram for explaining an acquisition wavelength range and a detection wavelength range under the observation conditions set on the setting screen 450 shown in FIG. 17. [Figure 19] 1 is a diagram illustrating a fluorescence spectral profile at a certain position of a specimen 210 detected by a microscope 101. FIG. [Figure 20] 20 is an example showing a display image 460 displaying an image acquired in the embodiment of FIGS. 17-19. [Figure 21] 18 is a schematic diagram for explaining the acquisition wavelength range and detection wavelength range under the observation conditions set on the setting screen 450 shown in FIG. 17 for each imaging channel. FIG. [Figure 22] 10 is a timing chart for executing an example of acquiring a plurality of fluorescence spectral distributions using the microscope 101. [Diagram 23] 13 shows a schematic diagram of another example of a rear observation system 142. [Figure 24]Another example of a rear observation system 144 is shown diagrammatically. [Diagram 25] A modified example of the observation system rear stage 140 is shown diagrammatically. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The present invention will be described below through embodiments of the invention, but the following embodiments do not limit the scope of the invention according to the claims. Furthermore, not all of the combinations of features described in the embodiments are necessarily essential to the solution of the invention.

[0010] 1 is a schematic diagram showing the structure of a microscope 101 for observing a specimen 210. The microscope 101 is a confocal microscope, and includes a light source 110, an illumination optical system 220, an observation optical system 240, a detection unit 160, an information processing device 170, and a control device 180. The illumination optical system 220 and the observation optical system 240 share some optical elements. Note that the microscope 101 does not need to have all of these components, and for example, may not have the light source 110, may not have the information processing device 170, or may not have the control device 180.

[0011] The light source 110 emits laser light of a wavelength used as excitation light when observing the specimen 210 by fluorescence. The excitation light emitted from the light source 110 is incident on the illumination optical system 220.

[0012] The illumination optical system 220 irradiates excitation light onto the specimen 210. The illumination optical system 220 has a dichroic mirror 121, a galvano scanner 130, a relay lens 122, a lens 192, and an objective lens 191. The dichroic mirror 121 has a property of reflecting the wavelength of the excitation light emitted from the light source 110 and transmitting light of other wavelengths. The excitation light incident from the light source 110 is reflected by the dichroic mirror 121, changes its propagation direction, and is incident on the galvano scanner 130.

[0013] The galvanometer scanner 130 has a pair of galvanometer mirrors 131 and 132 that reflect incident light. The galvanometer mirror 131 is rotatable around the x-axis in Fig. 1, and the galvanometer mirror 132 is rotatable around the y-axis. The excitation light incident on the galvanometer scanner 130 is reflected by the pair of galvanometer mirrors 131 and 132, and then passes through the relay lens 122 and the lens 192 and enters the objective lens 191.

[0014] The excitation light from the relay lens 122 is collimated by the lens 192 and then focused on the specimen 210 by the objective lens 191 .

[0015] The orientation of the galvanometer mirrors 131 and 132 of the galvanometer scanner 130 is controlled by the control unit 133, whereby the focusing position of the excitation light can be changed on the specimen 210. Thus, the excitation light is scanned two-dimensionally (in the x and y directions in FIG. 1) on the specimen 210 by the galvanometer scanner 130.

[0016] The specimen 210 contains, for example, a fluorescent substance, and in that case, fluorescence is emitted from the light-collecting position on the specimen 210. However, the emitted light emitted from the specimen 210 also contains components other than fluorescence, such as reflected light of the excitation light.

[0017] The radiation light emitted from the specimen 210 passes through the objective lens 191 and the lens 192 and is incident on the relay lens 122. The focal position of the lens 192 and the focusing position of the excitation light on the specimen 210 are optically conjugate. The radiation light incident on the relay lens 122 passes through the galvano scanner 130 and is incident on the dichroic mirror 121.

[0018] Of the emitted light, a component having the same wavelength as the excitation light is reflected by the dichroic mirror 121 and guided to the side of the light source 110. Of the emitted light, a component having a different wavelength from the excitation light is transmitted through the dichroic mirror 121.

[0019] It should be noted that the dichroic mirror 121 cannot completely remove the wavelength components of the excitation light, and therefore the emitted light that has passed through the dichroic mirror 121 still contains components of the excitation light wavelength.

[0020] The observation optical system 240 shares the dichroic mirror 121, the galvano scanner 130, the relay lens 122, the lens 192, and the objective lens 191 with the illumination optical system 220. The observation optical system 240 further includes a reflecting mirror 123, a condenser lens 124, a pinhole 125, a collimator lens 126, an observation system rear stage 140, and a detection unit 160.

[0021] The emitted light that passes through dichroic mirror 121 and is reflected by reflecting mirror 123 passes through condenser lens 124 and enters pinhole 125. Here, pinhole 125 is disposed at a position conjugate with the focal position of objective lens 191. For this reason, pinhole 125 passes only the light emitted from the condensing position, which is the focal point of objective lens 191, and blocks light from other points as noise.

[0022] The observation optical system 240 guides the fluorescence emitted from the specimen 210 to the detection unit 160. The detection unit 160 detects the fluorescence emitted from the specimen 210. The detection unit 160 outputs an electrical signal according to the intensity of the detected fluorescence to the information processing device 170. The details of the observation system rear stage 140 of the observation optical system 240 and the detection unit 160 will be described later.

[0023] The information processing device 170 has a control unit 171, a display unit 172, and input units 173 and 174. The control unit 171 has an interface with the detection unit 160, and executes image processing to generate an image from a signal obtained from the detection unit 160, and further stores and saves the generated image.

[0024] The display unit 172 is formed by an LCD panel, a CRT device, or the like, and in addition to displaying the generated image to the user, also displays a user interface when various settings are input to the microscope 101. The input units 173 and 174 include a character input device such as a keyboard and a pointing device such as a mouse, and are used when the user inputs settings, operation instructions, and the like to the microscope 101.

[0025] Furthermore, the information processing device 170 communicates with the control device 180 and is also used as a user interface for the control device 180. The control device 180 holds setting values ​​related to the operations of the galvano scanner 130, the observation system rear stage 140, the detection unit 160, and the like, and controls these operations. Furthermore, the control device 180 may execute all or part of the image processing and the like in the information processing device 170 for the purpose of reducing the load on the information processing device 170. That is, the control device 180 may execute all or part of the operations executed by the control unit 171 of the information processing device 170. Furthermore, the control unit 171 of the information processing device 170 may execute all or part of the operations executed by the control device 180.

[0026] 2 shows a schematic diagram of an example of the observation system rear stage 140 and the detection section 160. The observation system rear stage 140 has four wavelength selection units 151, 152, 153, and 154. Furthermore, the detection section 160 has four detectors 161, 162, 163, and 164 corresponding to the four wavelength selection units 151, 152, 153, and 154, respectively.

[0027] The wavelength selection unit 151 has an LVF (Linear Variable Filter) 250, a concave mirror 252, a pair of LVFs 254 and 256, and a condenser lens 258. Of these, the LVFs 250, 254, and 256 have a dielectric layer on a transparent substrate, the thickness of which changes along a predetermined direction (z direction in the figure), and the wavelength characteristics of transmission and reflection change depending on the position through which light passes.

[0028] FIG. 3 is a schematic diagram showing the function of the LVF 254 as an example. The LVF 254 has a dielectric layer on a transparent substrate, the thickness of which changes along a predetermined direction (z direction in the figure), and the wavelength characteristics of reflection and transmission change according to the position at which light is incident. More specifically, the boundary wavelength, which is the boundary between the wavelength of transmission and the wavelength of reflection, changes depending on the position in the z direction at which light is incident. Therefore, the LVF 254 is moved in the z direction by the driving unit 402 to change the wavelength characteristics for the incident light from a fixed optical path. The LVF 254 and the driving unit 402 form an optical filter 400 in which the wavelength characteristics of transmission and reflection are dynamically variable.

[0029] The graph shown on the right side of Fig. 3 shows the wavelength characteristics of the LVF 254. In the graph shown in Fig. 3, the vertical axis shows wavelength and the horizontal axis shows transmittance. The LVF 254 having the wavelength characteristics shown in Fig. 2 is a long-pass filter that transmits light with a wavelength longer than the boundary wavelength and reflects light with a wavelength shorter than the boundary wavelength.

[0030] Returning to Fig. 2, the LVF 250 into which the light from the collimator lens 126 is incident functions as a reflecting element whose reflection and transmission characteristics differ depending on the wavelength of the incident light, i.e., a dichroic mirror. The LVF 250 is a long-pass filter that reflects light with a wavelength shorter than the boundary wavelength and transmits light with a wavelength longer than the boundary wavelength. The optical path incident on the LVF 250 is split into two according to the wavelength.

[0031] The LVF 250 can be moved in the z direction by a driving unit, similar to the optical filter 400 shown in Fig. 3. This makes it possible to dynamically change the boundary wavelength for the incident light.

[0032] The LVF 250 is disposed inclined at an angle of less than 45 degrees, preferably 22.5 degrees or less, in the xy plane with respect to the light incident from the collimator lens 126. That is, the light receiving surface is rotated around the z axis so that the angle of incidence of the light to the LVF 250 (i.e., the angle between the normal to the incident surface of the LVF 250 and the direction of the principal ray) is less than 45 degrees, preferably 22.5 degrees or less. By reducing the incident angle in this way, the ellipticity of the spot shape incident on the LVF 250 is maintained at a value close to 1, and the area of ​​the spot shape is reduced. More specifically, the spot shape incident on the incident surface of the LVF 250 becomes an ellipse elongated in the direction in which the incident surface is tilted. The minor axis of this ellipse remains the light beam diameter D, while the major axis becomes D / cosθ (θ is the incident angle). Therefore, the area of ​​the spot shape is smaller when θ is 22.5 degrees than when θ is 45 degrees, and the ellipticity is also closer to 1. Therefore, it is possible to suppress the decrease in the spectral resolution of the incident spot at the LVF 250.

[0033] Furthermore, the incident surface of the LVF 250 is located at a position rotated around the z-axis. The direction in which the boundary wavelength changes in the LVF 250 is the z-direction. As a result, the major axis of the ellipse expands in a direction perpendicular to the z-axis, i.e., in a direction in which the boundary wavelength does not change. Therefore, it is possible to further suppress the decrease in the spectral resolution at the incident spot in the LVF 250.

[0034] Furthermore, the direction in which the LVF 250 is driven is also the z direction. Therefore, when the z direction is the vertical direction, i.e., the direction of gravity, any backlash in the mechanical system that drives the LVF 250 is shifted downward by its own weight, resulting in high position repeatability due to driving and high positioning accuracy.

[0035] The concave mirror 252 is disposed between the LVF 250 and the pair of LVFs 254 and 256 in the optical path of the wavelength selection unit 151. That is, the light on the short wavelength side reflected by the LVF 250 is incident on the concave mirror 252. The concave mirror 252 condenses the reflected light between the pair of LVFs 254 and 256. This reduces the spot diameter of the light beam incident on both of the pair of LVFs 254 and 256, thereby suppressing the deterioration of the spectral resolution in each of the LVFs 254 and 256. Furthermore, if the focal length of the collimating lens 126 is made smaller than the focal length of the concave mirror 252, the spot diameter when incident on each of the pair of LVFs 254 and 256 can be reduced, thereby further suppressing the deterioration of the spectral resolution.

[0036] LVF 254 is a long-pass filter arranged so that the boundary wavelength changes with respect to the position along the z direction. LVF 256 is a short-pass filter arranged so that the boundary wavelength changes with respect to the position along the z direction. The boundary wavelength of LVF 254 is shorter than the boundary wavelength of LVF 256. Since such a pair of LVFs 254 and 256 are arranged facing each other, they function as a band-pass filter that transmits a specific wavelength range.

[0037] Like the LVF 250, the LVFs 254 and 256 can be moved in the z direction by a driving unit, and can dynamically change the boundary wavelength for incident light whose incident position is fixed. The pair of LVFs 254 and 256 are preferably arranged so that the boundary wavelengths at positions along the z direction tend to be similar to each other. In other words, the LVFs 254 and 256 are preferably arranged so that the boundary wavelengths of both shift toward the shorter wavelength side (or the boundary wavelengths of both shift toward the longer wavelength side) the further they are in the +z direction.

[0038] The light transmitted through the pair of LVFs 254, 256 is collected by a collecting lens 258 and enters a detector 161. The detector 161 is a highly sensitive photoelectric conversion element such as a photomultiplier tube, and outputs an electrical signal corresponding to the detected fluorescence to the information processing device 170 via the control device 180.

[0039] 4 is a schematic diagram for explaining the wavelength range that can be transmitted through the wavelength selection unit 151 and detected by the detector 161. Here, with regard to the wavelength selection unit 151, attention should be paid to the reflectance rather than the transmittance of the LVF 250, so the vertical axis shows the reflectance.

[0040] 4, the LVF 250 reflects the short wavelength side, the LVF 254 cuts (blocks) the short wavelength side, and the LVF 256 cuts (blocks) the long wavelength side. Furthermore, the boundary wavelength of the LVF 254 is shorter than the boundary wavelengths of the LVF 250 and the LVF 256. This allows a predetermined wavelength range to be transmitted and set as the detectable range of the detector 161. Furthermore, by making the LVF 250 function as a dichroic mirror, the light transmitted through the LVF 250 can be transferred to a downstream stage such as the wavelength selection unit 152, and other wavelength bands can also be detected.

[0041] The wavelength selection units 152 and 153 are disposed after the wavelength selection unit 151. The wavelength selection unit 152 has an LVF 260, a concave mirror 262, a pair of LVFs 264 and 266, and a condenser lens 268. The wavelength selection unit 153 has an LVF 270, a concave mirror 272, a pair of LVFs 274 and 276, and a condenser lens 278. The wavelength selection units 152 and 153 have the same configuration as the wavelength selection unit 151 except for the points described below, and therefore their explanations are omitted.

[0042] A wavelength selection unit 154 is further disposed after the wavelength selection unit 153. The wavelength selection unit 154 has a concave mirror 282, a pair of LVFs 284 and 286, and a condenser lens 288. The wavelength selection unit 154 has the same configuration as the wavelength selection unit 151 except for the fact that it does not have the LVF 250 and for the points that will be described later, so a description thereof will be omitted. Note that the concave mirror 282 can also be said to be a total reflection mirror from the viewpoint that it does not transmit light of a specific wavelength.

[0043] The detection section 160 is provided with detectors 162, 163, and 164 that receive light from the wavelength selection units 152, 153, and 154, respectively. These detectors 162, 163, and 164 have the same configuration as the detector 161, so a description thereof will be omitted. In the embodiment shown in Fig. 2, light is incident on all of the detectors 161, 162, 163, and 164 from the y direction. In other words, the detection surfaces of the detectors 161, 162, 163, and 164 are all parallel to the zx plane and face the same direction.

[0044] 5 shows a schematic diagram of the detectable ranges of the four wavelength selection units 151, 152, 153, and 154. In order to simplify the drawing, the transmittance and reflectance of the LVFs 250, 260, and 270 functioning as dichroic mirrors are omitted.

[0045] 5, the detectable ranges of the four wavelength selection units 151, 152, 153, and 154 are set in order from the shortest wavelength side. That is, detectable range 1 is set by a pair of LVFs 254 and 256 of the wavelength selection unit 151, and detectable range 2 is set on the longer wavelength side by a pair of LVFs 264 and 266 of the wavelength selection unit 152. Similarly, detectable range 3 is set on the longer wavelength side of detection range 2 by a pair of LVFs 274 and 276 of the wavelength selection unit 153, and detectable range 4 is set on the longer wavelength side by a pair of LVFs 284 and 286 of the wavelength selection unit 154.

[0046] 5, the observation system rear stage 140 and the detection unit 160 can detect fluorescence from the specimen 210 in four different wavelength ranges. This can also be said to have four different detection channels. In the following description, these channels may be referred to as real channels, since each of them has a real optical system.

[0047] 6 is a schematic diagram showing how the detectable range is changed in the wavelength selection unit 151. In order to simplify the drawing, the transmittance and reflectance of the LVF 250 functioning as a dichroic mirror are not shown.

[0048] The LVFs 250, 254, and 256 included in the wavelength selection unit 151 are all movable along the z direction in which the boundary characteristics change. Therefore, for example, by arranging the LVFs 250, 254, and 256 at corresponding z positions, it is possible to set the detectable range A in Fig. 6, and by arranging the LVFs 250, 254, and 256 at other corresponding z positions, it is possible to set the detectable range B in Fig. 6. In other words, in the optical system of one wavelength selection unit 151, a plurality of different wavelength ranges can be detected in a time-division manner.

[0049] In this case, moving both LVF 254 and LVF 256 in the same direction by the same amount along the z axis shifts the detectable range to the shorter or longer wavelength side while roughly maintaining the bandwidth, whereas moving at least one of LVF 254 and LVF 256 in the opposite direction relative to each other along the z axis widens or narrows the bandwidth of the detectable range.

[0050] It should be noted that the other wavelength selection units 152, 153, and 154 can detect a plurality of different wavelength ranges in a time-division manner using a similar method. In the following description, each detection time when different wavelength ranges are detected in a time-division manner may be referred to as a pass.

[0051] 7 is a schematic diagram of the light source 110. The light source 110 has four laser light sources 111, 112, 113, and 114 that emit light of different wavelengths. For example, the wavelength of the laser light emitted from the laser light source 111 is 405 nm, the wavelength of the laser light emitted from the laser light source 112 is 488 nm, the wavelength of the laser light emitted from the laser light source 113 is 561 nm, and the wavelength of the laser light emitted from the laser light source 114 is 640 nm.

[0052] Mirror 115 reflects the laser light emitted from laser light source 114. Dichroic mirror 116 transmits the laser light reflected by mirror 115 and reflects the laser light emitted from laser light source 113. Dichroic mirror 117 transmits the laser light transmitted through and reflected by dichroic mirror 116 and reflects the laser light emitted from laser light source 112. Dichroic mirror 118 reflects the laser light transmitted through and reflected by dichroic mirror 117 and transmits the laser light emitted from laser light source 111.

[0053] The laser light emitted from the light source 110 is incident on the dichroic mirror 121 shown in Fig. 1. The dichroic mirror 121 may be switched according to the number of excitation lights simultaneously incident on the specimen surface and the setting change of the fluorescence acquisition wavelength range desired by the user, and several types are prepared and arranged on a wheel.

[0054] 8 is an example of a flowchart showing an observation procedure of the microscope 101. First, a user designates an area of ​​the specimen 210 on which fluorescence observation is to be performed by the microscope 101 (step S10).

[0055] Next, the user specifies a desired acquisition wavelength range, and the range is created and edited as an imaging channel (S12). The imaging channel will be described later.

[0056] A pair of corresponding LVFs is set based on the imaging channels (S14), and a combination of the imaging channel and the real channel is set (S16). After that, the laser intensity is set for each imaging channel (S18), and the sensitivity of the detector is set (S20).

[0057] After the setting is completed, the process waits until the screen capture button is pressed (S22: No). If the screen capture button is pressed (S22: Yes), an image is captured by internal control (S24), and the operation ends.

[0058] FIG. 9 is a flow chart showing details of step S12 for creating and editing an imaging channel, and FIG. 10 shows an example of a setting screen 300 in that case.

[0059] First, a setting screen 300 is displayed on the display unit 172 and accepts input from the user. An input field 301 is a field for selecting a fluorescent dye, and input fields 302 and 303 are fields for inputting wavelengths on the short wavelength side and long wavelength side, respectively, of the acquisition wavelength range.

[0060] When the tab in the input field 301 is selected, it is understood that the emission spectrum information is to be selected from a luminescent dye (S100: Yes), and a list of selectable fluorescent dyes is displayed in the input field 301. The selectable fluorescent dyes are stored in advance in the memory of the control unit 171 together with the corresponding acquisition wavelength ranges. A luminescent dye is selected by the user from the list (S102).

[0061] On the other hand, if the tab in the input field 301 is not selected, it is understood that the emission spectrum information is not to be selected from the luminescent dye (S100: No), and the acquisition wavelength range, i.e., the wavelengths on the long and short wavelength sides of the emission light, are accepted (S104) by the user inputting numerical values ​​into the input fields 302 and 303. After step S102 or S104, the imaging channel name is accepted in the input field 304 (S106).

[0062] As described above, an imaging channel is created and edited. At this point, the imaging channel is not linked to the actual optical system of the microscope 101, or more specifically, to a real channel, and can be considered a temporary channel. In the example of FIG. 10, no fluorescent dye is specified, and the acquisition wavelength range "400 nm to 450 nm" is set as an imaging channel specified by the name "IM_Ch1".

[0063] Fig. 11 shows an example of a setting screen 310 in step S14 for setting a pair of LVFs for the imaging channels. In the example of Fig. 11, for two imaging channels "IM_Ch1" and "IM_Ch2", the corresponding emission spectra 341, 345, excitation light 340, 343, and detectable ranges 342, 346 are shown diagrammatically against the wavelength on the horizontal axis. Furthermore, in the imaging channel "IM_Ch2", a detection-prohibited range 347 is displayed around the excitation light 343 in response to the fact that the excitation light 343 is within the detectable range 346.

[0064] The profiles of the emission spectra 341, 345 and the wavelengths of the excitation light 340, 343 are stored in advance together with the corresponding fluorescent dyes in the memory of the control unit 171. When the wavelengths in the acquisition wavelength range in the imaging channel are set as numerical values, the emission spectrum may be displayed on the setting screen 310 as a rectangle with the relevant range as the maximum value and the other ranges as the minimum value (typically zero).

[0065] Detectable ranges 342, 346 are displayed by default in correspondence with emission spectra 341, 345. For example, detectable ranges 342, 346 are initially set to be the half-widths of emission spectra 341, 345. Furthermore, the dashed lines on the left and right of detectable ranges 342, 346 can be moved from the default display positions by the user by dragging and dropping the mouse pointer, etc.

[0066] Here, the left dashed line corresponds to the boundary wavelength of the LVF of the longpass filter, and the right dashed line corresponds to the boundary wavelength of the LVF of the shortpass filter. Therefore, by pressing the OK button 305 after the user has set, for the imaging channel "IM_Ch1", the wavelength at the position of the left dashed line of the detectable range 342 at that time is set as the boundary wavelength of the LVF of the longpass filter, and the wavelength at the position of the right dashed line is set as the boundary wavelength of the LVF of the shortpass filter.

[0067] Furthermore, the boundary wavelength of the LVF functioning as a dichroic mirror is automatically set under a predetermined condition with respect to the boundary wavelength of the LVF of the short-pass filter, which determines the long wavelength side of the detectable range. For example, the boundary wavelength of the LVF functioning as a dichroic mirror is set to a wavelength 10 nm longer than the boundary wavelength of the LVF of the short-pass filter (the dashed line on the right side of the detectable range 342 in the figure).

[0068] The same setting is made for the imaging channel "IM_Ch2." By pressing the cancel button 306, the screen returns to the setting screen 300 in FIG. 10 for setting the imaging channel.

[0069] Fig. 12 is a flowchart showing details of step S16 for setting combinations of imaging channels and real channels, and Fig. 13 shows an example of a setting screen 320 in that case. Fig. 13 shows an example in which four imaging channels have been created, and correspondingly, the four imaging channel names "IM_Ch1" and the like are displayed in display field 352.

[0070] The display field 350 displays lasers that can be used with the microscope 101. In the example of Fig. 7, four laser light sources 111, 112, 113, and 114 are available, and correspondingly, the display field 350 of Fig. 13 shows the four laser light sources in a schematic manner together with their respective wavelengths, "405 nm," "488 nm," "561 nm," and "640 nm."

[0071] Detectors that can be used with the microscope 101 are displayed in the display field 356. In the example of Fig. 2, four detectors 161, 162, 163, and 164 are available, and correspondingly, four detectors are shown diagrammatically in the display field 356 of Fig. 13. In this case, the type of detector, for example, "PMT" indicating that it is a photomultiplier tube, may be displayed.

[0072] The linking of the laser light source and the imaging channel is accepted (S120). In this case, when any of the laser light sources in the display field 350 is dragged, a line is displayed from the laser light source to the dragged position, and when the laser light source is dropped on any of the imaging channels in the display field 352, a line is drawn between the laser light source and the imaging channel in the setting screen 320, indicating that they are linked.

[0073] In this case, the laser light source and the imaging channel can be linked as long as the laser light source has a shorter wavelength than the minimum wavelength value of the acquisition wavelength range when the imaging channel is set, and may be linked one-to-one, one-to-N, or N-to-1 (N is an integer of 2 or more). In the example of Fig. 13, the laser light source and the imaging channel are linked one-to-one. Note that the imaging channel may be selected first, and then the laser light source to be linked thereto may be selected later.

[0074] The association between the imaging channel and the detector is accepted (S122). In this case, the method of associating the imaging channel and the detector may be the same as the method of associating the laser light source and the imaging channel.

[0075] In this case, the imaging channels and the detectors may be associated with each other on a 1:1 basis, or may be associated with each other on a 1:N basis or an N:1 basis. In the example of Fig. 13, the imaging channels and the detectors are associated with each other on a 2:1 basis.

[0076] Here, if the imaging channel and the detector are associated with each other in a manner other than one-to-one, it may not be possible to simultaneously detect the fluorescence from the specimen 210 in time because of the control time required to drive the LVF, switch the filter cube with the filter wheel, and position the optical elements. In addition, in a situation where a dichroic mirror (LVF or a dichroic mirror with a built-in filter cube) is placed in the upstream optical path (optical path until it reaches the detector) of the associated detector, if the dichroic mirror has long-pass characteristics, the range detectable by the associated detector will be longer than the boundary wavelength of the dichroic mirror.

[0077] As an illustrative example, in the case of FIG. 2, in the case of detector 164 to be linked, the dichroic mirrors (LVF or dichroic mirror with built-in filter cube) are 270, 260, and 250 in the upstream optical path (optical path until reaching the detector).

[0078] Unless the imaging channel's acquisition wavelength range is longer than the boundary wavelength of the dichroic mirror (there may be multiple) placed in the upstream optical path of the detector to be linked, the imaging channel will not be linked to the detector to be linked (driving the LVF, switching the filter cube) to change the characteristics of the dichroic mirror. If there is a detector among the multiple detectors that does not receive fluorescence, the boundary wavelength is automatically moved to the shortest wavelength side in the case of a dichroic mirror by an LVF, or the wheel is rotated to a position where there is no filter cube in the case of a dichroic mirror built into a filter cube, so that the fluorescence incident on the nearest dichroic mirror in the upstream optical path of the detector is not dispersed (partially transmitted, the rest reflected) but is completely transmitted. If the imaging channel cannot be linked, an error is notified to the user.

[0079] Therefore, paths are set up to detect them in a time-division manner. In the example in Figure 13, two imaging channels, "IM_Ch1" and "IM_Ch2", are linked to one detector, "D_162". Therefore, two paths are set up, and in "Path 1", which is earlier in time, the fluorescence in imaging channel "IM_Ch1" is detected by detector "D_162", and in "Path 2", which is later in time, the fluorescence in imaging channel "IM_Ch2" is detected by detector "D_162".

[0080] In this way, the combination of the imaging channel and the real channel of the microscope 101 is set.

[0081] 14 is an example of a setting screen 330 for setting the laser intensity in step S18 and the detector sensitivity in step S20. In display fields 331, 332, and 333, the imaging channel and detection wavelength range that have been set up to that point are displayed.

[0082] The setting screen 330 further has a slider 334 for setting the detector sensitivity (e.g., applied voltage) corresponding to the imaging channel and a slider 335 for setting the intensity of the excitation light. The sensitivity and excitation light intensity for each imaging channel are set by dragging the sliders 334, 335 left and right using the input unit 174, etc. Furthermore, when the screen acquisition button 337 on the setting screen 330 is pressed, internal control for acquiring an image is performed based on step S22 in Fig. 8 (S24).

[0083] 15 is a flowchart showing details of step S24, which is an internal control for acquiring an image. First, in accordance with the imaging channel corresponding to the first pass, the optical system, such as the z-direction positions of LVFs 250, 260, 264, 266, etc., is set based on the combination with the real channel.

[0084] The control device 180 starts outputting excitation light from a designated laser light source 111, etc., of the light source 110 (S140), and drives the galvanometer mirrors 131, 132 so that the designated observation region is irradiated with the excitation light (S142, S144). Here, the laser light source 111, etc., is designated based on the imaging channel of the fluorescence to be detected. As a result, the detection unit 160 detects the fluorescence included in the detectable range corresponding to the imaging channel (S144).

[0085] Thus, when the intensity of the fluorescence generated from the focusing position is detected by the series of operations in steps S144 and S146, the control device 180 stores the detected intensity and then determines whether or not detection of the specified number of pixels on one scanning line in the originally specified observation area has been completed (S148).

[0086] If the number of detected pixels has not reached the designated number of pixels (S148: NO), the control device 180 increments the number of detections in the X coordinate (S150) and returns control to step S144. As a result, the microscope 101 drives the galvanometer mirror 131 again to move the focusing position of the excitation light to the position of another pixel on the same line (S144), and again detects the light intensity of the fluorescence (S146).

[0087] The operations of steps S144 and S146 are repeated, and when the number of pixels where the light intensity of the fluorescence has been detected reaches the designated number of pixels (S148: YES), the control device 180 judges whether or not the detection of the number of scanning lines in the observation area initially designated has been completed (S152). When the number of detected scanning lines has not reached the designated number of scanning lines (S152: NO), the control device 180 increments the number of detections in the Y coordinate (S154) and returns control to step S142. Then, the control device 180 drives the control unit 133 of the galvano mirror 130 to move the focusing position of the excitation light onto another scanning line, and causes the light intensity of the fluorescence to be detected again.

[0088] When the number of scanning lines where the light intensity of the fluorescence has been detected reaches the scanning line of the specified observation area (S152: YES), the control device 180 constructs an observation image of the observation area based on the value of the detected light intensity of the fluorescence (S156). The constructed observation image may be displayed in the display field 336 of the setting screen 330 of the display unit 172, or may be stored in a storage unit (not shown) provided in the information processing device 170.

[0089] After step S156, the control device 180 judges whether or not the detection of the designated number of paths has been completed (S158). If the number of detected paths has not reached the designated number of paths (S158: NO), the control device 180 increments the number of paths (S160), sets the optical system by driving the LVF 250 or the like based on the combination with the real channel in accordance with the imaging channel corresponding to the next path (S162), and returns control to step S140. Then, the control device 180 outputs excitation light from the laser light source 111 or the like designated by the imaging channel, and detects the light intensity of the fluorescence again.

[0090] When the detected number of passes reaches the designated number of passes (S162: YES), the control device 180 stops the output of the laser light source (S174), thereby ending the observation operation in the microscope 101.

[0091] As described above, according to the microscope 101 of this embodiment, multiband detection, i.e., simultaneous multicolor detection, is possible using a four-stage wavelength selection unit, i.e., four real channels. Furthermore, in at least one real channel, for example, a pair of LVFs 254 and 256 are used as bandpass filters, so that the transmitted wavelength band can be easily adjusted by changing the z-direction positions of the LVFs 254 and 256. This allows multipass detection, i.e., time-division multicolor detection, to be possible with one real channel.

[0092] In a real channel equipped with a pair of LVFs 254, 256, the width of the detection wavelength range detected at one time is narrowed and fluorescence images are similarly acquired continuously, thereby making it possible to obtain the fluorescence spectral distribution of the specimen 210.

[0093] Fig. 16 is a flow diagram for acquiring a fluorescence spectral distribution using the microscope 101, and Fig. 17 shows a setting screen 450 corresponding to Fig. 16. In Fig. 16 and Fig. 17, the same configurations and operations as those in Fig. 1 to Fig. 15 are given the same reference numerals and descriptions thereof will be omitted.

[0094] This observation pattern is a case where fluorescence is acquired multiple times by driving a pair of LVFs 254, 256 using the wavelength selection unit 151. Here, the wavelength range detected at one time (in one time) is 20 nm, and this is repeated 14 times to acquire a fluorescence spectral profile from 430 nm to 710 nm.

[0095] 17 is displayed on the display unit 172 when creating / editing an imaging channel in step S12 of Fig. 16. In the input field 411, the wavelength value of the light (excitation light) emitted by the light source 110 can be set, and in the illustrated example, 405 nm is specified. Input to the input field 411 can be made using the input units 173, 174 of the information processing device 170.

[0096] The input fields 412 and 414 are areas for inputting when setting, with numerical values, the acquisition wavelength range of the fluorescence spectrum emitted from the specimen 210. Input into the input fields 412 and 414 can be made using the input units 173 and 174 of the information processing device 170. The input field 412 is an area for inputting the short wavelength end of the acquisition wavelength range, and the input field 414 is an area for inputting the long wavelength end. In the illustrated example, the acquisition wavelength range is set to wavelengths from 430 nm to 710 nm.

[0097] The input field 451 sets the detection wavelength range for detecting the fluorescence spectrum at one time (in one go) in the acquisition wavelength range set in the input fields 412 and 414. The detection wavelength range here corresponds to the wavelength resolution of the fluorescence spectrum profile. In the illustrated example, it is set to detect the fluorescence intensity at 20 nm intervals. Therefore, in this example, detection of 14 bands is specified for the wavelength band from 430 nm to 710 nm. Instead of setting the detection wavelength range, the number of bands to be detected in the acquisition wavelength range set in the input fields 412 and 414 may be input. In this case, the wavelength resolution is automatically set based on the number of bands input.

[0098] The input field 413 graphically displays the acquisition wavelength range and wavelength resolution set in the input fields 451, 412, and 414, and also allows the user to input the wavelength range to be acquired by directly operating the bar displayed in the input field 413. The numerical values ​​in the input fields 412 and 414 and the bar displayed in the input field 413 are linked.

[0099] Fig. 18 is a schematic diagram for explaining the acquisition wavelength range and detection wavelength range under the observation conditions set on the setting screen 450 shown in Fig. 17. In acquiring a fluorescence spectrum in this case, excitation light with a wavelength of 405 nm specified in the input field 411 is irradiated onto the specimen 210. Furthermore, in the acquisition wavelength range of wavelengths from 430 nm to 710 nm specified in the input fields 412 and 414, fluorescence images are acquired 14 times at intervals of 20 nm with the wavelength resolution (detection wavelength range) specified in the input field 451.

[0100] In this case, in each detection wavelength range, the short wavelength side is cut (blocked) by LVF 254, and the long wavelength side is cut (blocked) by LVF 256. In other words, the control device 180 cuts (blocks) the wavelength band lower than the lower limit of each detection wavelength range by LVF 254, and cuts (blocks) the band higher than the upper limit of the same range by LVF 256. Furthermore, when a fluorescence image is acquired in one detection wavelength range, the control device 180 operates the driving units 402 of the LVFs 254 and 256, respectively, to shift the detection wavelength range to a band adjacent to the detection wavelength range in which the fluorescence intensity has already been detected (S222, S162 in FIG. 16).

[0101] FIG. 19 is a diagram illustrating a fluorescence spectral profile at a certain position of the specimen 210 detected by the microscope 101. The microscope 101 repeats the above-mentioned operation to detect the fluorescence intensity for each detection wavelength range for a plurality of detection wavelength ranges. Thus, by plotting the fluorescence intensity of the detected image on a graph with the horizontal axis being wavelength λ and the vertical axis being intensity, a spectral profile at each detection position of the specimen 210 can be generated as shown in FIG. 19. The spectral profile is displayed, for example, in area 420. A two-dimensional image obtained by adding up the fluorescence intensities (emission intensities) detected for each detection wavelength range may be further displayed in area 419 of the setting screen 450.

[0102] Fig. 20 is an example showing a display image 460 displaying an image acquired in the embodiment of Fig. 17 to Fig. 19. The observation image 448 is an observation image constructed in a predetermined detection wavelength range. The display image 460 has a plurality of image display fields 443 that display a plurality of observation images 448 constructed in different detection wavelength ranges in a tiled manner. This allows the user to view the observation images 448 in each detection wavelength range at once.

[0103] Furthermore, an example of acquiring multiple fluorescence spectral distributions using the microscope 101 will be described. This observation pattern is a case where the pair of LVFs 254, 256 and the pair of LVFs 264, 266 are driven using the wavelength selection units 151 and 152 to acquire fluorescence multiple times. The wavelength selection unit 151 detects a wavelength range of 20 nm at one time (in one time) and acquires a fluorescence spectral profile from 430 nm to 610 nm by repeating the fluorescence acquisition nine times, and the wavelength selection unit 152 detects a wavelength range of 10 nm at one time (in one time) and acquires a fluorescence spectral profile from 650 nm to 710 nm by repeating the fluorescence acquisition six times.

[0104] When creating / editing an imaging channel in step S12 of FIG. 16, a setting screen 450 of FIG. 17 is displayed on the display unit 172 for each imaging channel. In the input field 411 of the imaging channel "IM_Ch1", 405 nm is set, and in the input fields 412 and 414, an acquisition wavelength range of wavelengths from 430 nm to 610 nm is set. In the input field 451, it is set that the fluorescence intensity is detected at 20 nm intervals. Therefore, in the imaging channel "IM_Ch1", detection of nine bands is specified for the wavelength band from 430 nm to 610 nm. In addition, in the input field 411 of the imaging channel "IM_Ch2", 640 nm is set, and in the input fields 412 and 414, an acquisition wavelength range of wavelengths from 650 nm to 710 nm is set. In the input field 451, it is set that the fluorescence intensity is detected at 10 nm intervals. Therefore, in the imaging channel "IM_Ch2", detection of six bands is specified for the wavelength band from 650 nm to 710 nm.

[0105] When setting a combination of imaging channels and real channels in step S16 in Fig. 16, the setting screen 320 in Fig. 13 is displayed on the display unit 172. In this setting screen 320, the laser light source in display field 350, the image channel in display field 352, and the detector in display field 356 are linked. The imaging channel "IM_Ch1" is linked to the laser light source "405 nm" and the detector "D_161," and the imaging channel "IM_Ch2" is linked to the laser light source "640 nm" and the detector "D_162."

[0106] FIG. 21 is a schematic diagram for explaining the acquisition wavelength range and the detection wavelength range under the observation conditions set in the setting screen 450 shown in FIG. 17 for each imaging channel. In this case, in acquiring the fluorescence spectrum, the specimen 210 is irradiated with excitation light having a wavelength of 405 nm specified in the input field 411 of the imaging channel "IM_Ch1". In addition, in the acquisition wavelength range of wavelengths from 430 nm to 610 nm specified in the input fields 412 and 414, a fluorescence image is acquired nine times at intervals of 20 nm with the wavelength resolution (detection wavelength range) specified in the input field 451. In the acquisition wavelength range of wavelengths from 650 nm to 710 nm specified in the input fields 412 and 414, a fluorescence image is acquired six times at intervals of 10 nm with the wavelength resolution (detection wavelength range) specified in the input field 451.

[0107] 22 is a timing chart for executing an example of acquiring a plurality of fluorescence spectral distributions using the microscope 101. In period T1, the laser light source 111 irradiates the specimen 210 with excitation light having a wavelength of 405 nm, and in period T2, the detector "D_161" detects the fluorescence generated from the specimen 210. In period T3, the pair of LVFs 254 and 256 each drive the driving units 402 to shift the detection wavelength range to a band adjacent to the detection wavelength range in which the fluorescence intensity has already been detected.

[0108] In period T4, the laser light source 114 irradiates the specimen 210 with excitation light having a wavelength of 640 nm, and in period T5, the detector "D_162" detects the fluorescence generated from the specimen 210. In period T6, the pair of LVFs 264, 266 each drive the driving unit 402 to shift the detection wavelength range to a band adjacent to the detection wavelength range in which the fluorescence intensity has already been detected.

[0109] The start times of periods T4 and T5 are the end times of periods T1 and T2, and the start times of periods T1 and T2 are the end times of periods T4 and T5. The start time of period T3 is the end time of periods T1 and T2, and the start time of period T6 is the end time of periods T4 and T5. The start time of period T3 is the start time of periods T4 and T5, and the start time of period T6 is the start time of periods T1 and T2.

[0110] That is, taking into consideration the time it takes for the LVFs to be driven and stabilized, the wavelength selection unit 151 is used to irradiate the specimen 210 with excitation light of a wavelength of 405 nm, and the drive units 402 of the LVFs 264 and 266 of the wavelength selection unit 152 are driven during the period in which the fluorescence is detected by the detector "D_161", and the wavelength selection unit 152 is used to irradiate the specimen 210 with excitation light of a wavelength of 640 nm, and the drive units 402 of the LVFs 254 and 256 of the wavelength selection unit 151 are driven during the period in which the fluorescence is detected by the detector "D_162". This allows for efficient fluorescence detection.

[0111] Fig. 23 is a schematic diagram of another example of a rear stage observation system 142. In the rear stage observation system 142, the same components as those in the rear stage observation system 140 of Fig. 2 are given the same reference numerals and descriptions thereof will be omitted.

[0112] The wavelength selection units 155, 156, 157, and 158 of the observation system rear stage 142 have condenser lenses 259, 269, 279, and 289 in addition to the configuration of the wavelength selection units 151, 152, 153, and 154 of the observation system rear stage 140. The wavelength selection unit 155 will be described as a representative, and the other wavelength selection units 152, 153, and 154 will not be described because they have the same configuration.

[0113] The condenser lens 259 condenses the incident light, and is disposed so that the focal position of the condenser lens 259 coincides with the focal position of the concave mirror 252. As a result, the concave mirror 252 reflects the incident light to make it a parallel beam. Therefore, since parallel beams are incident on each of the pair of LVFs 254 and 256, the effective film thicknesses of the LVFs 254 and 256 are the same for all light rays in the beam, and the decrease in spectral resolution can be suppressed. Furthermore, if the focal length of the condenser lens 259 is made smaller than the focal length of the concave mirror 252, the spot diameter when incident on each of the pair of LVFs 254 and 256 can be made smaller, and the decrease in spectral resolution can be further suppressed.

[0114] Fig. 24 is a schematic diagram of yet another example of an observation system rear stage 144. In the observation system rear stage 144, the same components as those in the observation system rear stage 140 in Fig. 2 are given the same reference numerals and descriptions thereof will be omitted.

[0115] The observation system rear stage 144 has a wavelength selection unit 145 in a stage preceding the wavelength selection unit 151. The wavelength selection unit 145 has a dichroic mirror 230, bandpass filters 232 and 234, and a condenser lens 236.

[0116] The dichroic mirror 230 and the bandpass filters 232, 234 are attached to, for example, a filter cube, which as a unit reflects a predetermined wavelength band and transmits other predetermined wavelength regions. Furthermore, the filter cube may be mounted on a filter wheel together with other filter cubes with different wavelength bands of reflection and transmission, any one of which may be inserted into the optical path.

[0117] The light reflected by the dichroic mirror 230 and transmitted through the bandpass filter 234 is collected by the collecting lens 236 and detected by the detector 161. On the other hand, the light transmitted through the dichroic mirror 230 and the bandpass filter 232 is incident on the downstream wavelength selection unit 151, etc., and is detected as necessary.

[0118] When light passes through the dichroic mirror 230, the principal ray shifts in the y direction by an amount corresponding to the plate thickness of the dichroic mirror 230. However, the LVF 250 and the like that function as dichroic mirrors in the downstream wavelength selection unit 151 and the like are oriented so that the wavelength characteristics, i.e., the boundary wavelength, do not change in the y direction. Therefore, the reflection and transmission characteristics of the wavelength selection unit 151 and the like are hardly affected by the shift in the y direction.

[0119] The observation system rear stage 144 further includes a wavelength selection unit 146 located rearward of the wavelength selection unit 152. The wavelength selection unit 146 includes a dichroic mirror 290, bandpass filters 292 and 294, and a condenser lens 296. The wavelength selection unit 146 has a similar configuration to the wavelength selection unit 145, except that the wavelengths selected may be different, and therefore a description thereof will be omitted.

[0120] Fig. 25 is a schematic diagram showing a modified example of the observation system rear stage 140. In the observation system rear stage 140 of Fig. 25, the LVF 256 is arranged at a slight inclination, for example, about 1 degree, in the xy plane with respect to the principal ray. This makes it possible to avoid a problem in which light reflected without passing through the LVF 256 is incident on a different z position of the LVF 256 while being multiple-reflected between the pair of LVFs 254, 256, causing light of an undesired band to reach the detector 161.

[0121] The LVF 254 may be tilted in the xy plane instead of or in addition to tilting the LVF 256. Also, the other wavelength selection units 152 and the like may be similarly arranged.

[0122] In any of the above embodiments, multiband detection, i.e., simultaneous multicolor detection, is possible using multistage wavelength selection units 151, etc. Furthermore, in at least one wavelength selection unit 151, a pair of LVFs 254, 256, etc. are used as bandpass filters, so that the transmitted wavelength band can be easily changed by changing the z-direction positions of the LVFs 254, 256. This allows multipass detection, i.e., time-division multicolor detection, to be possible with one wavelength selection unit 151.

[0123] In any of the embodiments, the wavelength selection units 151, etc., are four stages. However, the number of stages is not limited to this and may be two or more stages, which enables multiband detection, that is, simultaneous multicolor detection.

[0124] In any of the above embodiments, the LVF 254 etc. of the pair of LVFs 254, 256 etc. is a long-pass filter, and the LVF 256 etc. is a short-pass filter. Alternatively, the LVF 254 etc. may be a short-pass filter, and the LVF 256 etc. may be a long-pass filter.

[0125] In addition, in any of the above embodiments, the wavelengths selected are longer as the wavelength selection unit 152 is located at the rear. Alternatively, the wavelengths selected may be shorter as the wavelength selection unit 152 is located at the rear. Furthermore, each wavelength selection unit may be, so to speak, unitized by storing the components constituting the unit, such as an LVF, in a housing or by attaching the components to a base member. In this case, each wavelength selection unit may be insertable into and detachable from the microscope 101.

[0126] Although the present invention has been described above using the embodiments, the technical scope of the present invention is not limited to the scope described in the above embodiments. It is clear to those skilled in the art that various modifications and improvements can be made to the above embodiments. It is clear from the description of the claims that such modifications and improvements can also be included in the technical scope of the present invention.

[0127] It should be noted that the order of execution of each process, such as operations, procedures, steps, and stages, in the devices, systems, programs, and methods shown in the claims, specifications, and drawings is not specifically stated as "before," "prior to," etc., and may be realized in any order unless the output of a previous process is used in a later process. Even if the operational flow in the claims, specifications, and drawings is explained using "first," "next," etc. for convenience, it does not mean that it is essential to perform the process in this order. [Explanation of symbols]

[0128] 101 microscope, 110 light source, 111, 112, 113, 114 laser light source, 115, 123 mirror, 116, 117, 118, 121, 230, 290 dichroic mirror, 122 relay lens, 124 condenser lens, 126 collimator lens, 125 pinhole, 130 galvanometer scanner, 131, 132 galvanometer mirror, 140, 142, 144 observation system rear stage, 146, 151, 152, 153, 154, 155, 156, 157, 158 wavelength selection unit, 160 detection unit, 161, 162, 163, 164 detector, 170 information processing device, 171 control unit, 172 display unit, 173, 174 input unit, 180 control device, 191 Objective lens, 192 Lens, 210 Specimen, 220 Illumination optical system, 230 Dichroic mirror, 232, 234, 292, 294 Bandpass filter, 236, 258, 259, 268, 269, 278, 279, 288, 289, 296 Condenser lens, 240 Observation optical system, 250, 254, 256, 260, 264, 266, 270, 274, 276, 284, 286 Level filter, 252, 262, 272, 282 Concave mirror, 300, 310, 330 Setting screen, 301, 302, 303, 304 Input field, 305 OK button, 306 Cancel button, 334, 335 slider, 331, 332, 333, 336, 350, 352 display column, 337 screen capture button, 340, 343 excitation light, 341, 345 emission spectrum, 342, 346 detectable range, 347 non-detectable range, 400 optical filter, 402 drive unit

Claims

1. A microscope, an illumination optical system that irradiates the specimen with excitation light; A detector for detecting fluorescence emitted from the specimen; an observation optical system that guides the fluorescent light to the detector; The observation optical system includes: a first optical filter whose wavelength characteristics of reflection and transmission are variable depending on the position at which light is incident; a second optical filter that is disposed in an optical path of the light reflected by the first optical filter, the second optical filter having a boundary wavelength of transmission that changes with respect to a position along a first direction, and that transmits light having a wavelength longer than the first boundary wavelength at a position where the reflected light is incident; a third optical filter that is disposed in an optical path of the light reflected by the first optical filter, the third optical filter having a boundary wavelength of transmission that changes with respect to a position along the first direction and that transmits light having a wavelength shorter than the second boundary wavelength at a position where the reflected light is incident; Including, A microscope, wherein the first boundary wavelength is shorter than the second boundary wavelength.

2. The microscope according to claim 1 , wherein the wavelength characteristics of the first optical filter vary depending on a position along the first direction.

3. 3. The microscope according to claim 2, wherein the first optical filter is disposed at an angle of less than 45 degrees to the incident light within a plane intersecting the first direction.

4. The microscope according to claim 1 , wherein the second optical filter and the third optical filter are movable along the first direction.

5. The microscope according to claim 1 , wherein the first optical filter is movable along the first direction.

6. The microscope according to claim 1 , wherein the first direction is a direction of gravity.

7. 7. The microscope according to claim 1, wherein one of the second optical filter and the third optical filter is disposed at an angle to the other in a plane intersecting the first direction.

8. 8. The microscope according to claim 1, wherein the observation optical system further includes a concave mirror that focuses light reflected by the first optical filter between the second optical filter and the third optical filter.

9. 8. The microscope according to claim 1, wherein the observation optical system further includes a concave mirror that collimates the light reflected by the first optical filter and causes the light to be incident on the second optical filter and the third optical filter.

10. The observation optical system includes: a reflecting element that receives the light transmitted through the first optical filter and reflects at least a portion of the light; a fourth optical filter that is disposed in an optical path of the light reflected by the reflecting element, the fourth optical filter having a boundary wavelength that changes with respect to a position along the first direction, and that transmits light having a wavelength longer than a third boundary wavelength at a position where the reflected light is incident; a fifth optical filter that is disposed in an optical path of the light reflected by the reflecting element, the fifth optical filter having a boundary wavelength that changes with respect to a position along the first direction and that transmits light having a wavelength longer than a fourth boundary wavelength at a position where the reflected light is incident; Further comprising: The microscope according to claim 1 , wherein the third boundary wavelength is shorter than the fourth boundary wavelength.

11. 11. The microscope according to claim 10, wherein the reflecting element is a sixth optical filter whose reflecting and transmitting wavelength characteristics vary depending on the position at which light is incident, or a total reflection mirror.

12. the first optical filter, the second optical filter, and the third optical filter are housed within a first unit; the sixth optical filter or the reflective element, the fourth optical filter, and the fifth optical filter are housed in a second unit; The first unit and the second unit are configured to be insertable and detachable. A microscope according to claim 10 or 11.

13. a first detector that receives a portion of the light reflected by the first optical filter, the light passing through the second optical filter and a third optical filter; a second detector that receives a portion of the light transmitted through the first optical filter; Further equipped with The microscope according to claim 1 , wherein the light receiving surface of the first detector and the light receiving surface of the second detector face in the same direction.

Images