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 band-pass 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 include an illumination optical system that irradiates excitation light onto a specimen. It may include a detector that detects fluorescence emitted from the specimen. It may include an observation optical system that guides the fluorescence to the detector. The observation optical system may include a first optical filter whose reflection and transmission wavelength characteristics vary depending on the position at which light is incident. It may include 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 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. It may also include 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 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 its position along the first direction. The first optical filter may be tilted at an angle of 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 optical filter may be movable along the first direction. The first direction may be the direction of gravity. One of the second optical filter and the third optical filter may be tilted relative to the other in a plane intersecting the first direction. The observation optical system may further include a concave mirror that focuses 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 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.

[0005] The observation optical system may include a reflecting element that receives 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, the fourth optical filter having a boundary wavelength that changes with position along the first direction and that transmits light having a wavelength longer than a 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, the fifth optical filter having a boundary wavelength that changes with position along the first direction and that 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 reflective element may be a sixth optical filter whose reflection and transmission wavelength characteristics vary depending on the position of incidence of light, or a total reflection mirror. The first, second, and third optical filters may be housed in the first unit, and the sixth optical filter or reflective element, fourth, and fifth optical filters may be housed in the second unit. The first and second units may be configured to be detachable. The optical filter may include a first detector that receives a portion of the light reflected by the first optical filter and that has passed through the second and third optical filters, and a second detector that receives a portion of the light that has passed through the first optical filter. The light receiving surfaces of the first and second detectors may face the same direction.

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

[0008] [Figure 1] 1 is a schematic diagram showing the structure of a microscope 101 for observing a specimen 210. FIG. [Figure 2] An example of the observation system downstream 140 and the detection unit 160 is shown schematically. [Figure 3] FIG. 10 is a schematic diagram showing the function of the LVF 254 as an example. [Figure 4] 10 is a schematic diagram illustrating a wavelength range that is transmitted through the wavelength selection unit 151 and can be detected by the detector 161. FIG. [Figure 5] The detectable ranges of the four wavelength selection units 151, 152, 153, and 154 are shown schematically. [Figure 6] 10A and 10B show a schematic diagram of changing the detectable range in the wavelength selection unit 151. [Figure 7] The light source 110 is shown schematically. [Figure 8] 1 is an example of a flowchart showing an observation procedure of the microscope 101. [Figure 9]10 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] 10 shows an example of a setting screen 310 in step S14 for setting the LVF etc. for the imaging channel. [Figure 12] 10 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] 10 shows an example of a setting screen 330 for step S18 for setting the laser intensity and step S18 for setting the sensitivity of the detector. [Figure 15] 10 is a flowchart showing details of step S24, which is an internal control for acquiring an image. [Figure 16] 1 is a flow chart for acquiring a fluorescence spectral distribution using a microscope 101. [Figure 17] A corresponding setting screen 450 is shown in FIG. [Figure 18] 18 is a schematic diagram illustrating an acquisition wavelength range and a detection wavelength range under the observation conditions set on the setting screen 450 shown in FIG. 17. FIG. [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 illustrating 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 illustrating an example of acquiring a plurality of fluorescence spectral distributions using the microscope 101. [Figure 23] 10 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 schematically. [Figure 25] A modified example of the observation system rear stage 140 is shown schematically. DETAILED DESCRIPTION OF THE INVENTION

[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; for example, it may not have the light source 110, the information processing device 170, or the control device 180.

[0011] The light source 110 emits laser light of a wavelength that is used as excitation light when observing the fluorescence of the specimen 210. 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 the 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 then 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 a lens 192 and then focused onto the specimen 210 by an 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, thereby changing the focusing position of the excitation light on the specimen 210. Therefore, 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 enters 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 that enters the relay lens 122 passes through the galvano scanner 130 and enters 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 light source 110 side. Of the emitted light, a component having a wavelength different from that of 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 component 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, a post-observation system 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. Therefore, 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 corresponding to the intensity of the detected fluorescence to the information processing device 170. Details of the observation system post-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 also executes image processing to generate an image from a signal acquired 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, etc., and in addition to displaying the generated image to the user, it also displays the 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, etc. 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 galvanometer scanner 130, the observation system downstream 140, the detection unit 160, etc., and controls these operations. Furthermore, the control device 180 may execute all or part of the image processing, etc., in the information processing device 170 in order to reduce the load on the information processing device 170. In other words, 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 schematically shows 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 film thickness of which changes along a predetermined direction (z direction in the drawing), 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 whose film thickness varies along a predetermined direction (the z direction in the figure), and the wavelength characteristics of reflection and transmission change depending on 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 a driver 402 to change the wavelength characteristics of incident light from a fixed optical path. The LVF 254 and driver 402 form an optical filter 400 whose wavelength characteristics of transmission and reflection are dynamically variable.

[0029] The graph 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 represents wavelength and the horizontal axis represents transmittance. The LVF 254 having the wavelength characteristics shown in Fig. 2 is a long-pass filter that transmits light with wavelengths longer than the boundary wavelength and reflects light with wavelengths shorter than the boundary wavelength.

[0030] Returning to Figure 2, the LVF 250, into which light from the collimator lens 126 enters, functions as a reflective element whose reflection and transmission characteristics vary 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 of the incident light is split into two by the LVF 250 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 incident light.

[0032] The LVF 250 is tilted in the xy plane at an angle of less than 45 degrees, preferably 22.5 degrees or less, relative to the light incident from the collimating lens 126. That is, the light-receiving surface is rotated around the z-axis so that the angle of incidence of 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 chief 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 close to 1, thereby reducing the area of ​​the spot shape. More specifically, the spot shape incident on the incident surface of the LVF 250 becomes an ellipse elongated in the direction of tilting the incident surface. The minor axis of this ellipse remains the luminous flux diameter D, while the major axis is D / cosθ (θ is the incident angle). Therefore, the area of ​​the spot shape is smaller and the ellipticity is closer to 1 when θ is 22.5 degrees than when θ is 45 degrees. This suppresses a decrease in the spectral resolution of the incident spot on the LVF 250.

[0033] Furthermore, the incident surface of the LVF 250 is positioned rotated around the z-axis. The direction in which the boundary wavelength changes in the LVF 250 is the z-direction. This causes the major axis of the ellipse to expand in a direction perpendicular to the z-axis, i.e., in a direction in which the boundary wavelength does not change. This further reduces the degradation of 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 play in the mechanical system that drives the LVF 250 is pulled downward by its own weight, resulting in high position repeatability and high positioning accuracy due to the drive.

[0035] The concave mirror 252 is disposed in the optical path of the wavelength selection unit 151 between the LVF 250 and the pair of LVFs 254 and 256. That is, 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 beams incident on both of the pair of LVFs 254 and 256, thereby suppressing a decrease in spectral resolution at each of the LVFs 254 and 256. Furthermore, if the focal length of the collimating lens 126 is made shorter 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 a decrease in spectral resolution.

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

[0037] Like the LVF 250, the LVFs 254 and 256 are each movable in the z direction by a drive unit, allowing dynamic change of 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 have similar trends. That is, the LVFs 254 and 256 are preferably arranged so that the boundary wavelengths of both LVFs shift toward shorter wavelengths (or both LVFs shift toward longer wavelengths) as they move further 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 illustrating 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, LVF 250 reflects the short wavelength side, LVF 254 cuts (blocks) the short wavelength side, and LVF 256 cuts (blocks) the long wavelength side. Furthermore, the boundary wavelength of LVF 254 is shorter than the boundary wavelengths of LVF 250 and LVF 256. This allows a predetermined wavelength range to be transmitted, making it possible for detector 161 to detect light. Furthermore, by making LVF 250 function as a dichroic mirror, light transmitted through LVF 250 can be passed to a downstream stage such as wavelength selection unit 152, allowing other wavelength bands to be detected.

[0041] Wavelength selection units 152 and 153 are arranged downstream of 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 will be omitted.

[0042] A wavelength selection unit 154 is further arranged 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 considered a total reflection mirror because 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 configuration 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 schematically shows the detectable ranges of the four wavelength selection units 151, 152, 153, and 154. To simplify the drawing, the transmittance and reflectance of the LVFs 250, 260, and 270, which function as dichroic mirrors, are not shown.

[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 the pair of LVFs 254 and 256 of the wavelength selection unit 151, and detectable range 2 is set on the longer wavelength side by the pair of LVFs 264 and 266 of the wavelength selection unit 152. Similarly, detectable range 3 is set on the longer wavelength side of detectable range 2 by the pair of LVFs 274 and 276 of the wavelength selection unit 153, and detectable range 4 is set on the longer wavelength side by the pair of LVFs 284 and 286 of the wavelength selection unit 154.

[0046] 5, the observation system downstream 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 schematically shows how the detectable range is changed in the wavelength selection unit 151. To simplify the drawing, the transmittance and reflectance of the LVF 250, which functions as a dichroic mirror, are not shown.

[0048] Each of the LVFs 250, 254, and 256 included in the wavelength selection unit 151 is movable along the z direction, along 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 Figure 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 Figure 6. In other words, in the optical system of one wavelength selection unit 151, it is possible to detect a plurality of different wavelength ranges in a time-division manner.

[0049] In this case, if both LVF 254 and LVF 256 are moved in the same direction along the z axis by the same amount, the detectable range shifts to the shorter or longer wavelength side while maintaining the same bandwidth. On the other hand, if at least one of LVF 254 and LVF 256 is moved in the opposite direction along the z axis, the bandwidth of the detectable range becomes wider or narrower.

[0050] Note that a plurality of different wavelength ranges can be detected in a time-division manner in the other wavelength selection units 152, 153, and 154 in a similar manner. 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 schematically shows 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 that has passed through and been reflected by dichroic mirror 116 and reflects the laser light emitted from laser light source 112. Dichroic mirror 118 reflects the laser light that has passed through and been 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 can be switched depending on 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 the observation procedure of the microscope 101. First, the user designates an area of ​​the specimen 210 on which fluorescence observation is to be performed in the microscope 101 (step S10).

[0055] Next, the user specifies a desired acquisition wavelength range, which is then 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 detector sensitivity is set (S20).

[0057] After the setting is complete, the system waits until the screen capture button is pressed (S22: No). If the screen capture button is pressed (S22: Yes), the system captures an image through internal control (S24), and the operation ends.

[0058] FIG. 9 is a flowchart showing the 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 of the acquisition wavelength range, respectively.

[0060] When the tab in the input field 301 is selected, it is interpreted as selection of emission spectrum information 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 along with the corresponding acquisition wavelength ranges. The user is then allowed to select a luminescent dye from the list (S102).

[0061] On the other hand, if the tab in input field 301 is not selected, it is interpreted that the emission spectrum information is not 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, are accepted (S104) by the user inputting numerical values ​​into input fields 302 and 303. After step S102 or S104, the imaging channel name is accepted in input field 304 (S106).

[0062] An imaging channel is created and edited as described above. 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; it can be considered a temporary channel. In the example of Figure 10, no fluorescent dye is specified, and the acquisition wavelength range "400 nm to 450 nm" is set as the imaging channel specified by the name "IM_Ch1."

[0063] Figure 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 Figure 11, for two imaging channels "IM_Ch1" and "IM_Ch2," the corresponding emission spectra 341 and 345, excitation light beams 340 and 343, and detectable ranges 342 and 346 are schematically shown against the wavelength on the horizontal axis. Furthermore, for imaging channel "IM_Ch2," a non-detection range 347 is displayed around the excitation light beam 343 because the excitation light beam 343 falls within the detectable range 346.

[0064] The profiles of the emission spectra 341 and 345 and the wavelengths of the excitation light 340 and 343, together with the corresponding fluorescent dyes, are stored in advance 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 and 346 are displayed by default in correspondence with emission spectra 341 and 345. For example, detectable ranges 342 and 346 are initially set to be the half-widths of emission spectra 341 and 345. Furthermore, the dashed lines on the left and right of detectable ranges 342 and 346 can be moved by the user from the default display positions by dragging and dropping with the mouse pointer, for example.

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

[0067] Furthermore, the boundary wavelength of the LVF functioning as a dichroic mirror is automatically set under predetermined conditions relative 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 the 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, four imaging channel names such as "IM_Ch1" are displayed in display field 352.

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

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

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

[0073] In this case, a laser light source and an imaging channel can be linked as long as the laser light source has a wavelength shorter than the minimum wavelength value of the acquisition wavelength range when the imaging channel is set, and they 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 to it 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 one to one, or may be associated with one to N or N to 1. In the example of Fig. 13, the imaging channels and the detectors are associated with two to one.

[0076] Here, if the imaging channels and detectors are associated in a manner other than one-to-one, the control time required to drive the LVF, switch the filter cube using the filter wheel, and position the optical elements may make it impossible to simultaneously detect the fluorescence from the specimen 210. Furthermore, if a dichroic mirror (LVF or a dichroic mirror with a built-in filter cube) is located in the upstream optical path (the optical path up to the detector) of the associated detector, and if that 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] The imaging channel and the target detector will not be linked unless the dichroic mirror's characteristics are changed (by driving the LVF or switching the filter cube) until the acquisition wavelength range of the imaging channel becomes longer than the boundary wavelength of the dichroic mirror (there may be multiple) located in the upstream optical path of the detector to be linked. 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 with 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 incident fluorescence is not dispersed (partially transmitted, the rest reflected) by the nearest dichroic mirror in the upstream optical path of that detector and is completely transmitted. If linking is not possible, an error is notified to the user.

[0079] Therefore, paths are set up to detect these 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 the earlier "Path 1," the fluorescence in imaging channel "IM_Ch1" is detected by detector "D_162," and in the later "Path 2," 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 shows an example of a setting screen 330 for setting the laser intensity in step S18 and the detector sensitivity in step S20. Display fields 331, 332, and 333 display the imaging channel and detection wavelength range that have been set up to that point.

[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 capture 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 the details of step S24, which is the internal control for acquiring an image. First, for 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 or the like among the light sources 110 (S140), and drives the galvanometer mirrors 131 and 132 so that the excitation light is irradiated onto the designated observation region (S142, S144). Here, the laser light source 111 or the like is designated based on the imaging channel of the fluorescence to be detected. As a result, the detection unit 160 detects fluorescence that falls within the detectable range corresponding to that imaging channel (S144).

[0085] Thus, when the intensity of the fluorescence emitted 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 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 specified number of pixels (S148: NO), the control device 180 increments the number of detected pixels 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 determines whether or not detection of the number of scan lines in the initially designated observation area has been completed (S152). If the number of detected scan lines has not reached the designated number of scan 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 galvanometer mirror 130 to move the focusing position of the excitation light onto another scan line and again detect the light intensity of the fluorescence.

[0088] If the number of scanning lines where the fluorescence light intensity has been detected reaches the number of scanning lines of the specified observation area (S152: YES), the control device 180 constructs an observation image of the observation area based on the detected fluorescence light intensity values ​​(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 memory unit (not shown) provided in the information processing device 170.

[0089] After step S156, the control device 180 determines whether detection of the specified number of paths has been completed (S158). If the number of detected paths has not reached the specified 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 actual channel for 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 specified for that imaging channel, and again detects the light intensity of the fluorescence.

[0090] If 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, the microscope 101 of this embodiment enables multiband detection, i.e., simultaneous multicolor detection, 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 enables multipath detection, i.e., time-division multicolor detection, with one real channel.

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

[0093] Fig. 16 is a flow chart 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 denoted by the same reference numerals, and descriptions thereof will be omitted.

[0094] This observation pattern involves acquiring fluorescence multiple times by driving a pair of LVFs 254 and 256 using the wavelength selection unit 151. Here, the wavelength range detected at one time (in one pass) is set to 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 and 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 example shown, 405 nm is specified. Input into the input field 411 can be made using the input units 173 and 174 of the information processing device 170.

[0096] The input fields 412 and 414 are areas for inputting values ​​when setting 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] Input field 451 sets the detection wavelength range for detecting a fluorescence spectrum at one time (in one go) within 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 spectral profile. In the illustrated example, it is set to detect 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, it is also possible to input the number of bands to be detected within the acquisition wavelength range set in the input fields 412 and 414. 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 411, 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 illustrating the acquisition wavelength range and detection wavelength range under the observation conditions set on the setting screen 450 shown in Fig. 17. In this case, when acquiring a fluorescence spectrum, excitation light with a wavelength of 405 nm specified in input field 411 is irradiated onto the specimen 210. Furthermore, within the acquisition wavelength range of 430 nm to 710 nm specified in input fields 412 and 414, fluorescence images are acquired 14 times at 20 nm intervals with the wavelength resolution (detection wavelength range) specified in 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, control device 180 cuts (blocks) the wavelength band below the lower limit of each detection wavelength range by LVF 254, and cuts (blocks) the band above the upper limit of the same range by LVF 256. Furthermore, when a fluorescence image is acquired in one detection wavelength range, control device 180 operates drive units 402 of LVFs 254 and 256, respectively, to shift the detection wavelength range to a band adjacent to the detection wavelength range in which 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 on the specimen 210 detected by the microscope 101. By repeating the above-described operations, the microscope 101 detects the fluorescence intensity for each of a plurality of detection wavelength ranges. Therefore, by plotting the fluorescence intensity of the detected image on a graph with wavelength λ on the horizontal axis and intensity on the vertical axis, a spectral profile at each detection position on 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 also be displayed in area 419 on the setting screen 450.

[0102] 20 is an example of a display image 460 displaying an image acquired in the embodiments of FIGS. 17 to 19. The observation image 448 is an observation image constructed in a predetermined detection wavelength range. The display image 460 has multiple image display fields 443 that display multiple observation images 448 constructed in different detection wavelength ranges in a tiled manner. This allows the user to view the observation images 448 for each detection wavelength range at the same time.

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

[0104] When creating or 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. For imaging channel "IM_Ch1," 405 nm is set in input field 411, and an acquisition wavelength range of 430 nm to 610 nm is set in input fields 412 and 414. The input field 451 is set to detect fluorescence intensity at 20 nm intervals. Therefore, for imaging channel "IM_Ch1," detection of nine bands is specified for the wavelength band of 430 nm to 610 nm. For imaging channel "IM_Ch2," 640 nm is set in input field 411, and an acquisition wavelength range of 650 nm to 710 nm is set in input fields 412 and 414. The input field 451 is set to detect fluorescence intensity at 10 nm intervals. Therefore, for imaging channel "IM_Ch2," detection of six bands is specified for the wavelength band of 650 nm to 710 nm.

[0105] When setting a combination of imaging channels and real channels in step S16 of Fig. 16, the setting screen 320 of Fig. 13 is displayed on the display unit 172. On 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 illustrating the acquisition wavelength range and detection wavelength range under the observation conditions set for each imaging channel on the setting screen 450 shown in FIG. 17. In this case, fluorescence spectrum acquisition is performed by irradiating the specimen 210 with excitation light at a wavelength of 405 nm, specified in the input field 411 for the imaging channel "IM_Ch1." Furthermore, within the acquisition wavelength range of 430 nm to 610 nm, specified in the input fields 412 and 414, fluorescence images are acquired nine times at intervals of 20 nm, with the wavelength resolution (detection wavelength range) specified in the input field 451. Furthermore, within the acquisition wavelength range of 650 nm to 710 nm, specified in the input fields 412 and 414, fluorescence images are acquired six times at intervals of 10 nm.

[0107] 22 is a timing chart showing an example of acquiring multiple fluorescence spectral distributions using the microscope 101. During period T1, the laser light source 111 irradiates the specimen 210 with excitation light having a wavelength of 405 nm, and during period T2, the detector "D_161" detects the fluorescence emitted from the specimen 210. During 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] During period T4, the laser light source 114 irradiates the specimen 210 with excitation light having a wavelength of 640 nm, and during period T5, the detector "D_162" detects the fluorescence emitted from the specimen 210. During period T6, the pair of LVFs 264, 266 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.

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

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

[0111] 23 is a schematic diagram of another example of a rear observation system 142. In the rear observation system 142, the same components as those in the rear observation system 140 of FIG. 2 are designated by the same reference numerals, and a description 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 positioned 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 into a parallel beam. Therefore, because 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, thereby suppressing a decrease in spectral resolution. Furthermore, if the focal length of the condenser lens 259 is made shorter 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, further suppressing a decrease in spectral resolution.

[0114] 24 is a schematic diagram of yet another example of a post-stage observation system 144. In the post-stage observation system 144, the same components as those in the post-stage observation system 140 of 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 before 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 another predetermined wavelength range. Furthermore, the filter cube may be mounted on a filter wheel together with other filter cubes with different reflection and transmission wavelength bands, and any of them 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 condenser 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 wavelength selection unit 151 or the like in the subsequent stage and is detected as necessary.

[0118] When light passes through the dichroic mirror 230, the chief ray shifts in the y direction by an amount corresponding to the thickness of the dichroic mirror 230. However, the LVF 250 and the like that function as dichroic mirrors in the wavelength selection unit 151 and the like at the subsequent stage 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 downstream 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 the same configuration as the wavelength selection unit 145, except that the wavelengths selected may be different, and therefore a description thereof will be omitted.

[0120] 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 disposed at a slight inclination, for example, of about 1 degree, in the xy plane with respect to the chief ray. This makes it possible to avoid a problem in which light that is 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. The other wavelength selection units 152 and the like may also be similarly arranged.

[0122] As described above, in any of the 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., is used as a bandpass filter, so that the transmitted wavelength band can be easily changed by changing the z-direction positions of the LVFs 254, 256. This enables multipath detection, i.e., time-division multicolor detection, with one wavelength selection unit 151.

[0123] In any of the embodiments, the wavelength selection unit 151 etc. has four stages. However, the number of stages is not limited to this and may be two or more stages, which allows 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] Furthermore, in any of the above embodiments, the wavelengths selected by the wavelength selection units 152 at the later stages are longer wavelengths. Alternatively, the wavelengths selected by the wavelength selection units 152 at the later stages may be shorter wavelengths. Furthermore, each wavelength selection unit may be unitized, so to speak, by storing components such as an LVF constituting the unit 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 embodiments, the technical scope of the present invention is not limited to the scope described in the above embodiments. It will be apparent to those skilled in the art that various modifications and improvements can be made to the above embodiments. It is clear from the claims that such modifications and improvements can also be included within the technical scope of the present invention.

[0127] It should be noted that the execution order 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 that the processes can be performed in any order unless the output of a previous process is used in a subsequent process. Even if the operational flow in the claims, specifications, and drawings is described using "first," "next," etc. for convenience, this does not mean that the processes must be performed 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 excitation light onto the specimen; a detector for detecting fluorescence emitted from the specimen; an observation optical system that guides the fluorescence to the detector; Has, The observation optical system includes: a first optical filter whose wavelength characteristics for reflection and transmission vary 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 transmission boundary wavelength 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 transmission boundary wavelength 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, the first boundary wavelength is shorter than the second boundary wavelength, The first optical filter has an incident surface at a position rotated around the first direction, the wavelength characteristics vary depending on the position along the first direction, and is arranged in a plane intersecting the first direction at an angle of less than 45 degrees with respect to the incident light, and the beam diameter of the light incident on the incident surface in the first direction is maintained at the incident surface.

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

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

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

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

6. 6. 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.

7. 6. The microscope according to claim 1, wherein the observation optical system further includes a concave mirror that converts light reflected by the first optical filter into a parallel beam and causes the parallel beam to be incident on the second optical filter and the third optical filter.

8. The observation optical system includes: a reflecting element that receives the light that has passed 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 varies with 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 the fourth boundary wavelength at a position where the reflected light is incident; further comprising 8. The microscope according to claim 1, wherein the third boundary wavelength is shorter than the fourth boundary wavelength.

9. 9. The microscope according to claim 8, wherein the reflecting element is a sixth optical filter whose wavelength characteristics for reflection and transmission vary depending on the position at which light is incident, or a total reflection mirror.

10. the first optical filter, the second optical filter, and the third optical filter are housed in 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; 10. The microscope according to claim 9, wherein the first unit and the second unit are configured to be detachable.

11. 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 that has passed through the first optical filter; Furthermore, 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