FLUORESCENCE MICROSCOPE DEVICE, OBSERVATION CONDITION OPTIMIZATION METHOD, AND PROGRAM

Abstract

To provide an art that enables highly sensitive optical detection over a whole area of a wavelength region of fluorochrome to observe, even if an optical detector is not provided that is highest in wavelength sensitivity over the whole area of the wavelength region of the fluorochrome.SOLUTION: A fluorescence microscope device comprises: a plurality of optical detectors that is different in a wavelength sensitivity characteristic; a detection wavelength changing unit that can change a detection wavelength of each optical detector; an input device that inputs fluorochrome or wavelength range serving as an observation object; a memory that stores first information on the wavelength sensitivity characteristic of each optical detector; and an observation condition optimization unit that determines and selects an optimal single optical detector or a plurality of optimal optical detectors to be used in observation of the observation object from the plurality of optical detectors on the basis of the fluorochrome or wavelength range serving as the observation object input by the input device, and the first information stored in the memory, and optimizes the detection wavelength change unit for the observation of the observation object. When using the plurality of optical detectors in the observation of the observation object, the fluorescence microscope device is configured to aggregate an intensity signal of fluorescence detected by each optical detector.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The disclosure of the present specification relates to a fluorescence microscope apparatus, an observation condition optimization method, and a program. [Background technology]

[0002] Conventionally, among fluorescence microscope devices equipped with a plurality of photodetectors, there are known ones that have a function of automatically selecting the photodetector, etc. to be used for observation based on information about the fluorescent dye (fluorescent reagent, fluorescent dye) to be observed (see, for example, Patent Documents 1 and 2). Also, among fluorescence microscope devices equipped with a plurality of photodetectors with different wavelength sensitivity characteristics, there are known ones that have a function of automatically selecting the photodetector, etc. to be used for observation based on information about the wavelength range of the fluorescent dye to be observed and information about the wavelength sensitivity characteristics of the photodetectors (see, for example, Patent Document 3). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Patent No. 4145421 [Patent Document 2] Patent No. 5011076 [Patent Document 3] Patent No. 6456617 Summary of the Invention [Problem to be solved by the invention]

[0004] In a fluorescence microscope equipped with multiple photodetectors with different wavelength sensitivity characteristics, the optimal photodetector for observation may differ between the short-wavelength and long-wavelength ends of the wavelength range of the fluorescent dye being observed. For example, in a fluorescence microscope equipped with photodetector A and photodetector B with different wavelength sensitivity characteristics, if the wavelength range of the fluorescent dye being observed is 500 to 600 nm, photodetector A may be more sensitive than photodetector B in the 500 to 545 nm wavelength range, and photodetector B may be more sensitive than photodetector A in the 545 to 600 nm wavelength range. In this case, neither photodetector A nor photodetector B can be said to be the optimal photodetector for observing this fluorescent dye, but the observation will currently be performed using one of the photodetectors (in this example, photodetector B, which has a wider sensitive wavelength range).

[0005] In light of the above-described circumstances, an object of one aspect of the present invention is to provide a technology that enables highly sensitive light detection across the entire wavelength range of the fluorescent dye being observed in a fluorescence microscope apparatus equipped with multiple photodetectors with different wavelength sensitivity characteristics, even if the apparatus does not have a photodetector with the highest wavelength sensitivity across the entire wavelength range of the fluorescent dye being observed. [Means for solving the problem]

[0006] A fluorescence microscope apparatus according to one aspect of the present invention includes a plurality of photodetectors with different wavelength sensitivity characteristics, a detection wavelength changing unit that can change the detection wavelength of each of the plurality of photodetectors, and a fluorescent dye or To be observed an input device for inputting a wavelength range; a memory for storing first information relating to the wavelength sensitivity characteristics of each of the plurality of photodetectors; Kihotaru Photopigment or The aforementioned an observation condition optimization unit that determines and selects one or more optimal photodetectors to be used for observing the observation object from among the plurality of photodetectors based on a wavelength range and the first information stored in the memory, and optimizes the detection wavelength change unit for observing the observation object, the observation condition optimization unit determines whether to use one photodetector or multiple photodetectors for observing the observation object by comparing an intensity signal obtained when the single photodetector is used for the fluorescent dye or the wavelength range input by the input device with an intensity signal obtained by adding up the intensity signals obtained when the multiple photodetectors are used for the fluorescent dye or the wavelength range input by the input device; When a plurality of photodetectors are used to observe the object, the intensity signals of the fluorescence detected by each of the plurality of photodetectors are summed.

[0007] A method according to another aspect of the present invention is a method for optimizing observation conditions for a fluorescence microscope apparatus including a plurality of photodetectors with different wavelength sensitivity characteristics, a detection wavelength changing unit capable of changing the detection wavelength of each of the plurality of photodetectors, and a memory for storing first information relating to the wavelength sensitivity characteristics of each of the plurality of photodetectors, the method comprising: To be observed determining and selecting one or more photodetectors that are optimal for use in observing the object of observation from among the plurality of photodetectors based on the wavelength range and the first information stored in the memory, and optimizing the detection wavelength changing unit for observing the object of observation; In the determination, an intensity signal obtained when the one photodetector is used for the input fluorescent dye or wavelength range is compared with an intensity signal obtained by adding up the intensity signals obtained when the multiple photodetectors are used for the input fluorescent dye or wavelength range, thereby determining whether to use one photodetector or multiple photodetectors for observing the object of observation; When a plurality of photodetectors are used to observe the object, the intensity signals of the fluorescence detected by each of the plurality of photodetectors are summed.

[0008] A program according to another aspect of the present invention is a fluorescence microscope apparatus including a plurality of photodetectors with different wavelength sensitivity characteristics, a detection wavelength changing unit capable of changing the detection wavelength of each of the plurality of photodetectors, and a memory that stores first information relating to the wavelength sensitivity characteristics of each of the plurality of photodetectors, the program including: To be observed determining and selecting one or more photodetectors that are optimal for use in observing the object of observation from among the plurality of photodetectors based on the wavelength range and the first information stored in the memory, and optimizing the detection wavelength changing unit for observing the object of observation; In the determination, an intensity signal obtained when the one photodetector is used for the input fluorescent dye or wavelength range is compared with an intensity signal obtained by adding up the intensity signals obtained when the multiple photodetectors are used for the input fluorescent dye or wavelength range, thereby determining whether to use one photodetector or multiple photodetectors for observing the object of observation; When a plurality of photodetectors are used to observe the object, the intensity signals of the fluorescence detected by each of the plurality of photodetectors are summed. [Effects of the Invention]

[0009] According to the above aspect, in a fluorescence microscope device equipped with multiple photodetectors with different wavelength sensitivity characteristics, it is possible to provide a technology that enables highly sensitive photodetection across the entire wavelength range of the fluorescent dye being observed, even if the device does not have a photodetector with the highest wavelength sensitivity across the entire wavelength range. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a diagram illustrating an example of the configuration of a fluorescence microscope apparatus 100 according to an embodiment. [Figure 2] 1 is a flowchart illustrating an example of an observation condition optimization process performed by a fluorescence microscope apparatus 100 according to an embodiment. [Figure 3] 10 is a flowchart illustrating the details of the process of step S20. [Figure 4] FIG. 10 is a diagram illustrating an example of a fluorescent dye input screen. [Figure 5] FIG. 10 is a diagram illustrating an example of a setting input screen. DETAILED DESCRIPTION OF THE INVENTION

[0011] Fig. 1 is a diagram illustrating the configuration of a fluorescence microscope apparatus 100 according to one embodiment. As illustrated in Fig. 1, the fluorescence microscope apparatus 100 includes a scan unit 1 that performs two-dimensional scanning with laser light, and a first detection unit 2 and a second detection unit 3 that detect fluorescence that is generated in a sample A when irradiated with the laser light and that enters via the scan unit 1.

[0012] The scan unit 1 includes a laser light source 11 that emits laser light, and an illumination optical system 12 that guides the laser light from the laser light source 11 to the sample A. The laser light source 11 includes a plurality of laser light sources with different oscillation wavelengths, for example, 405 nm, 488 nm, and 543 nm, and includes an AOTF (Acousto-Optics Tunable Filter) that can control the emission of laser light of each oscillation wavelength. The illumination optical system 12 includes an optical fiber 13 that guides the laser light from the laser light source 11, and a collimating lens 14.

[0013] The scan unit 1 also includes an objective lens 15 that collects fluorescence from the sample A, an imaging lens 16 that forms an image of the fluorescence collected by the objective lens 15, a scanner 17, a pupil projection lens 18 that converts the fluorescence imaged by the imaging lens 16 into approximately parallel light, an excitation dichroic mirror 19 that branches the approximately parallel light of the fluorescence from the optical path of the laser light, a confocal lens 20 that collects the branched fluorescence, and a confocal pinhole 21 that passes only the fluorescence generated from the focal position of the objective lens 15 out of the collected fluorescence.

[0014] Multiple excitation dichroic mirrors 19 with different spectra (spectral characteristics) are fixed to a rotatable excitation turret 22, and by rotating the excitation turret 22, the excitation dichroic mirror 19 inserted into the optical path can be changed.

[0015] The first detection unit 2 is equipped with two photometric dichroic mirrors 31A and 31B that separate (disperse) the fluorescence incident from the scan unit 1 (fluorescence that has passed through the confocal pinhole 21) into two optical paths according to the wavelength range, a wavelength selection mechanism 32A that selects the wavelength to be detected from the fluorescence in one of the optical paths separated by the photometric dichroic mirror 31B, a photodetector 33A that detects the wavelength selected by the wavelength selection mechanism 32A, a wavelength selection mechanism 32B that selects the wavelength to be detected from the fluorescence in the other optical path separated by the photometric dichroic mirror 31B, and a photodetector 33B that detects the wavelength selected by the wavelength selection mechanism 32B.

[0016] Photometric dichroic mirrors 31A, 31B with different spectra are fixed to rotatable photometric turrets 34A, 34B, respectively, and by rotating the photometric turrets 34A, 34B, the photometric dichroic mirror 31A, 31B inserted into the optical path can be changed.

[0017] The photometric dichroic mirror 31A is configured to transmit the fluorescence from the scan unit 1 toward the second detection unit 3 or reflect it toward the photometric dichroic mirror 31B depending on the wavelength range. The photometric dichroic mirror 31B is configured to transmit the fluorescence from the photometric dichroic mirror 31A toward the wavelength selection mechanism 32A or reflect it toward the wavelength selection mechanism 32B depending on the wavelength range.

[0018] The wavelength selection mechanism 32A includes a diffraction grating (VPH (Volume Phase Holographic)) 35A that separates the fluorescence into spectral components, a oscillating mirror 36A that reflects the fluorescence separated by the diffraction grating 35A, an imaging lens 37A that focuses the fluorescence reflected by the oscillating mirror 36A on the light receiving surface of the photodetector 33A, and a slit (light-shielding slit) 38A that partially blocks the fluorescence focused by the imaging lens 37A.

[0019] Diffraction grating 35A is configured to split the spectral components of the fluorescence transmitted through photometric dichroic mirror 31B into one direction. Oscillating mirror 36A is provided so as to be able to swing about a swing axis perpendicular to the direction of the spectral sequence split by diffraction grating 35A. Oscillating mirror 36A is configured so as to change the spectral components that pass through slit 38A according to the swing angle.

[0020] Slit 38A includes fixed member 39A and movable member 40A disposed with a gap from fixed member 39A in the direction of the spectral series. Movable member 40A is provided so as to be movable relative to fixed member 39A in the direction of the spectral series, and is capable of widening or narrowing the gap between itself and fixed member 39A, i.e., the opening through which fluorescence passes. Photodetector 33A has wavelength sensitivity characteristics that are more sensitive on the short wavelength side than photodetectors 33C and 33D, which will be described later.

[0021] The wavelength selection mechanism 32B has the same configuration as the wavelength selection mechanism 32A, that is, the wavelength selection mechanism 32B includes a diffraction grating (VPH) 35B, a swing mirror 36B, an imaging lens 37B, and a slit (light-shielding slit) 38B.

[0022] Diffraction grating 35B is configured to disperse the spectral components of the fluorescence reflected by photometric dichroic mirror 31B in one direction. Oscillating mirror 36B is provided so as to be swingable about an oscillation axis perpendicular to the direction of the spectral sequence dispersed by diffraction grating 35B, and is capable of changing the spectral components that pass through slit 38B according to the oscillation angle. Slit 38B includes a fixed member 39B and a movable member 40B. Photodetector 33B has the same wavelength sensitivity characteristics as photodetector 33A.

[0023] The second detection unit 3 has the same configuration as the first detection unit 2. That is, the second detection unit 3 includes two photometric dichroic mirrors 31C and 31D, a wavelength selection mechanism 32C, a photodetector 33C, a wavelength selection mechanism 32D, and a photodetector 33D.

[0024] Similar to photometric dichroic mirrors 31A and 31B, photometric dichroic mirrors 31C and 31D have multiple mirrors with different spectra fixed to rotatable photometric turrets 34C and 34D, respectively, and by rotating photometric turrets 34C and 34D, it is possible to change the photometric dichroic mirror 31C or 31D inserted into the optical path.

[0025] Photometric dichroic mirror 31C is configured to transmit or reflect the fluorescence from photometric dichroic mirror 31A of first detection unit 2 toward photometric dichroic mirror 31D depending on the wavelength range. Photometric dichroic mirror 31D is configured to transmit or reflect the fluorescence from photometric dichroic mirror 31C toward wavelength selection mechanism 32C depending on the wavelength range.

[0026] The wavelength selection mechanism 32C is configured to select a wavelength to be detected from the fluorescence that has passed through the photometric dichroic mirror 31D, and includes a diffraction grating (VPH) 35C, a swing mirror 36C, an imaging lens 37C, and a slit (light-shielding slit) 38C.

[0027] Diffraction grating 35C is configured to split the spectral components of the fluorescence from photometric dichroic mirror 31D in one direction. Oscillating mirror 36C is provided so as to be able to swing about a swing axis perpendicular to the direction of the spectral sequence split by diffraction grating 35C, and is capable of changing the spectral components that pass through slit 38C according to the swing angle.

[0028] Slit 38C includes fixed member 39C and movable member 40C. Photodetector 33C detects the wavelength selected by wavelength selection mechanism 32C. Photodetector 33C has wavelength sensitivity characteristics that make it more sensitive to longer wavelengths than photodetectors 33A and 33B.

[0029] The wavelength selection mechanism 32D is configured to select a wavelength to be detected from the fluorescence reflected by the photometric dichroic mirror 31 D. The wavelength selection mechanism 32D includes a diffraction grating (VPH) 35D, a swing mirror 36D, an imaging lens 37D, and a slit (light-shielding slit) 38D.

[0030] Diffraction grating 35D is configured to split the spectral components of the fluorescence from photometric dichroic mirror 31D in one direction. Oscillating mirror 36D is provided so as to be able to swing about a swing axis perpendicular to the direction of the spectral sequence split by diffraction grating 35D, and is capable of changing the spectral components that pass through slit 38D according to the swing angle.

[0031] Slit 38D includes a fixed member 39D and a movable member 40D. Photodetector 33D detects the wavelength selected by wavelength selection mechanism 32D. Photodetector 33D has the same wavelength sensitivity characteristics as photodetector 33C.

[0032] Note that photodetector 33A or 33B and photodetector 33C or 33D are examples of photodetectors with different wavelength sensitivity characteristics. The excitation turret 22 on which multiple excitation dichroic mirrors 19 with different spectra are fixed, the photometry turret 34A on which multiple photometry dichroic mirrors 31A with different spectra are fixed, the photometry turret 34B on which multiple photometry dichroic mirrors 31B with different spectra are fixed, the wavelength selection mechanisms 32A and 32B, the photometry turret 34C on which multiple photometry dichroic mirrors 31C with different spectra are fixed, the photometry turret 34D on which multiple photometry dichroic mirrors 31D with different spectra are fixed, and the wavelength selection mechanisms 32C and 32D are examples of a detection wavelength changing unit that can change the detection wavelength of each of the multiple photodetectors with different wavelength sensitivity characteristics. Hereinafter, the term "detection wavelength changing unit" refers to the excitation turret 22, the photometry turret 34, and the wavelength selection mechanism 32. The excitation dichroic mirror 19 and the photometric dichroic mirrors 31A, 31B, 31C, and 31D are examples of spectroscopic optical elements.

[0033] The fluorescence microscope device 100 also includes an input device 4, a display device 5, and a control device 6. The input device 4 receives various inputs in response to user input operations, such as input of the fluorescent dye or wavelength range to be observed, and input of settings related to the photodetector 33 used to observe the observation target. The input device 4 is, for example, a keyboard, a mouse, or a touch panel.

[0034] The display device 5 displays various types of information, such as a fluorescent dye input screen that enables input of the fluorescent dye to be observed, a setting input screen that enables input of settings related to the photodetector 33 used for observing the observation target, and an image. The display device 5 is, for example, an LCD (Liquid Crystal Display).

[0035] The control device 6 controls each part of the fluorescence microscope device 100. For example, the control device 6 controls the laser light source 11, the scanner 17, and the photometric turret 22 of the scan unit 1, the photometric turrets 34A and 34B, the wavelength selection mechanisms 32A and 32B, and the photodetectors 33A and 33B of the first detection unit 2, the photometric turrets 34C and 34D, the wavelength selection mechanisms 32C and 32D, and the photodetectors 33C and 33D of the second detection unit 3, and the display device 5.

[0036] The control device 6 also functions as an observation condition optimization unit and an image construction unit. The observation condition optimization unit functions to determine and select one or more optimal photodetectors 33 to be used for observing the object of observation from among multiple photodetectors 33 with different wavelength sensitivity characteristics, and to optimize the detection wavelength change unit for observing the object of observation. When selecting the photodetectors 33, the first detector 33A may be selected preferentially over the second detector 33B within the first detection unit 2, and the third detector 33B may be selected preferentially over the fourth detector 33D within the second detection unit 3. The image construction unit functions to construct an image based on the fluorescence intensity signals detected by one or more photodetectors 33 selected by the observation condition optimization unit. If multiple photodetectors 33 are selected, the image construction unit constructs an image based on the sum of the fluorescence intensity signals detected by each of the selected photodetectors.

[0037] The control device 6 is, for example, a PC (Personal Computer) and includes a processor 6a and a memory 6b. The above-described controls and functions of the control device 6 may be realized, for example, by the processor 6a executing a program stored in the memory 6b (so-called software processing), or by hardware processing, or by a combination of software processing and hardware processing. The processor 6a includes, for example, one or more integrated circuits. The integrated circuits may be, for example, a CPU (Central Processing Unit), a GPU (Graphics Processing Unit), a DSP (Digital Signal Processor), an ASIC (Application Specific Integrated Circuit), an FPGA (Field-Programmable Gate Array), etc.

[0038] The memory 6b stores a program executed by the processor 6a. The memory 6b also stores first information, second information, and third information. The first information is information regarding the wavelength sensitivity characteristics of each of the photodetectors 33A, 33B, 33C, and 33D. The second information is information regarding the spectrum of each excitation dichroic mirror 19, the spectrum of each photometering dichroic mirror 31A, the spectrum of each photometering dichroic mirror 31B, the spectrum of each photometering dichroic mirror 31C, and the spectrum of each photometering dichroic mirror 31D. The third information is information regarding the spectrum of a fluorescent dye that can be observed. The memory 9 also stores information regarding the oscillation wavelength (spectrum) of each laser light that can be emitted by the laser light source 11. The memory 9 also stores images.

[0039] The memory 6b includes a non-transitory computer-readable medium that stores a program executed by the processor 11. The memory 6b may include, for example, one or more semiconductor memories and one or more other storage devices. The semiconductor memory includes, for example, volatile memory such as RAM (Random Access Memory), and non-volatile memory such as ROM (Read Only Memory), programmable ROM, and flash memory. The RAM may include, for example, DRAM (Dynamic Random Access Memory), SRAM (Static Random Access Memory), etc. The other storage devices may include, for example, magnetic storage devices including, for example, magnetic disks as computer-readable media, and optical storage devices including, for example, optical disks as computer-readable media, etc.

[0040] In the fluorescence microscope apparatus 100 configured as described above, prior to observation, the user inputs the fluorescent dye or wavelength range to be observed using the input device 4. One or more optimal photodetectors 33 to be used for observing the observation target are determined and selected from among multiple photodetectors 33 with different wavelength sensitivity characteristics, and the detection wavelength change unit is optimized for observation of the observation target. During observation, an image is constructed based on the fluorescence intensity signals detected by the selected one or more photodetectors 33. For example, if multiple photodetectors 33 are selected, an image is constructed based on the result of adding up the fluorescence intensity signals detected by each of the multiple photodetectors 33. The image is then displayed on the display device 5 and / or stored in the memory 6b. The processing performed by this fluorescence microscope apparatus 100 is described in detail below.

[0041] Fig. 2 is a flowchart illustrating an example of the observation condition optimization process performed by the fluorescence microscope device 100. Fig. 3 is a flowchart illustrating the details of the process of step S20.

[0042] 2, when the user inputs the fluorescent dye to be observed using the input device 4 (step S10), the control device 6 derives the optimum observation conditions for observing the fluorescent dye to be observed (step S20). Specifically, the observation conditions derived include one or more photodetectors 33 to be used, the oscillation wavelength of the laser light emitted by the laser light source 11, the excitation dichroic mirror 19 and the photometric dichroic mirror 31 to be inserted in the optical path from the sample A to the one or more photodetectors 33 to be used, and the wavelengths to be selected by the wavelength selection mechanism 32 that selects the wavelengths to be detected by the one or more photodetectors 33 to be used.

[0043] More specifically, the processing of step S20 is performed, for example, by the processing illustrated in Fig. 3. First, the control device 6 simulates all possible combinations of intensity signals that can be obtained from a single photodetector 33 when observation is performed using the single photodetector 33 (step S21). Here, all possible combinations are combinations of the single photodetector 33 to be used, the oscillation wavelength of the laser light, the excitation dichroic mirror 19 and the photometric dichroic mirror 31 inserted in the optical path from the sample A to the single photodetector 33 to be used, and the wavelengths selected by the wavelength selection mechanism 32 that selects the wavelengths to be detected by the single photodetector 33 to be used. However, the wavelengths selected by the wavelength selection mechanism 32 at this time are set to a wavelength range that is previously associated with the fluorescent dye to be observed (the wavelength range of the fluorescent dye to be observed).

[0044] The simulation of step S21 can be performed based on the first information, second information, and third information stored in memory 6b. More specifically, the intensity signal obtained for each combination can be calculated by summing up the products, for each wavelength, of the fluorescence spectrum of the fluorescent dye to be observed (included in the third information), the wavelength sensitivity characteristics of the one photodetector 33 to be used (included in the first information), and the spectra (transmission spectrum or reflection spectrum) (included in the second information) of the excitation dichroic mirror 19 and the photometric dichroic mirror 31 inserted in the optical path from sample A to the one photodetector 33 to be used. The intensity signal obtained may also be calculated by multiplying this by the excitation efficiency of the fluorescent dye to be observed at the oscillation wavelength of the laser light.

[0045] In addition, when simulating step S21, combinations that are known from the beginning to be unable to perform fluorescence observation, such as combinations that use a laser light oscillation wavelength that results in an excitation efficiency of the fluorescent dye to be observed below a certain level, or combinations that use an excitation dichroic mirror 19 that cannot reflect the laser light oscillation wavelength, may be excluded from the simulation. This allows the processing time required for the simulation of step S21 to be further reduced.

[0046] When the simulation in step S21 is completed, the control device 6 acquires the combination that maximizes the intensity signal obtained from the simulation result and the intensity signal at that time (step S22). Note that if there are multiple combinations that maximize the intensity signal obtained from the simulation result, any one of the combinations may be acquired.

[0047] Next, the control device 6 simulates, for all possible combinations (step S23), how much intensity signal can be obtained by adding up the intensity signals of the fluorescence detected by each of the photodetectors 33 when observation is performed using a plurality of (two in this example) photodetectors 33 with different wavelength sensitivity characteristics. Here, all possible combinations are combinations of the two photodetectors 33 to be used, the oscillation wavelength of the laser light, the excitation dichroic mirror 19 and the photometric dichroic mirror 31 inserted in the optical path from the sample A to the two photodetectors 33 to be used, and the wavelengths selected by the wavelength selection mechanism 32 that selects the wavelengths to be detected by each of the two photodetectors 33 to be used.

[0048] However, the wavelengths selected by each wavelength selection mechanism 32 at this time are wavelength ranges obtained by dividing the wavelength range previously associated with the fluorescent dye to be observed by wavelengths at which the transmittance (or reflectance) in the spectrum of the photometric dichroic mirror 31 that disperses light between the two photodetectors 33 to be used is 500 to 600 nm. For example, if the wavelength range previously associated with the fluorescent dye to be observed is 500 to 600 nm, the photometric dichroic mirror 31 that disperses light between the two photodetectors 33 to be used reflects light with wavelengths of 530 nm or shorter (reflectance of 80% or higher) and transmits light with wavelengths of 550 nm or longer (transmittance of 80% or higher), and the wavelength at which the transmittance (or reflectance) in the spectrum is 50% is 540 nm, then the wavelengths selected by the wavelength selection mechanism 32 on the reflection side of the photometric dichroic mirror 31 are 500 to 540 nm, and the wavelengths selected by the wavelength selection mechanism 32 on the transmission side are 540 to 600 nm.

[0049] In this way, the wavelengths selected by each wavelength selection mechanism 32 do not overlap, but the selected wavelengths may overlap by the wavelength range of the rising edge (or falling edge) of the transmittance (or reflectance) in the spectrum of the photometric dichroic mirror 31. For example, in the above example, if the wavelength range of the rising edge is 530 to 550 nm, the wavelength selection mechanism 32 on the reflection side may select a wavelength of 500 to 550 nm, and the wavelength selection mechanism 32 on the transmission side may select a wavelength of 530 to 600 nm. This allows the two photodetectors 33 to detect all fluorescence in the wavelength range of the rising edge (or falling edge).

[0050] The simulation in step S23 can be performed based on the first information, second information, and third information stored in the memory 6b, similarly to step S21. More specifically, the intensity signal obtained for each combination can be determined by calculating the sum of the products, for each wavelength, of the fluorescence spectrum of the fluorescent dye to be observed (included in the third information), the wavelength sensitivity characteristics of one of the two photodetectors 33 used (included in the first information), and the spectrum (transmission spectrum or reflection spectrum) (included in the second information) of the excitation dichroic mirror 19 and the photometric dichroic mirror 31 inserted in the optical path from the sample A to that one photodetector 33; and by calculating the sum of the products, for each wavelength, of the fluorescence spectrum of the fluorescent dye to be observed (included in the third information), the wavelength sensitivity characteristics (included in the first information) of the other of the two photodetectors 33 used, and the spectrum (transmission spectrum or reflection spectrum) (included in the second information) of the excitation dichroic mirror 19 and the photometric dichroic mirror 31 inserted in the optical path from the sample A to the other photodetector 33, and adding up each sum. Alternatively, the sum of each may be further multiplied by the excitation efficiency of the fluorescent dye to be observed at the oscillation wavelength of the laser light, and then added together to obtain an intensity signal.

[0051] In the simulation of step S23, as in step S21, combinations that are known from the beginning to be unable to perform fluorescence observation, such as combinations that use a laser light oscillation wavelength that results in an excitation efficiency of the fluorescent dye to be observed below a certain level, or combinations that use an excitation dichroic mirror 19 that cannot reflect the laser light oscillation wavelength, may be excluded from the simulation. This allows the processing time required for the simulation of step S23 to be further reduced.

[0052] When the simulation in step S23 is completed, the control device 6 acquires the combination that maximizes the intensity signal obtained from the simulation result and the intensity signal at that time (step S24). Note that if there are multiple combinations that maximize the intensity signal obtained from the simulation result, any one of the combinations may be acquired.

[0053] Next, the control device 6 compares the intensity signal for the combination acquired in step S22 (a combination using one photodetector 33) with the intensity signal for the combination acquired in step S24 (a combination using two photodetectors 33), and determines whether the ratio of the intensity signal for the combination acquired in step S24 to the intensity signal for the combination acquired in step S22 is greater than or equal to a predetermined ratio (e.g., 110%) (step S25).

[0054] If the determination result in step S25 is YES, the control device 6 selects the combination acquired in step S24 as the optimum observation conditions for observation (step S26). On the other hand, if the determination result in step S25 is NO, the control device 6 selects the combination acquired in step S22 as the optimum observation conditions for observation (step S27).

[0055] In the judgment and selection of steps S25 to S27, the combination with the higher intensity signal is not simply compared and selected because when multiple photodetectors 33 are used, the number of photodetectors used increases compared to when only one photodetector is used, and it is expected that noise components will also be added together.

[0056] Once the optimum observation conditions for observing the fluorescent dye to be observed have been derived in this way (when step S20 in FIG. 2 is completed), the control device 6 sets one or more photodetectors 33 to be used in accordance with the observation conditions, and controls the laser light source 11, excitation turret 22, photometric turret 34, and wavelength selection mechanism 32 to set the oscillation wavelength of the laser light emitted by the laser light source 11, the excitation dichroic mirror 19 and photometric dichroic mirror 31 to be inserted in the optical path from the sample A to the one or more photodetectors 33 to be used, and the wavelength selected by the wavelength selection mechanism 32, which selects the wavelength to be detected by the one or more photodetectors to be used (step S30). In this way, the optimum observation conditions for observing the fluorescent dye to be observed are set.

[0057] Then, when observing a fluorescent dye to be observed under the set observation conditions, the observation is performed as follows. When one photodetector 33 is set (selected) as the photodetector 33 to be used, the scanner 17 scans the sample A with laser light emitted from the laser light source 11, and the one photodetector 33 detects fluorescence from the sample A. Then, a fluorescence intensity signal detected by the one photodetector 33 and the scanning position by the scanner 17 are output to the control device 6. The control device 6 performs AD conversion on the fluorescence intensity signal detected by the one photodetector 33, and constructs (generates) an image based on the AD-converted intensity signal and the corresponding scanning position. Then, the constructed image is displayed on the display device 8 or stored in memory 6b.

[0058] On the other hand, when multiple photodetectors 33 are set (selected) as the photodetectors 33 to be used, the scanner 17 scans the sample A with the laser light emitted from the laser light source 11, and the multiple photodetectors 33 detect the fluorescence from the sample A. Then, the fluorescence intensity signals detected by each of the multiple photodetectors 33 and the scanning position by the scanner 17 are output to the control device 6. The control device 6 adds up the fluorescence intensity signals detected by each of the multiple photodetectors 33 after AD conversion, and constructs an image based on the combined intensity signal and the corresponding scanning position. Then, the constructed image is displayed on the display device 5 or stored in memory 6b.

[0059] The AD conversion described above may be performed before the intensity signals are input to the control device 6. In this case, the AD conversion may be performed by an AD conversion circuit provided in each photodetector 33, or by an AD conversion circuit connected between each photodetector 33 and the control device 6. The order of the AD conversion and summation described above may also be reversed. That is, the intensity signals may be summed before AD conversion. In this case, the signal summation is performed by a summation circuit provided on the photodetector side relative to the AD conversion circuit.

[0060] The fluorescence microscope apparatus 100 described above enables highly sensitive photodetection across the entire wavelength range of the fluorescent dye being observed, even if the apparatus does not include a photodetector 33 with the highest wavelength sensitivity across the entire wavelength range of the fluorescent dye being observed. For example, when the wavelength range of the fluorescent dye being observed is 500 to 600 nm, photodetector 33A (or 33B) is more sensitive than photodetector 33C (or 33D) in the 500 to 545 nm wavelength range, and photodetector 33C (or 33D) is more sensitive than photodetector 33A (or 33B) in the 545 to 600 nm wavelength range. In this case, conventionally, photodetection was performed using only one of the photodetectors 33 (for example, photodetector 33C (or 33D) with a wider sensitive wavelength range), making it impossible to perform highly sensitive photodetection across the entire wavelength range being observed. In contrast to this, in this embodiment, photodetection is performed using photodetector 33A (or 33B) for the wavelength range of 500 to 545 nm, and photodetector 33c (or 33D) for the wavelength range of 545 to 600 nm, and the intensity signals obtained by each photodetector 33 are added together, thereby enabling highly sensitive photodetection across the entire wavelength range to be observed.

[0061] 2, the fluorescent dye to be observed in step S10 may be input, for example, from a fluorescent dye input screen displayed on the display device 5 by the control device 6. Fig. 4 is a diagram illustrating the fluorescent dye input screen.

[0062] As shown in FIG. 4, the fluorescent dye input screen includes a list display field 201 that displays a list of fluorescent dyes that the user can select as observation targets, an observation target display field 202 that displays the fluorescent dyes to be observed, an “Add” button 203, and the like.

[0063] On the fluorescent dye input screen, the user can use the input device 4 to add the desired fluorescent dye to the observation target display field 202 (input it as an observation target) by double-clicking the desired fluorescent dye displayed in the list display field 201, or by selecting it and pressing the ``Add'' button 203.

[0064] In this way, when a fluorescent dye to be observed is input from the fluorescent dye input screen, once the fluorescent dye is added to the observation target display field 202, the process of step S20 (derivation of optimal observation conditions) described above is performed with the fluorescent dye displayed in the observation target display field 202 as the fluorescent dye to be observed. However, if the addition of a fluorescent dye to the observation target display field 202 results in multiple fluorescent dyes being displayed in the observation target display field 202, the process of step S20 described above is performed for each of the fluorescent dyes. In this case, simultaneous observation of the multiple fluorescent dyes may be assumed, and optimal observation conditions for each fluorescent dye may be derived. Then, for each fluorescent dye to be observed, the channel and the device ID of one or more photodetectors 33 used for the observation obtained in the process of step S20 are additionally displayed in the observation target display field 202. In the example of Figure 4, for the fluorescent dye "DAPI", the channel "CH1" and the device ID "SD1" are additionally displayed, and for the fluorescent dye "Alexa Fluor 488", the channel "CH2" and the device ID "SD2, HSD3" are additionally displayed.

[0065] Thereafter, when the user presses the "OK" button 204 on the fluorescent dye input screen using the input device 4, the processing of step S30 described above (setting of optimal observation conditions) is performed. However, if multiple fluorescent dyes are displayed in the observation target display field 202, optimal observation conditions for each fluorescent dye may be set, for example, derived assuming simultaneous observation of the multiple fluorescent dyes.

[0066] Furthermore, when the "OK" button 204 on the fluorescent dye input screen is pressed, the control device 6 may further display a setting input screen on the display device 5. The setting input screen is an input screen that enables input of settings related to the photodetector 33 and the like used for observing the observation target, and also serves as a screen for adjusting the brightness of each channel. Figure 5 is a diagram illustrating an example of the setting input screen.

[0067] The setting input screen displays the fluorescent dye to be observed for each channel and the device ID of one or more photodetectors 33 to be used, as shown in Fig. 5. In the example of Fig. 5, the fluorescent dye "DAPI" and device ID "SD1" are displayed for channel "CH1," and the fluorescent dye "Alexa Fluor 488" and device ID "SD2, HSD3" are displayed for channel "CH2."

[0068] The setting input screen allows the user to set (change) the wavelength range for observing the fluorescent dye of each channel. This setting can be performed by directly entering values ​​using the input device 4 in the setting input field 301. For example, the wavelength range of 500-600 nm for observing the fluorescent dye "Alexa Fluor 488" of channel "CH2" can be changed to 510-600 nm. However, if the wavelength range detected by the photodetector 33 (e.g., 33B) with device ID "SD2" is set to 500-540 nm and the wavelength range detected by the photodetector 33 (e.g., 33C) with device ID "HSD3" is set to 540-600 nm, the wavelength range detected by the photodetector 33 with device ID "SD2" is changed to 510-540 nm, and the wavelength range detected by the photodetector 33 with device ID "HSD3" remains unchanged. Alternatively, if the observation wavelength range is changed to 540 to 600 nm, the photodetector 33 with the device ID "SD2" will not be used, and only the photodetector 33 with the device ID "HDS3" will be used.

[0069] Furthermore, on the setting input screen, the user can set (change) the oscillation wavelength and light intensity of the laser light used for observation for each channel, the HV (High Voltage) of the PMT, which is the photodetector 33, the gain of the intensity signal, and the offset of the intensity signal. This setting can be performed using the input device 4 in the setting input field 302 by moving a slide bar (e.g., 302a), pressing the left and right buttons (e.g., 302b), or directly inputting a numerical value.

[0070] When setting the photodetector 33 in the setting input field 302, even if multiple photodetectors 33 are used, such as the channel "CH2" illustrated in FIG. 5, the setting is performed as a setting for one pseudo photodetector 33. Then, when the setting for the pseudo photodetector 33 is performed, the setting is set in the same way for each of the multiple photodetectors 33 to be used. Therefore, when multiple photodetectors 33 are used, the user does not need to perform the setting for each photodetector 33 individually.

[0071] The setting input screen also displays the fluorescence spectrum of the fluorescent dye to be observed, the oscillation wavelength (spectrum) of the laser light to be used, and the wavelength range in which the fluorescent dye is observed. These are displayed in a display field 303.

[0072] When multiple photodetectors 33 are used, there may be differences in sensitivity among the photodetectors 33. In such cases, the sensitivity of one or more of the multiple photodetectors 33 used may be corrected so that the sensitivity of each photodetector 33 matches.

[0073] For example, even if the settings for the multiple photodetectors 33 used are the same, if there is a difference in the output intensity signal per incident photon between the respective photodetectors 33, the sensitivity (HV or gain) of one or more of the multiple photodetectors 33 used may be corrected so that the output intensity signals per incident photon match.

[0074] In this case, for example, fourth information regarding the output intensity signal per incident photon for each photodetector 33 (or the ratio between the number of incident photons and the output intensity signal) may be stored in advance in the memory 6b, and the sensitivity (HV or gain) of one or more of the multiple photodetectors 33 used may be corrected based on the fourth information so that the output intensity signal per incident photon for each of the multiple photodetectors 33 used matches. Alternatively, the intensity signal may be corrected before the above-mentioned summation based on the fourth information. For example, when the photodetectors 33 used are photodetector 33A and photodetector 33C, if the output intensity signal per incident photon of photodetector 33C is twice as large as that of photodetector 33A, the output intensity signal of photodetector A before summation may be doubled or the output intensity signal of photodetector 33C before summation may be halved.

[0075] Furthermore, for example, fifth information relating to the intensity signal output from each photodetector 33 when no photons are incident may be stored in advance in memory 6b, and when multiple (N) photodetectors 33 are used and image construction is performed based on the result of adding up the intensity signals of the fluorescence detected by each of the N photodetectors 33, the intensity signals of the fluorescence detected by each of the N photodetectors 33 may be added up, and then, based on the fifth information, a signal obtained by multiplying the intensity signal output from each photodetector 33 when no photons are incident by N-1 may be subtracted to construct the image. This makes it possible to prevent the offset level of the intensity signal from increasing due to the addition.

[0076] Furthermore, in step S10 illustrated in FIG. 2 above, the user may input a wavelength range to be observed instead of inputting the fluorescent dye to be observed. In this case, the control device 6 may display an input screen on the display device 5 that allows the user to input the wavelength range to be observed, and the user may input the wavelength range to be observed from that input screen. In this case, in step S20, instead of the fluorescence spectrum of the fluorescent dye to be observed, a spectrum with maximum intensity (100%) across the entire wavelength range to be observed is used. Furthermore, the input screen may be configured to have an input field that allows the user to input the fluorescent dye to be observed and an input field that allows the user to input the wavelength range to be observed, so that either input can be accommodated.

[0077] In the fluorescence microscope device 100, a specific example of the multiple photodetectors 33 with different wavelength sensitivity characteristics may be, for example, a combination of a PMT (Photomultiplier-Tube) that has high sensitivity on the short wavelength side and uses GaAsP (Gallium Arsenide Phosphide) for its photocathode, and a PMT that has high sensitivity on the long wavelength side and uses GaAs (Gallium Arsenide) for its photocathode. Alternatively, for example, multiple SiPMs (Silicon Photomultipliers) with different wavelength sensitivity characteristics may be used. Multiple SiPMs with different wavelength sensitivity characteristics are more effective in that the output intensity signal of each SiPM is quantitative (the ratio between the number of incident photons and the output intensity signal can be kept constant) even if the wavelength sensitivity characteristics are different, and therefore the quantitativeness is not lost when the output intensity signals of each SiPM are added together.

[0078] Furthermore, in the fluorescence microscope device 100, a gradation dichroic mirror may be provided in place of each of the photometric turret 34A to which multiple photometric dichroic mirrors 31A with different spectra are fixed, the photometric turret 34B to which multiple photometric dichroic mirrors 31B with different spectra are fixed, the photometric turret 34C to which multiple photometric dichroic mirrors 31C with different spectra are fixed, and the photometric turret 34D to which multiple photometric dichroic mirrors 31D with different spectra are fixed. The gradation dichroic mirror is provided so as to be movable in a direction along the incident surface onto which the fluorescence is incident, and by changing the incident position of the fluorescence on the incident surface, the wavelength range to be resolved can be changed without limit. In this case, for example, instead of the combination of photometric dichroic mirrors 31 used in each photometric turret 34, a combination of the incident positions of the fluorescence used for each gradation dichroic mirror may be used.

[0079] Furthermore, the fluorescence microscope device 100 may be provided with three or more photodetectors with different wavelength sensitivity characteristics by adding one or more detection units adjacent to the second detection unit 3. In this case, the added detection unit has the same configuration as the first detection unit 2 (or the second detection unit 3), but the wavelength sensitivity characteristics of the two photodetectors included in the added detection unit are different from the wavelength sensitivity characteristics of the two photodetectors included in the other detection units.

[0080] The above-described embodiments are illustrative examples for the purpose of facilitating understanding of the invention, and the present invention is not limited to these embodiments. Various modifications and changes can be made to the present invention without departing from the scope of the claims. [Explanation of symbols]

[0081] 1 Scan Unit 2 First detection unit 3 Second detection unit 4 Input Devices 5 Display device 6. Control device 6a processor 6b memory 11 Laser light source 12 Illumination optical system 13 Optical Fiber 14 Collimating lens 15 Objective Lens 16 Imaging lens 17. Scanner 18 Pupil projection lens 19 Excitation dichroic mirror 20 Confocal Lens 21 Confocal Pinhole 22 Excitation Turret 31A, 31B, 31C, 31D Photometric Dichroic Mirrors 32A, 32B, 32C, 32D Wavelength selection mechanism 33A, 33B, 33C, 33D Photodetectors 34A, 34B, 34C, 34D photometric turret 35A, 35B, 35C, 35D Diffraction Gratings 36A, 36B, 36C, 36D Oscillating mirror 37A, 37B, 37C, 37D Imaging lenses 38A, 38B, 38C, 38D Slit 39A, 39B, 39C, 39D Fixed parts 40A, 40B, 40C, 40D Movable parts 100 Fluorescence microscope equipment 201 List display column 202 Observation target display column 203, 204 buttons 301, 302 setting input field 303 Display field

Claims

1. a plurality of photodetectors with different wavelength sensitivity characteristics; a detection wavelength changing unit capable of changing the detection wavelength of each of the plurality of photodetectors; an input device for inputting a fluorescent dye to be observed or a wavelength range to be observed; a memory that stores first information relating to the wavelength sensitivity characteristics of each of the plurality of photodetectors; an observation condition optimization unit that determines and selects one or more photodetectors that are optimal for use in observing the object of observation from among the plurality of photodetectors, based on the fluorescent dye or the wavelength range input by the input device and the first information stored in the memory, and optimizes the detection wavelength change unit for observing the object of observation; Equipped with the observation condition optimization unit determines whether to use one photodetector or multiple photodetectors for observing the observation object by comparing an intensity signal obtained when the single photodetector is used for the fluorescent dye or the wavelength range input by the input device with an intensity signal obtained by adding up the multiple photodetectors for the fluorescent dye or the wavelength range input by the input device; When a plurality of photodetectors are used to observe the object of observation, intensity signals of the fluorescence detected by each of the plurality of photodetectors are summed. A fluorescence microscope apparatus characterized by:

2. the memory further stores second information relating to a spectrum of a spectroscopic optical element included in the detection wavelength changing unit; the observation condition optimization unit determines and selects one or more photodetectors that are optimal for use in observing the observation target from among the plurality of photodetectors, based on the fluorescent dye or wavelength range of the observation target input by the input device and the first information and the second information stored in the memory, and optimizes the detection wavelength change unit for observing the observation target.

2. The fluorescence microscope apparatus according to claim 1.

3. the memory further stores third information relating to the spectrum of a fluorescent dye that can be observed; the observation condition optimization unit determines and selects one or more photodetectors that are optimal for use in observing the observation target from among the plurality of photodetectors, based on the fluorescent dye to be observed that is input by the input device and the first information and the third information stored in the memory, or based on the fluorescent dye to be observed that is input by the input device and the first information, the second information, and the third information stored in the memory, and optimizes the detection wavelength changing unit for observing the observation target.

3. The fluorescence microscope apparatus according to claim 2.

4. an image construction unit that constructs an image based on the intensity signals of the fluorescence detected by the one or more photodetectors selected by the observation condition optimization unit; when a plurality of photodetectors are selected by the observation condition optimization unit, the image construction unit constructs an image based on a result of summing up the intensity signals of the fluorescence detected by each of the plurality of photodetectors; 4. The fluorescence microscope apparatus according to claim 1, wherein the fluorescence microscope apparatus comprises: a first light source;

5. a display device that displays a setting input screen that enables settings related to the one or more photodetectors selected by the observation condition optimization unit to be input by the input device, When a plurality of photodetectors are selected by the observation condition optimization unit, an input field that allows input of settings for one pseudo photodetector is displayed on the setting input screen displayed by the display device as an input field that allows input of settings for the plurality of photodetectors selected by the observation condition optimization unit, and settings for the plurality of photodetectors selected by the observation condition optimization unit are made based on the settings for the one pseudo photodetector that are input into the input field.

5. The fluorescence microscope apparatus according to claim 1, wherein the fluorescence microscope apparatus comprises: a first light source;

6. Each of the plurality of photodetectors having different wavelength sensitivity characteristics is a SiPM (Silicon Photomultiplier).

6. The fluorescence microscope apparatus according to claim 1, wherein the fluorescence microscope apparatus comprises: a first light source;

7. When a plurality of photodetectors are used for observing the observation target, the observation condition optimization unit overlaps a wavelength range of a rising portion or a falling portion of a spectrum of a spectroscopic optical element that separates the light between the photodetectors to be used as part of a detection wavelength in optimization of the detection wavelength change unit.

7. The fluorescence microscope apparatus according to claim 1, wherein the fluorescence microscope apparatus comprises: a first light source;

8. the observation condition optimization unit, when a ratio of a maximum intensity signal obtained by adding together a plurality of photodetectors used to observe the observation object to a maximum intensity signal obtained when one photodetector is used to observe the observation object is equal to or greater than a predetermined ratio, determines and selects the plurality of photodetectors that obtain the maximum intensity signal by adding together as the optimal photodetectors to use for observing the observation object; 8. The fluorescence microscope apparatus according to claim 1, wherein the fluorescence microscope apparatus comprises: a first light source;

9. the memory further stores fourth information relating to an output intensity signal per incident photon for each of the plurality of photodetectors having different wavelength sensitivity characteristics; When a plurality of photodetectors are selected by the observation condition optimization unit, the sensitivity of one or more of the plurality of photodetectors is corrected based on the fourth information stored in the memory so that output intensity signals per incident photon for each of the plurality of photodetectors are consistent.

9. The fluorescence microscope apparatus according to claim 1, wherein the fluorescence microscope apparatus comprises: a first light source;

10. the memory further stores fifth information relating to an intensity signal output from each photodetector when no photons are incident thereon; When the observation condition optimization unit selects N photodetectors as a plurality of photodetectors, the image construction unit sums up the intensity signals of the fluorescence detected by each of the N photodetectors, and then, based on the fifth information stored in the memory, subtracts a signal obtained by multiplying (N-1) the intensity signal output by each photodetector when no photons are incident, thereby constructing an image.

5. The fluorescence microscope apparatus according to claim 4.

11. 1. A method for optimizing observation conditions for a fluorescence microscope apparatus including a plurality of photodetectors having different wavelength sensitivity characteristics, a detection wavelength changing unit capable of changing the detection wavelength of each of the plurality of photodetectors, and a memory that stores first information relating to the wavelength sensitivity characteristics of each of the plurality of photodetectors, the method comprising: determining and selecting one or more photodetectors that are optimal for use in observing the observation target from among the plurality of photodetectors based on the input fluorescent dye or wavelength range of the observation target and the first information stored in the memory, and optimizing the detection wavelength changing unit for observing the observation target; In the determination, an intensity signal obtained when the single photodetector is used for the input fluorescent dye or wavelength range is compared with an intensity signal obtained by adding together the intensity signals obtained when the multiple photodetectors are used for the input fluorescent dye or wavelength range, thereby determining whether to use one photodetector or multiple photodetectors for observing the object of observation; When a plurality of photodetectors are used to observe the object of observation, intensity signals of the fluorescence detected by each of the plurality of photodetectors are summed.

2. A method for optimizing observation conditions.

12. a detection wavelength changing unit that can change the detection wavelength of each of the plurality of photodetectors; and a memory that stores first information relating to the wavelength sensitivity characteristics of each of the plurality of photodetectors; determining and selecting one or more photodetectors that are optimal for use in observing the observation target from among the plurality of photodetectors based on the input fluorescent dye or wavelength range of the observation target and the first information stored in the memory, and optimizing the detection wavelength changing unit for observing the observation target; In the determination, an intensity signal obtained when the single photodetector is used for the input fluorescent dye or wavelength range is compared with an intensity signal obtained by adding together the intensity signals obtained when the multiple photodetectors are used for the input fluorescent dye or wavelength range, thereby determining whether to use one photodetector or multiple photodetectors for observing the object of observation; When a plurality of photodetectors are used to observe the object of observation, intensity signals of the fluorescence detected by each of the plurality of photodetectors are summed. A program characterized by executing the process.

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