JP2023008411A - Fluorescence microscope device, observation condition optimization method and program - Google Patents

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

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

Conventionally, a fluorescence microscope apparatus equipped with multiple photodetectors has a function of automatically selecting a photodetector or the like to be used for observation based on information on a fluorescent dye (fluorescent reagent, fluorescent dye) to be observed. known (see, for example, Patent Documents 1 and 2). In addition, in a fluorescence microscope apparatus equipped with a plurality of photodetectors with different wavelength sensitivity characteristics, light detection used for observation based on information on the wavelength range of the fluorescent dye to be observed and information on the wavelength sensitivity characteristics of the photodetector A device having a function of automatically selecting a vessel or the like is also known (see Patent Document 3, for example).

Japanese Patent No. 4145421 Japanese Patent No. 5011076 Japanese Patent No. 6456617

In a fluorescence microscope apparatus having a plurality of photodetectors with different wavelength sensitivity characteristics, the optimum photodetector for observation may differ between the short wavelength side and the long wavelength side in the wavelength range of the fluorescent dye to be observed. For example, in a fluorescence microscope device equipped with a photodetector A and a photodetector B having different wavelength sensitivity characteristics, when the wavelength range of the fluorescent dye to be observed is 500 to 600 nm, the wavelength range of 500 to 545 nm is photodetection. In this case, the sensitivity of the device A is higher than that of the photodetector B, and the sensitivity of the photodetector B is higher than that of the photodetector A in the wavelength range of 545 to 600 nm. In this case, neither the photodetector A nor the photodetector B can be said to be the optimum photodetector for observing this fluorescent dye. Observation will be performed using a photodetector B) with a wider high wavelength range.

Based on the above circumstances, an object according to one aspect of the present invention is to provide a fluorescence microscope apparatus having a plurality of photodetectors with different wavelength sensitivity characteristics, wherein the wavelength sensitivity is maintained over the entire wavelength range of the fluorescent dye to be observed. It is an object of the present invention to provide a technique that enables high-sensitivity photodetection over the entire wavelength range even if the highest photodetector is not provided.

A fluorescence microscope apparatus according to one aspect of the present invention includes 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 fluorescence to be observed. an input device for inputting a dye or wavelength range; a memory for storing first information about wavelength sensitivity characteristics of each of the plurality of photodetectors; and the fluorescent dye or wavelength range to be observed input by the input device. and the first information stored in the memory, determining and selecting one or more optimum photodetectors to be used for observing the observation target from among the plurality of photodetectors. and an observation condition optimizing unit that optimizes the detection wavelength changing unit for observation of the observation target, and when a plurality of photodetectors are used to observe the observation target, the plurality of light The fluorescence intensity signals detected by each of the detectors are summed.

A method according to another aspect of the present invention includes a plurality of photodetectors having different wavelength sensitivity characteristics, a detection wavelength changing unit capable of changing detection wavelengths of each of the plurality of photodetectors, and the plurality of photodetectors. A method for optimizing observation conditions for a fluorescence microscope apparatus comprising a memory for storing first information about wavelength sensitivity characteristics of each instrument, wherein an input fluorescent dye or wavelength range to be observed and stored in the memory. determining and selecting, from among the plurality of photodetectors, one or more photodetectors most suitable for use in observing the observation target, based on the obtained first information; and changing the detection wavelength. unit is optimized for observation of the observation target, and when a plurality of photodetectors are used for observation of the observation target, fluorescence intensity signals detected by each of the plurality of photodetectors are summed. .

A program according to another aspect of the present invention includes a plurality of photodetectors having different wavelength sensitivity characteristics, a detection wavelength changing unit capable of changing detection wavelengths of each of the plurality of photodetectors, and the plurality of photodetectors. Fluorescent dyes or wavelength ranges to be observed and the first information stored in the memory are input to a computer of a fluorescence microscope apparatus comprising a memory for storing first information about wavelength sensitivity characteristics of each of the instruments. and determining and selecting one or more optimum photodetectors to be used for observing the observation target from among the plurality of photodetectors, and setting the detection wavelength changing unit to the observation target. Optimizing the observation, and when using a plurality of photodetectors for observing the observation target, performing a process of summing the fluorescence intensity signals detected by each of the plurality of photodetectors. .

According to the above aspect, in a fluorescence microscope device having a plurality of photodetectors with different wavelength sensitivity characteristics, even if the photodetector with the highest wavelength sensitivity over the entire wavelength range of the fluorescent dye to be observed is not provided , it is possible to provide a technique that enables highly sensitive photodetection over the entire wavelength range.

1 is a diagram illustrating the configuration of a fluorescence microscope device 100 according to an embodiment; FIG. 4 is a flowchart illustrating observation condition optimization processing performed by the fluorescence microscope apparatus 100 according to one embodiment. 4 is a flowchart illustrating details of processing in step S20. FIG. 11 is a diagram illustrating a fluorescent dye input screen; It is a figure which illustrates a setting input screen.

FIG. 1 is a diagram illustrating the configuration of a fluorescence microscope device 100 according to one embodiment. As illustrated in FIG. 1 , the fluorescence microscope apparatus 100 includes a scan unit 1 that scans a laser beam two-dimensionally, and a laser beam that is generated in a sample A by irradiation and enters via the scan unit 1 . It has a first detection unit 2 and a second detection unit 3 that detect fluorescence.

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. As shown in FIG. The laser light source 11 has a plurality of different oscillation wavelengths such as 405 nm, 488 nm, and 543 nm, and has an AOTF (Acousto-Optics tunable filter) capable of controlling 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 .

The scanning unit 1 includes an objective lens 15 for condensing the fluorescence from the sample A, an imaging lens 16 for forming an image of the fluorescence condensed by the objective lens 15, a scanner 17, and the imaging lens 16. A pupil projection lens 18 that converts the imaged fluorescence into approximately parallel light, an excitation dichroic mirror 19 that branches the approximately parallel fluorescence from the optical path of the laser beam, and a confocal lens 20 that collects the branched fluorescence. , and a confocal pinhole 21 for passing only the fluorescence emitted from the focal position of the objective lens 15 among the collected fluorescence.

A plurality of excitation dichroic mirrors 19 having different spectra (spectral characteristics) are fixed to a rotatable excitation turret 22. By rotating the excitation turret 22, the excitation dichroic mirror 19 to be inserted into the optical path can be changed. It has become.

The first detection unit 2 includes two photometric dichroic mirrors 31A and 31B that separate (spectrospect) 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 a wavelength to be detected from the fluorescence of one optical path resolved by the photometric dichroic mirror 31B, a photodetector 33A that detects the wavelength selected by the wavelength selection mechanism 32A, and a photometric dichroic mirror 31B. A wavelength selection mechanism 32B for selecting a wavelength to be detected from the fluorescence in the other optical path, and a photodetector 33B for detecting the wavelength selected by the wavelength selection mechanism 32B.

A plurality of photometric dichroic mirrors 31A and 31B with different spectra are fixed to rotatable photometric turrets 34A and 34B, respectively. You can do it.

The photometric dichroic mirror 31A transmits the fluorescence from the scan unit 1 toward the second detection unit 3 or reflects it toward the photometric dichroic mirror 31B depending on the wavelength range. The photometric dichroic mirror 31B transmits the fluorescence from the photometric dichroic mirror 31A toward the wavelength selection mechanism 32A or reflects it toward the wavelength selection mechanism 32B depending on the wavelength range.

The wavelength selection mechanism 32A includes a diffraction grating (VPH (Volume Phase Holographic)) 35A that disperses fluorescence into spectral components, a swinging mirror 36A that reflects the fluorescence split by the diffraction grating 35A, and a fluorescence reflected by the swinging mirror 36A. is provided on the light-receiving surface of the photodetector 33A, and a slit (light-shielding slit) 38A for partially shielding the fluorescence condensed by the imaging lens 37A.

The diffraction grating 35A splits the spectral components of the fluorescence transmitted through the photometric dichroic mirror 31B in one direction. The swinging mirror 36A is provided so as to be swingable about a swinging axis orthogonal to the arrangement direction of the spectral trains separated by the diffraction grating 35A. The swinging mirror 36A can change the spectral components passed through the slit 38A according to the swinging angle.

The slit 38A includes a fixed member 39A and a movable member 40A that is spaced apart from the fixed member 39A in the direction in which the spectral arrays are arranged. The movable member 40A is provided so as to be movable relative to the fixed member 39A in the arrangement direction of the spectrum array, and can widen or narrow the gap between the fixed member 39A, that is, the opening through which the fluorescence passes. It's like The photodetector 33A has a wavelength sensitivity characteristic of higher sensitivity on the short wavelength side than the photodetectors 33C and 33D, which will be described later.

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.

The diffraction grating 35B splits the spectral components of the fluorescence reflected by the photometric dichroic mirror 31B in one direction. The swinging mirror 36B is provided so as to be swingable around a swinging axis perpendicular to the arrangement direction of the spectral trains separated by the diffraction grating 35B, and the spectral components passed through the slit 38B can be changed according to the swinging angle. is now possible. The slit 38B has a fixed member 39B and a movable member 40B. The photodetector 33B has the same wavelength sensitivity characteristics as the photodetector 33A.

The second detection unit 3 has a configuration similar to that of the first detection unit 2 . Specifically, 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.

Similar to the photometric dichroic mirrors 31A and 31B, the photometric dichroic mirrors 31C and 31D have a plurality of different spectra fixed to rotatable photometric turrets 34C and 34D, respectively. The photometric dichroic mirrors 31C and 31D to be inserted can be changed.

The photometric dichroic mirror 31C transmits or reflects the fluorescence from the photometric dichroic mirror 31A of the first detection unit 2 to the photometric dichroic mirror 31D depending on the wavelength range. The photometric dichroic mirror 31D transmits the fluorescence from the photometric dichroic mirror 31C toward the wavelength selection mechanism 32C or reflects it toward the wavelength selection mechanism 32D depending on the wavelength range.

The wavelength selection mechanism 32C selects a wavelength to be detected from fluorescence that has passed through the photometric dichroic mirror 31D. This wavelength selection mechanism 32C includes a diffraction grating (VPH) 35C, a swinging mirror 36C, an imaging lens 37C, and a slit (light shielding slit) 38C.

The diffraction grating 35C unidirectionally disperses the spectral component of fluorescence from the photometric dichroic mirror 31D. The swinging mirror 36C is provided so as to be swingable around a swinging axis perpendicular to the arrangement direction of the spectral trains separated by the diffraction grating 35C, and the spectral components passed through the slit 38C can be changed according to the swinging angle. is now possible.

The slit 38C has a fixed member 39C and a movable member 40C. The photodetector 33C detects the wavelength selected by the wavelength selection mechanism 32C. The photodetector 33C has a wavelength sensitivity characteristic of higher sensitivity on the longer wavelength side than the photodetectors 33A and 33B.

The wavelength selection mechanism 32D selects a wavelength to be detected from fluorescence reflected by the photometric dichroic mirror 31D. This wavelength selection mechanism 32D includes a diffraction grating (VPH) 35D, a swinging mirror 36D, an imaging lens 37D, and a slit (light shielding slit) 38D.

The diffraction grating 35D splits the spectral component of fluorescence from the photometric dichroic mirror 31D in one direction. The swinging mirror 36D is provided so as to be swingable around a swinging axis perpendicular to the arrangement direction of the spectral trains separated by the diffraction grating 35D, and the spectral components passed through the slit 38D can be changed according to the swinging angle. is now possible.

The slit 38D has a fixed member 39D and a movable member 40D. The photodetector 33D detects the wavelength selected by the wavelength selection mechanism 32D. This photodetector 33D has the same wavelength sensitivity characteristics as the photodetector 33C.

The photodetector 33A or photodetector 33B and the photodetector 33C or photodetector 33D are examples of a plurality of photodetectors having different wavelength sensitivity characteristics. An excitation turret 22 to which a plurality of excitation dichroic mirrors 19 with different spectra are fixed, a photometry turret 34A to which a plurality of photometry dichroic mirrors 31A with different spectra are fixed, and a photometry turret 34B to which a plurality of photometry dichroic mirrors 31B with different spectra are fixed. , wavelength selection mechanisms 32A and 32B, a photometry turret 34C to which a plurality of photometry dichroic mirrors 31C with different spectra are fixed, a photometry turret 34D to which a plurality of photometry dichroic mirrors 31D with different spectra are fixed, and the wavelength selection mechanisms 32C and 32D are 1 is an example of a detection wavelength changing unit capable of changing the detection wavelength of each of a plurality of photodetectors having different wavelength sensitivity characteristics. In the following description, the detection wavelength changing section refers to the excitation turret 22, the photometry turret 34, and the wavelength selection mechanism 32. FIG. The excitation dichroic mirror 19 and the photometric dichroic mirrors 31A, 31B, 31C, and 31D are examples of spectral optical elements.

The fluorescence microscope device 100 also includes an input device 4 , a display device 5 and a control device 6 . The input device 4 performs various inputs such as input of a fluorescent dye or wavelength range to be observed and input of settings related to the photodetector 33 and the like used for observation of the observation target, according to the user's input operation. The input device 4 is, for example, a keyboard, mouse, or touch panel.

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

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

The control device 6 also has functions as an observation condition optimization section and an image construction section. The function of the observation condition optimization unit is to determine and select one or more optimum photodetectors 33 to be used for observation of an observation target from among a plurality of photodetectors 33 having different wavelength sensitivity characteristics; This is a function for optimizing the detection wavelength changer for observation of the observation target. In the selection of the photodetector 33, the first detector 33A is preferentially selected over the second detector 33B within the first detection unit 2, and the second detector 33D is selected over the fourth detector 33D within the second detection unit 3. The third detector 33B may be preferentially selected. The function as the image construction unit is a function of constructing an image based on the fluorescence intensity signal detected by one or more photodetectors 33 selected by the function as the observation condition optimization unit. When there are a plurality of photodetectors 33, an image is constructed based on the sum of intensity signals of fluorescence detected by each of them.

The control device 6 is, for example, a PC (Personal Computer) and includes a processor 6a and a memory 6b. The above-described control 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 may be realized by hardware processing. , may be implemented by a combination of software processing and hardware processing. Processor 6a includes, for example, one or more integrated circuits. The integrated circuit 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), or the like.

The memory 6b stores programs executed by the processor 6a. The memory 6b also stores first information, second information, and third information. The first information is information on wavelength sensitivity characteristics of each of the photodetectors 33A, 33B, 33C, and 33D. The second information is information on the spectrum of each excitation dichroic mirror 19, the spectrum of each photometric dichroic mirror 31A, the spectrum of each photometric dichroic mirror 31B, the spectrum of each photometric dichroic mirror 31C, and the spectrum of each photometric dichroic mirror 31D. The third information is information on the spectrum of the fluorescent dye that can be observed. The memory 9 also stores information about the oscillation wavelength (spectrum) of each laser beam that the laser light source 11 can emit. The memory 9 also stores images.

Memory 6b includes non-transitory computer-readable media storing programs executed by processor 11 . Memory 6b may include, for example, any one or more semiconductor memories, or one or more other storage devices. Semiconductor memory includes, for example, volatile memory such as RAM (Random Access Memory), ROM (Read Only Memory), programmable ROM, and non-volatile memory such as flash memory. The RAM may include, for example, DRAM (Dynamic Random Access Memory), SRAM (Static Random Access Memory), and the like. Other storage devices may include, for example, computer-readable media such as magnetic storage devices including, for example, magnetic disks, and computer-readable media such as optical storage devices including, for example, optical discs.

In the fluorescence microscope apparatus 100 having such a configuration, prior to observation, when the user inputs the fluorescent dye or wavelength range to be observed using the input device 4, the plurality of photodetectors 33 having different wavelength sensitivity characteristics are selected. , the optimum one or more photodetectors 33 to be used for observation of the observation object are determined and selected, and the detection wavelength changing section is optimized for observation of the observation object. During observation, an image is constructed based on intensity signals of fluorescence detected by one or more selected photodetectors 33 . For example, when a plurality of photodetectors 33 are selected, image construction is performed based on the sum of intensity signals of fluorescence detected by each of the plurality of photodetectors 33 . Then, the image is displayed on the display device 5 or stored in the memory 6b. Processing performed by such a fluorescence microscope apparatus 100 will be described in detail below.

FIG. 2 is a flowchart illustrating an observation condition optimization process performed by the fluorescence microscope apparatus 100. FIG. FIG. 3 is a flowchart illustrating the details of the process of step S20.

In the observation condition optimization process, as illustrated in FIG. 2, when the user inputs a fluorescent dye to be observed using the input device 4 (step S10), the control device 6 selects the fluorescent dye to be observed. An optimum observation condition for observation is derived (step S20). Specifically, the observation conditions are the one or more photodetectors 33 to be used, the oscillation wavelength of the laser light emitted from the laser light source 11, and the one or more photodetectors 33 to be used from the sample A. An excitation dichroic mirror 19 and a photometric dichroic mirror 31 inserted into the optical path leading to , and wavelengths selected by a wavelength selection mechanism 32 for selecting wavelengths detected by one or more photodetectors 33 used are derived.

More specifically, the process of step S20 is performed by the process illustrated in FIG. 3, for example. First, the control device 6 determines how much intensity signal can be obtained by the one photodetector 33 when observation is performed using the one photodetector 33, in all possible combinations. is simulated (step S21). Here, all conceivable combinations include the one photodetector 33 to be used, the oscillation wavelength of the laser light, and the excitation dichroic mirror 19 inserted in the optical path from the sample A to the one photodetector 33 to be used. and a combination of the photometric dichroic mirror 31 and the wavelength selected by the wavelength selection mechanism 32 that selects the wavelength detected by the single photodetector 33 used. However, the wavelength selected by the wavelength selection mechanism 32 at this time is the wavelength range (wavelength range of the observed fluorescent dye) pre-associated with the fluorescent dye to be observed.

The simulation of step S21 can be performed based on the first information, the second information, and the third information stored in the memory 6b. More specifically, the intensity signal obtained by each combination is the fluorescence spectrum of the fluorochrome to be observed (included in the third information) and the wavelength sensitivity characteristic of one photodetector 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 the one photodetector 33 used. , can be obtained from the sum of products for each wavelength. Further, the obtained intensity signal may be obtained by further multiplying this by the excitation efficiency of the fluorescent dye to be observed at the oscillation wavelength of the laser light.

When simulating in step S21, a combination that uses an oscillation wavelength of laser light at which the excitation efficiency of the fluorescent dye to be observed is below a certain level, or an excitation dichroic that cannot reflect the oscillation wavelength of laser light. A combination that is known from the beginning to be impossible for fluorescence observation, such as a combination using the mirror 19, may be excluded from simulation targets. Thereby, the processing time for the simulation in step S21 can be further shortened.

After 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). In the simulation result, if there are a plurality of combinations that maximize the obtained intensity signal, any one of the combinations may be obtained.

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

However, the wavelength selected by each wavelength selection mechanism 32 at this time is the wavelength range previously associated with the fluorescent dye to be observed. It is defined as a wavelength range divided by wavelengths at which the transmittance (or reflectance) in the spectrum becomes 50%. For example, the wavelength range previously associated with the fluorescent dye to be observed is 500 to 600 nm, and the photometric dichroic mirror 31 that disperses light between the two photodetectors 33 used reflects light with a wavelength of 530 nm or less. (reflectance of 80% or more), transmits light with a wavelength of 550 nm or more (transmittance of 80% or more), and the wavelength at which the transmittance (or reflectance) in the spectrum becomes 50% is 540 nm, the photometric dichroic The wavelength selected by the wavelength selection mechanism 32 on the reflection side of the mirror 31 is 500 to 540 nm, and the wavelength selected by the wavelength selection mechanism 32 on the transmission side is 540 to 600 nm.

In this way, the wavelengths selected by each wavelength selection mechanism 32 do not overlap, but the selected wavelengths are equal to the wavelength range of the rising section (or falling section) of the transmittance (or reflectance) in the spectrum of the photometric dichroic mirror 31. may be duplicated. For example, in the above example, if the wavelength range of the rising section is 530 to 550 m, the wavelength selected by the wavelength selection mechanism 32 on the reflection side is 500 to 550 nm, and the wavelength selected by the wavelength selection mechanism 32 on the transmission side is It may be 530 to 600 nm. As a result, the two photodetectors 33 can detect the fluorescence in the wavelength range of the rising section (or the falling section) without omission.

The simulation in step S23 can be performed based on the first information, second information, and third information stored in the memory 6b, as in step S21. More specifically, the intensity signal obtained by each combination is the fluorescence spectrum of the fluorescent dye to be observed (included in the third information) and the wavelength sensitivity characteristic of one of the two photodetectors 33 used (the third information). 1 information) and the respective spectra (transmission spectra or reflection spectra) of the excitation dichroic mirror 19 and the photometric dichroic mirror 31 inserted in the optical path from the sample A to one of the photodetectors 33 (included in the second information ), the sum of products for each wavelength is obtained, and the fluorescence spectrum of the fluorescent dye to be observed (included in the third information) and the wavelength sensitivity characteristic of the other of the two photodetectors 33 to be used ( the spectrum (transmission spectrum or reflection spectrum) of each 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 (included in the first information) (included in the second information) included) for each wavelength, and adding up the respective sums. Alternatively, each sum may be further multiplied by the excitation efficiency of the fluorescent dye to be observed at the oscillation wavelength of the laser beam, and then summed to obtain the intensity signal.

In the simulation of step S23, as in step S21, a combination using the oscillation wavelength of the laser light at which the excitation efficiency of the fluorescent dye to be observed is below a certain level, or the oscillation wavelength of the laser light is reflected. Combinations that are known from the beginning to be incapable of fluorescence observation, such as combinations that use the excitation dichroic mirror 19 that cannot be used, may be excluded from simulation targets. As a result, the processing time for the simulation in step S23 can be further shortened.

When the simulation of step S23 is completed, the control device 6 acquires the combination that maximizes the strength signal obtained from the simulation result and the strength signal at that time (step S24). In addition, in the simulation result, if there are a plurality of combinations that maximize the obtained intensity signal, any one of the combinations may be acquired.

Next, the control device 6 combines the intensity signal of the combination obtained in step S22 (the combination in which one photodetector 33 is used) and the combination obtained in step S24 (the number of photodetectors 33 to be used is two). combination), and the ratio of the intensity signal of the combination acquired in step S24 to the intensity signal of the combination acquired in step S22 is a predetermined ratio (for example, 110%) or more. is determined (step S25).

If the determination result in step S25 is YES, the control device 6 selects the combination obtained in step S24 as the optimum observation condition 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 condition for observation (step S27).

In the judgment and selection in steps S25 to S27, the reason why the combination with the higher intensity signal is not simply compared is that when a plurality of photodetectors 33 are used, the photodetectors to be used This is because the number of photodetectors to be used increases compared to the case of using only one, so it is assumed that noise components are added accordingly.

After deriving the optimal observation conditions for observing the fluorescent dye to be observed in this way (when step S20 in FIG. 2 ends), the control device 6 selects one or more light detectors to be used according to the observation conditions. In addition to setting the device 33, the laser light source 11, the excitation turret 22, the photometry turret 34, and the wavelength selection mechanism 32 are controlled to determine the oscillation wavelength of the laser light emitted by the laser light source 11 and the wavelength to be used from the sample A. Selection is made by an excitation dichroic mirror 19 and a photometric dichroic mirror 31 that are inserted into the optical path leading to one or more photodetectors 33, and a wavelength selection mechanism 32 that selects the wavelengths detected by the one or more photodetectors used. A wavelength is set (step S30). Thereby, the optimum observation conditions for observation of the fluorescent dye to be observed are set.

Observation of the fluorescent dye to be observed under the set observation conditions 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 , the fluorescence from sample A is detected. Then, the fluorescence intensity signal detected by one photodetector 33 and the scanning position by the scanner 17 are output to the control device 6 . The controller 6 AD-converts the fluorescence intensity signal detected by 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 the memory 6b.

On the other hand, when a plurality of photodetectors 33 are set (selected) as the photodetectors 33 to be used, the scanner 17 scans the sample A with laser light emitted from the laser light source 11, and the plurality of photodetectors 33 detects the fluorescence from sample A; Then, fluorescence intensity signals detected by each of the plurality of photodetectors 33 and scanning positions by the scanner 17 are output to the control device 6 . The controller 6 AD-converts and adds up the fluorescence intensity signals detected by each of the plurality of photodetectors 33, and constructs an image based on the intensity signals after the addition and the corresponding scanning positions. Then, the constructed image is displayed on the display device 5 or stored in the memory 6b.

Note that the AD conversion described above may be performed before the intensity signal is 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 may be performed by an AD conversion circuit connected between each photodetector 33 and the control device 6. can be Also, the order of the AD conversion and summation described above may be reversed. That is, the AD conversion may be performed after summing the intensity signals. In this case, the summing of the signals is performed by a summing circuit provided on the photodetector side of the AD conversion circuit.

According to the fluorescence microscope apparatus 100 described above, even if the photodetector 33 having the highest wavelength sensitivity over the entire wavelength range of the fluorescent dye to be observed is not provided, light with high sensitivity over the entire wavelength range detection becomes possible. For example, when the wavelength range of the fluorescent dye to be observed is 500 to 600 nm, the photodetector 33A (or 33B) has higher sensitivity than the photodetector 33C (or 33D) in the wavelength range of 500 to 545 nm, In the case where the photodetector 33c (or 33D) has higher sensitivity than the photodetector 33A (or 33B) in the wavelength range of 545 to 600 nm, conventionally, one of the photodetectors 33 (for example, Since light detection is performed using the photodetector 33C (or 33D) having a wider wavelength range with high sensitivity, high sensitivity light detection could not be performed over the entire wavelength range to be observed. On the other hand, in this embodiment, the wavelength region of 500 to 545 nm uses the photodetector 33A (or 33B), and the wavelength region of 545 to 600 nm uses the photodetector 33c (or 33D). By combining the intensity signals obtained by the respective photodetectors 33, highly sensitive photodetection can be performed over the entire wavelength range to be observed.

In the process illustrated in FIG. 2 described above, the input of the fluorescent dye to be observed in step S10 may be performed from the fluorescent dye input screen displayed on the display device 5 by the control device 6, for example. FIG. 4 is a diagram illustrating a fluorescent dye input screen.

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, and an observation target display field that displays fluorescent dyes to be observed. 202, an "Add" button 203, and the like.

On the fluorescent dye input screen, the user uses the input device 4 to double-click the desired fluorescent dye displayed in the list display field 201, or select it and press the "Add" button 203, A desired fluorescent dye can be added (input as an observation target) to the observation target display field 202 .

In this way, when a fluorescent dye to be observed is input from the fluorescent dye input screen, when a fluorescent dye is added to the observation target display column 202, the fluorescent dye displayed in the observation target display column 202 is displayed. The above-described processing of step S20 (derivation of optimum observation conditions) is performed for the fluorescent dye to be observed. However, when a plurality of fluorescent dyes are displayed in the observation target display column 202 as a result of adding the fluorescent dye to the observation target display column 202, the processing of step S20 is performed for each of the fluorescent dyes. will be In this case, assuming simultaneous observation of the plurality of fluorescent dyes, optimum observation conditions may be derived for each fluorescent dye. Then, in the observation target display field 202, for each fluorescent dye to be observed, the channel and the device ID of one or more photodetectors 33 used for observation obtained in the processing of step S20 are additionally displayed. be done. In the example of FIG. 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 IDs "SD2, HSD3" are displayed. is additionally displayed.

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

Further, when the “OK” button 204 on the fluorescent dye input screen is pressed, the control device 6 may further display the 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 observation of the observation target, and is also a brightness adjustment screen for each channel. FIG. 5 is a diagram illustrating a setting input screen.

As illustrated in FIG. 5, the setting input screen displays the fluorescent dye to be observed for each channel and the device ID of one or a plurality of photodetectors 33 to be used. In the example of FIG. 5, the fluorescent dye "DAPI" and the device ID "SD1" for the channel "CH1" are displayed, and the fluorescent dye "Alexa Fluor 488" and the device ID "SD2, HSD3" for the channel "CH2" are displayed. is displayed.

On the setting input screen, the user can set (change) the wavelength range for observing the fluorescent dye of each channel. This setting can be performed by directly inputting a numerical value or the like using the input device 4 from the setting input field 301 . For example, 500 to 600 nm set as the wavelength range for observing the fluorescent dye "Alexa Fluor 488" in the channel "CH2" can be changed to 510 to 600 nm. However, at this time, the wavelength range detected by the photodetector 33 (eg, 33B) with the device ID “SD2” is set to 500 to 540 nm, and the wavelength detected by the photodetector 33 (eg, 33C) with the device ID “HSD3” is If the range was 540 to 600 nm, the wavelength range detected by the photodetector 33 with the device ID "SD2" is changed to 510 to 540 nm, and the wavelength range detected by the photodetector 33 with the device ID "HSD3" is changed. is left as is. Alternatively, when the wavelength range to be observed is changed to 540 to 600 nm, the photodetector 33 with the device ID "SD2" is not used, and only the photodetector 33 with the device ID "HDS3" is used. .

In addition, on the setting input screen, the user can set the oscillation wavelength and light intensity of the laser beam used for observation, the HV (High Voltage) of the PMT that is the photodetector 33, the gain of the intensity signal, and the intensity of each channel. A signal offset can be set (changed). This setting can be performed by moving a slide bar (for example 302a), pressing left and right buttons (for example 302b), or directly inputting numerical values using the input device 4 from the setting input field 302. FIG.

In the setting for the photodetector 33 in the setting input field 302, even if there are a plurality of photodetectors 33 to be used, such as the channel "CH2" illustrated in FIG. will be That is, the setting is performed as a setting for one pseudo photodetector 33 . Then, when the settings for the one pseudo photodetector 33 are made, the same settings are made for each of the plurality of photodetectors 33 to be used. Therefore, when a plurality of photodetectors 33 are used, the user does not need to individually set the photodetectors 33 .

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 for observing the fluorescent dye. These are displayed in the display column 303 .

When a plurality of photodetectors 33 are used, there may be a difference in sensitivity between the photodetectors 33 . In such a case, the sensitivity of one or more photodetectors 33 among the plurality of photodetectors 33 to be used may be corrected so that the sensitivities of the respective photodetectors 33 match.

For example, even if the settings for the plurality of photodetectors 33 used are the same, if there is a difference in the output intensity signal per incident photon in each photodetector 33, the output intensity per incident photon is The sensitivity (HV or gain) of one or more photodetectors 33 among the plurality of photodetectors 33 used may be corrected so that the signals match.

In this case, for example, the fourth information about the output intensity signal per incident photon (or the ratio of the number of incident photons to the output intensity signal) for each photodetector 33 is stored in advance in the memory 6b, One or more of the plurality of photodetectors 33 to be used based on the fourth information so that the output intensity signal per incident photon for each of the plurality of photodetectors 33 to be used matches The sensitivity (HV or gain) of the detector 33 may be corrected. Alternatively, the intensity signal may be corrected before the above summation based on the fourth information. For example, when the photodetectors 33 to be used are the photodetector 33A and the photodetector 33C, the output intensity signal per incident photon is twice as large for the photodetector 33C as for the photodetector 33A. , the output intensity signal of the photodetector A before summation should be doubled, or the output intensity signal of the photodetector 33C before summation should be halved.

Further, for example, the fifth information about the intensity signal output by each photodetector 33 when no photons are incident is stored in advance in the memory 6b, and the number of photodetectors 33 to be used is plural (N). Fluorescence detected by each of the N photodetectors 33 when constructing an image based on the result of summing the intensity signals of the fluorescence detected by each of the N photodetectors 33 After adding up the intensity signals of , based on the fifth information, an image is constructed by subtracting a signal obtained by multiplying the intensity signal output by each photodetector 33 when no photons are incident by N-1. can be As a result, it is possible to prevent the offset level of the intensity signal from increasing due to the summation.

Further, in step S10 illustrated in FIG. 2, 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 to enable input of the wavelength range to be observed, and the wavelength range to be observed may be input from the input screen. good. In this case, in step S20, instead of the fluorescence spectrum of the fluorochrome to be observed, the spectrum with the maximum intensity (100%) over the entire wavelength range to be observed is used. In addition, the input screen is an input screen having an input field that enables input of the fluorescent dye to be observed and an input field that enables input of the wavelength range to be observed. You may make it possible.

In the fluorescence microscope apparatus 100, a specific example of the plurality of photodetectors 33 having different wavelength sensitivity characteristics is, for example, a PMT having high sensitivity on the short wavelength side and using GaAsP (gallium arsenide phosphide) as a photocathode. (Photomultiplier-Tube) and a PMT having high sensitivity on the long wavelength side and using GaAs (gallium arsenide) for the photocathode may be used. Alternatively, for example, a plurality of SiPMs (Silicon Photomultipliers) having different wavelength sensitivity characteristics may be used. In a plurality of SiPMs with different wavelength sensitivity characteristics, even if the wavelength sensitivity characteristics are different, the output intensity signal of each SiPM has quantitative properties (the ratio of the number of incident photons and the output intensity signal can be kept constant). This is more effective in that the quantification is not lost even if the output intensity signals are summed.

In the fluorescence microscope apparatus 100, a photometric turret 34A to which a plurality of photometric dichroic mirrors 31A with different spectra are fixed, a photometric turret 34B to which a plurality of photometric dichroic mirrors 31B with different spectra are fixed, and a plurality of photometric dichroic mirrors with different spectra. A gradation dichroic mirror may be provided instead of each of the photometric turret 34C to which 31C is fixed and the photometric turret 34D to which a plurality of photometric dichroic mirrors 31D having different spectra are fixed. The gradation dichroic mirror is provided so as to be movable in the direction along the incident surface on 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 limitation. . In this case, for example, instead of the combination of the photometric dichroic mirrors 31 used in the photometric turrets 34, the combination of fluorescence incident positions used in the gradation dichroic mirrors may be used.

Moreover, the fluorescence microscope apparatus 100 may be provided with three or more photodetectors having different wavelength sensitivity characteristics by adding one or more detection units adjacent to the second detection unit 3 . In this case, the additional 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 additional detection unit are different. are different from the wavelength sensitivity characteristics of the two photodetectors provided in the detection unit.

The above-described embodiments are specific examples for easy understanding of the invention, and the 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.

1 scanning unit 2 first detection unit 3 second detection unit 4 input device 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 turrets 31A, 31B, 31C, 31D photometric dichroic mirrors 32A, 32B, 32C, 32D wavelength selection mechanism 33A, 33B, 33C, 33D photodetector 34A, 34B, 34C, 34D Photometric turrets 35A, 35B, 35C, 35D Diffraction gratings 36A, 36B, 36C, 36D Oscillating mirrors 37A, 37B, 37C, 37D Imaging lenses 38A, 38B, 38C, 38D Slits 39A, 39B, 39C, 39D Fixed members 40A, 40B, 40C, 40D Movable member 100 Fluorescence microscope device 201 List display column 202 Observation object display columns 203, 204 Buttons 301, 302 Setting input column 303 Display column

Claims (12)

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 or wavelength range to be observed;
a memory that stores first information about wavelength sensitivity characteristics of each of the plurality of photodetectors;
one of the plurality of photodetectors to be used for observing the observation target based on the fluorescent dye or wavelength range to be observed input by the input device and the first information stored in the memory an observation condition optimizing unit that determines and selects one or more photodetectors that are most suitable for observing and optimizing the detection wavelength changing unit for observation of the observation target;
with
summing fluorescence intensity signals detected by each of the plurality of photodetectors when using a plurality of photodetectors to observe the observation target;
A fluorescence microscope device characterized by: the memory further stores second information about the spectrum of the spectroscopic optical element included in the detection wavelength changing unit;
The observation condition optimization unit, based on the fluorescent dye or wavelength range to be observed input by the input device, and the first information and the second information stored in the memory, Determining and selecting one or more photodetectors that are most suitable for observing the observation target from among the photodetectors, and optimizing the detection wavelength changing unit for observing the observation target. ,
2. The fluorescence microscope apparatus according to claim 1, wherein: The memory further stores third information about the spectrum of the fluorescent dye that can be observed,
The observation condition optimization unit is configured to optimize the observation condition based on the fluorescent dye to be observed input by the input device and the first information and the third information stored in the memory, or by the input device the observation target from among the plurality of photodetectors based on the inputted fluorescent dye as the observation target and the first information, the second information, and the third information stored in the memory; Determining and selecting the optimum one or more photodetectors to be used for observation of and optimizing the detection wavelength changing unit for observation of the observation target;
3. The fluorescence microscope apparatus according to claim 1, wherein: Further comprising an image construction unit that constructs an image based on intensity signals of fluorescence detected by one or more photodetectors selected by the observation condition optimization unit,
When the observation condition optimization unit selects a plurality of photodetectors, the image construction unit constructs an image based on a result of summing intensity signals of fluorescence detected by each of the plurality of photodetectors. I do,
4. The fluorescence microscope apparatus according to any one of claims 1 to 3, characterized in that: further comprising a display device for displaying a setting input screen that allows the input device to input settings related to one or more photodetectors selected by the viewing condition optimization unit;
When a plurality of photodetectors are selected by the viewing condition optimizing unit, the setting input screen displayed by the display device includes settings for the plurality of photodetectors selected by the viewing condition optimizing unit. As an input field for enabling input, an input field for enabling input of settings related to one pseudo photodetector is displayed, and based on the settings related to the one pseudo photodetector entered in the input field settings are made for the plurality of photodetectors selected by the viewing condition optimization unit,
5. The fluorescence microscope apparatus according to any one of claims 1 to 4, characterized in that: Each of the plurality of photodetectors with different wavelength sensitivity characteristics is SiPM (Silicon Photomultiplier),
6. The fluorescence microscope apparatus according to any one of claims 1 to 5, characterized in that: When a plurality of photodetectors are used for observing the observation target, the observation condition optimizing unit optimizes the detection wavelength changing unit by optimizing the spectrum of the spectroscopic optical element that separates the photodetectors to be used. overlap between the photodetectors as part of the detection wavelength;
7. The fluorescence microscope apparatus according to any one of claims 1 to 6, characterized in that: When a plurality of photodetectors are used to observe the observation target, the observation condition optimization unit is configured to reduce the maximum intensity signal obtained when a single photodetector is used to observe the observation target. When the ratio of the maximum intensity signals obtained by summing is equal to or greater than a predetermined ratio, a plurality of photodetectors when the maximum intensity signals are obtained by summation are used for observation of the observation target. judging and selecting as a vessel,
8. The fluorescence microscope apparatus according to any one of claims 1 to 7, characterized in that: The memory further stores fourth information regarding 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 viewing condition optimizing unit, the output intensity signals per incident photon for each of the plurality of photodetectors are stored in the memory so as to match correcting one or more sensitivities of the plurality of photodetectors based on the fourth information;
9. The fluorescence microscope apparatus according to any one of claims 1 to 8, characterized in that: The memory further stores fifth information about an intensity signal output by each photodetector when no photons are incident,
When N photodetectors, which are a plurality of photodetectors, are selected by the observation condition optimization unit, the image construction unit generates intensity signals of fluorescence detected by each of the N photodetectors is added, and then, based on the fifth information stored in the memory, a signal obtained by multiplying the intensity signal output by each photodetector when the photon is not incident is subtracted by N−1. perform image construction,
10. The fluorescence microscope apparatus according to any one of claims 1 to 9, characterized in that: 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 first information about the wavelength sensitivity characteristics of each of the plurality of photodetectors. A method for optimizing observation conditions for a fluorescence microscope apparatus comprising a memory for storing
Based on the input fluorescent dye or wavelength range to be observed and the first information stored in the memory, one of the plurality of photodetectors that is most suitable for observation of the observation target is selected. or determining and selecting a plurality of photodetectors and optimizing the detection wavelength changing unit for observation of the observation target,
summing fluorescence intensity signals detected by each of the plurality of photodetectors when using a plurality of photodetectors to observe the observation target;
An observation condition optimization method characterized by: 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 first information about the wavelength sensitivity characteristics of each of the plurality of photodetectors. in a computer of a fluorescence microscope apparatus comprising a memory for storing
Based on the input fluorescent dye or wavelength range to be observed and the first information stored in the memory, one of the plurality of photodetectors that is most suitable for observation of the observation target is selected. or determining and selecting a plurality of photodetectors and optimizing the detection wavelength changing unit for observation of the observation target,
summing fluorescence intensity signals detected by each of the plurality of photodetectors when using a plurality of photodetectors to observe the observation target;
A program characterized by executing the process of

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