Confocal microscope unit, confocal microscope, and method for controlling confocal microscope unit

Abstract

A confocal microscope unit 1 comprises: a light source device 10a having a light emitting element 21, a light detector 22, a driving unit 23, and a control unit 24; and a scanning mirror 4 that scans using an excitation light output by the light source device 10a. In a configuration mode, the control unit 24 executes a first control for adjusting and outputting a driving signal that drives the driving unit 23 on the basis of a detection signal from the light detector 22 and a control signal, and generates and stores a data table indicating a correspondence between a driving current and a quantity of the excitation light by executing the first control while changing the value of the control signal. In an operation mode, the control unit 24 executes a second control for outputting the control signal as the driving signal, reads out the data table, executes the second control using the control signal corresponding to the driving current corresponding to a target quantity of light on the basis of the data table, and executes a control for stopping the driving current if the quantity of light indicated by the detection signal is greater than a prescribed value.

Description

[Technical Field]

[0001] The present disclosure relates to a confocal microscope unit that constitutes a confocal microscope, and to the confocal microscope. [Background technology]

[0002] Conventionally, confocal microscope units that constitute a confocal microscope by being attached to a connection port of a microscope having a microscope optical system have been known (see, for example, Patent Documents 1 to 3 listed below). Such confocal microscope units make it easy to realize confocal imaging using a microscope. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2020 / 196783 [Patent Document 2] International Publication No. 2020 / 196782 [Patent Document 3] International Publication No. 2020 / 196784 Summary of the Invention [Problem to be solved by the invention]

[0004] In conventional confocal microscope units such as those described above, an optical image of a sample is acquired by scanning the sample with excitation light. When scanning the sample two-dimensionally with excitation light, the scanning speed on the sample must be changed. Even in such cases, it is desirable to uniformize the irradiation energy of the excitation light per unit area (unit length) of the sample.

[0005] Therefore, one aspect of the embodiment has been made in consideration of such problems, and aims to provide a confocal microscope unit and a confocal microscope that can accurately uniformize the irradiation energy of excitation light on a sample. [Means for solving the problem]

[0006] A confocal microscope unit according to one aspect of the embodiment is a confocal microscope unit that constitutes a confocal microscope by being attached to a connection port of a microscope having a microscope optical system, and includes a light source unit that outputs excitation light, a photodetector that detects the excitation light output from the light source and outputs a detection signal, a driver that supplies a drive current to the light source, and a control unit that outputs a drive signal to the driver in accordance with a control signal indicating a target light intensity, a scan mirror that scans the excitation light output from the light source unit over a sample, and a housing that is configured to be attachable to the connection port and to which the scan mirror and the light source unit are fixed, and the control unit has two functions: a setting mode and an operation mode, and in the setting mode, the control unit performs a first control that adjusts and outputs a drive signal based on the control signal and the detection signal, and performs the first control while changing the value of the control signal, thereby generating and storing a data table indicating the correspondence between the drive current and the light intensity of the excitation light, and in the operation mode, the control unit performs a second control that outputs the control signal as a drive signal, reads the data table, and performs the second control using a control signal corresponding to a drive current corresponding to the target light intensity based on the data table, and performs control that stops the drive current when the light intensity indicated by the detection signal exceeds a predetermined value.

[0007] Alternatively, a confocal microscope according to another aspect of the embodiment includes the confocal microscope unit according to the above aspect, and a microscope having a microscope optical system and a connection port to which the confocal microscope unit is attached.

[0008] Alternatively, a control method for a confocal microscope unit according to another aspect of the embodiment is a control method using a confocal microscope unit that constitutes a confocal microscope by being attached to a connection port of a microscope having a microscope optical system, the control method including a light source unit that outputs excitation light, a photodetector that detects the excitation light output from the light source and outputs a detection signal, a driver unit that supplies a drive current to the light source, and a controller that outputs a drive signal to the driver unit in accordance with a control signal indicating a target light intensity, a scan mirror that scans the excitation light output from the light source unit over a sample, and a housing that is configured to be attachable to the connection port and to which the scan mirror and the light source unit are fixed, the control method including the steps of: executing a first control that adjusts and outputs the drive signal based on the control signal and the detection signal, and executing the first control while changing the value of the control signal to generate and store a data table indicating the correspondence between the drive current and the intensity of the excitation light; executing a second control that outputs the control signal as a drive signal, reading the data table, and executing the second control using a control signal corresponding to a drive current corresponding to the target light intensity based on the data table, and executing control that stops the drive current when the light intensity indicated by the detection signal exceeds a predetermined value.

[0009] According to one aspect, another aspect, or another aspect, excitation light output from a light source in a light source unit is scanned onto a sample by a scan mirror, and a corresponding image of the sample can be observed through a microscope. In the light source unit, a setting mode function of the control unit generates and stores in advance a data table indicating the correspondence between the drive current supplied to the light source and the intensity of the excitation light. Based on the data table, an operation mode function of the control unit outputs a control signal corresponding to a drive current corresponding to a target light intensity as a drive signal to a drive unit that supplies the drive current to the light source. Additionally, the operation mode function stops the drive current when the intensity of the excitation light exceeds a predetermined value. This allows the light source to be driven quickly and accurately so that the intensity of the excitation light changes accordingly when a target light intensity is set so that the intensity of the excitation light changes in response to changes in the scanning speed of the excitation light on the sample. Furthermore, control is performed to stop the excitation light when the intensity of the excitation light exceeds a predetermined value, allowing the confocal microscope unit to be used safely. As a result, the irradiation energy of the excitation light on the sample can be precisely uniformed, and highly safe confocal imaging can be achieved. [Effects of the Invention]

[0010] According to one aspect of the present disclosure, the irradiation energy of excitation light on a sample can be made uniform with high precision. [Brief explanation of the drawings]

[0011] [Figure 1] 1 is a schematic configuration diagram of a confocal microscope A according to an embodiment. [Figure 2] FIG. 2 is a block diagram showing a schematic configuration of a light source device 10a of FIG. [Figure 3] FIG. 2 is a diagram showing a detailed circuit configuration of the light source device 10a of FIG. [Figure 4] 10 is a circuit diagram showing a switching state of the light source device 10a when operating in a setting mode. FIG. [Figure 5]10 is a circuit diagram showing a switching state of a light source device 10a when operating in an operation mode. FIG. [Figure 6] 10 is a flowchart showing the operation procedure of the function of the setting mode of the light source device 10a. [Figure 7] 10 is a flowchart showing the operation procedure of the functions in the operation modes of the light source device 10a. [Figure 8] 1 is a plan view showing an example of a scanning pattern of a first excitation light on a sample M using a confocal microscope A. FIG. [Figure 9] 9 is a graph showing the change over time in the amount of laser light output from the light source device 10a in accordance with the scanning mode shown in FIG. 8. [Figure 10] 10 is a graph showing the change over time in the amount of light in FIG. 9 in response to the sweep waveform of the scan mirror 4. [Figure 11] FIG. 10 is a diagram showing a circuit configuration of a light source device 110a according to a modified example. DETAILED DESCRIPTION OF THE INVENTION

[0012] Hereinafter, embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. In the description, the same elements or elements having the same functions will be denoted by the same reference numerals, and redundant description will be omitted.

[0013] Fig. 1 is a schematic diagram of a confocal microscope A according to an embodiment. The confocal microscope A shown in Fig. 1 constitutes a confocal microscope that acquires images that enable the construction of an optical tomographic image of a sample M to be observed, and is configured by connecting a confocal microscope unit 1 to a connection port P1 of a microscope 50 for connecting to an external unit. This confocal microscope unit 1 is a device that irradiates excitation light onto a sample M placed on a stage of the microscope 50 via a microscope optical system such as an imaging lens 51 and an objective lens 52 within the microscope 50, receives (detects) fluorescence generated from the sample M in response to the excitation light, and generates and outputs an optical tomographic image.

[0014] In detail, the confocal microscope unit 1 includes a main housing 2, a lens barrel 3 that forms part of the main housing 2 and is detachably connected to a connection port P1 of the microscope 50, a scan mirror 4, a fixed mirror 5, first to fourth subunits 6a to 6d that are fixed within the main housing 2, and a scan lens 7 that is fixed within the lens barrel 3. Each of the components of the confocal microscope unit 1 will be described in detail below.

[0015] The scan lens 7 in the lens barrel 3 is an optical element that relays the reflecting surface of the scan mirror 4 to the pupil position of the objective lens 52, and at the same time, has the function of forming a spot on the primary image plane of the microscope optical system of the microscope 50. The scan lens 7 guides the excitation light scanned by the scan mirror 4 to the microscope optical system, thereby irradiating the sample M, and guides the fluorescence generated from the sample M to the scan mirror 4 in response.

[0016] The scan mirror 4 in the main housing 2 is an optical scanning element such as a MEMS (Micro Electro Mechanical System) mirror configured so that a reflector can be tilted on two axes. The scan mirror 4 has the role of scanning the excitation light output from the first to fourth subunits 6a to 6d onto the sample M by continuously changing the reflection angle, and guiding the fluorescence generated in response to the excitation light toward the first to fourth subunits 6a to 6d.

[0017] The fixed mirror 5 is a light reflecting element fixed inside the main housing 2, and reflects the excitation light output from the first to fourth subunits 6a to 6d toward the scan mirror 4, and reflects the fluorescence reflected by the scan mirror 4 toward the first to fourth subunits 6a to 6d coaxially with the excitation light.

[0018] The first subunit 6a includes a base plate 8a, a dichroic mirror (first beam splitter) 9a arranged on the base plate 8a, a light source device (light source unit) 10a, a dichroic mirror 11a, a pinhole plate (first diaphragm member) 12a, and a photodetector (first photodetector) 13a. The dichroic mirror 9a is fixed in the fluorescence reflection direction of the fixed mirror 5 and is a beam splitter that reflects the first excitation light of wavelength λ1 irradiated by the first subunit 6a and the first fluorescence of wavelength range Δλ1 generated from the sample M in response to the first excitation light, and transmits light of longer wavelengths than the first excitation light and the first fluorescence. The dichroic mirror 11a is arranged in the first fluorescence reflection direction of the dichroic mirror 9a and is a beam splitter that transmits the first fluorescence of wavelength range Δλ1 and reflects the first excitation light of wavelength λ1 shorter than the wavelength range Δλ1. The light source device 10a incorporates a light-emitting element (e.g., a laser diode) that outputs first excitation light (e.g., laser light) with a wavelength λ1, and is positioned so that the first excitation light is reflected by the dichroic mirror 11a toward the dichroic mirror 9a coaxially with the first fluorescence. The pinhole plate 12a is positioned so that its pinhole position coincides with a conjugate position of the spot of the first excitation light on the sample M. It is an aperture member that limits the light flux of the first fluorescence, and together with the light source device 10a and the like, constitutes a confocal optical system. The pinhole diameter of the pinhole plate 12a can be adjusted externally, making it possible to change the resolution and signal intensity of the image detected by the photodetector 13a. The photodetector 13a is positioned so that its detection surface faces the pinhole plate 12a and receives and detects the first fluorescence that has passed through the pinhole plate 12a. The photodetector 13a is a photomultiplier tube, a photodiode, an avalanche photodiode, a multi-pixel photon counter (MPPC), a hybrid photodetector (HPD), an area image sensor, or the like.

[0019] The second to fourth subunits 6b to 6d have the same configuration as the first subunit 6a.

[0020] That is, the second subunit 6b includes a base plate 8b, a dichroic mirror (second beam splitter) 9b, a light source device 10b, a dichroic mirror 11b, a pinhole plate (second diaphragm member) 12b, and a photodetector (second photodetector) 13b. The dichroic mirror 9b reflects the second excitation light of wavelength λ2 (>λ1) emitted by the second subunit 6b and the second fluorescence of wavelength range Δλ2 generated from the sample M in response thereto, and transmits light of longer wavelengths than the second excitation light and the second fluorescence. The dichroic mirror 11b transmits the second fluorescence of wavelength range Δλ2 and reflects the second excitation light of wavelength λ2 shorter than the wavelength range Δλ2. The light source device 10b is a device incorporating a light-emitting element that outputs the second excitation light of wavelength λ2. The pinhole plate 12b is an aperture member that limits the light flux of the second fluorescence and is disposed so that the pinhole position coincides with the conjugate position of the spot of the second excitation light on the sample M. The photodetector 13b is disposed so that its detection surface faces the pinhole plate 12b and receives and detects the second fluorescence that has passed through the pinhole plate 12b. The photodetector 13b may be a photomultiplier tube, a photodiode, an avalanche photodiode, a multi-pixel photon counter (MPPC), a hybrid photodetector (HPD), an area image sensor, or the like.

[0021] The third subunit 6c includes a base plate 8c, a dichroic mirror (third beam splitter) 9c, a light source device 10c, a dichroic mirror 11c, a pinhole plate (third aperture member) 12c, and a photodetector (third photodetector) 13c. The dichroic mirror 9c reflects the third excitation light of wavelength λ3 (>λ2) emitted by the third subunit 6c and the third fluorescence of wavelength range Δλ3 generated from the sample M in response thereto, while transmitting light of longer wavelengths than the third excitation light and the third fluorescence. The dichroic mirror 11c transmits the third fluorescence of wavelength range Δλ3 and reflects the third excitation light of wavelength λ3 shorter than the wavelength range Δλ3. The light source device 10c is a device incorporating a light-emitting element that outputs the third excitation light of wavelength λ3. The pinhole plate 12c is an aperture member that limits the light flux of the third fluorescence and is disposed so that the pinhole position coincides with the conjugate position of the spot of the third excitation light on the sample M. The photodetector 13c is disposed so that its detection surface faces the pinhole plate 12c and receives and detects the third fluorescence that has passed through the pinhole plate 12c. The photodetector 13c may be a photomultiplier tube, a photodiode, an avalanche photodiode, a multi-pixel photon counter (MPPC), a hybrid photodetector (HPD), an area image sensor, or the like.

[0022] The fourth subunit 6d includes a base plate 8d, a total reflection mirror 9d, a light source device 10d, a dichroic mirror 11d, a pinhole plate (fourth diaphragm member) 12d, and a photodetector (fourth photodetector) 13d. The total reflection mirror 9d reflects the fourth excitation light having a wavelength λ4 (>λ3) emitted by the fourth subunit 6d and the fourth fluorescence having a wavelength range Δλ4 generated from the sample M in response to the fourth excitation light. The dichroic mirror 11d transmits the fourth fluorescence having a wavelength range Δλ4 and reflects the fourth excitation light having a wavelength λ4 shorter than the wavelength range Δλ4. The light source device 10d incorporates a light-emitting element that outputs the fourth excitation light having a wavelength λ4. The pinhole plate 12d is an diaphragm member that is positioned so that its pinhole position coincides with the conjugate position of the spot of the fourth excitation light on the sample M and limits the beam of the fourth fluorescence. The photodetector 13d is disposed with its detection surface facing the pinhole plate 12d, and receives and detects the fourth fluorescence that has passed through the pinhole plate 12d. The photodetector 13d is a photomultiplier tube, a photodiode, an avalanche photodiode, an MPPC (Multi-Pixel Photon Counter), an HPD (Hybrid Photo Detector), an area image sensor, or the like.

[0023] Next, the configuration of the light source device 10a will be described in detail. Fig. 2 is a block diagram showing a schematic configuration of the light source device 10a.

[0024] The light source device 10a includes a light-emitting element (light source) 21 that outputs laser light, which is a first excitation light having a wavelength λ1; a photodetector 22 that detects the laser light output from the light-emitting element 21 and outputs a detection signal; a driver 23 that supplies a drive current to the light-emitting element 21; and a controller 24 that outputs a drive signal to the driver 23 to control the drive current according to a target light intensity. The light-emitting element 21 is, for example, a laser diode (hereinafter simply referred to as "LD"). However, the light-emitting element 21 may be another light-emitting element such as a light-emitting diode (LED). The photodetector 22 is, for example, a photomultiplier tube, a photodiode, an avalanche photodiode, a multi-pixel photon counter (MPPC), a hybrid photodetector (HPD), an area image sensor, or the like. The driver 23 is a voltage-controlled current source including a transistor that generates a drive current according to the drive signal, which is a voltage signal.

[0025] The control unit 24 built into the light source device 10a includes a microcomputer including a processor (Central Processing Unit) such as a CPU, a recording medium such as a random access memory (RAM) or a read-only memory (ROM), a communication module, and an input / output module. The control unit 24 loads a program into hardware such as the CPU and RAM, and operates the communication module and the input / output module under the control of the CPU, while also reading and writing data from and to the RAM, thereby achieving the functions described below. The CPU may be a standalone piece of hardware or may be implemented in a programmable logic device such as an FPGA, like a software processor. The RAM and ROM may also be standalone pieces of hardware or may be built into a programmable logic device such as an FPGA. All of the various data required to execute the computer program and the various data generated by the execution of the computer program are stored in built-in memory such as the ROM or RAM, or in a storage medium such as a hard disk drive.

[0026] The light source devices 10b, 10c, and 10d have the same configuration as the light source device 10a, and differ only in the wavelength of the laser light output from the light emitting element 21. Only the detailed configuration of the light source device 10a will be described below.

[0027] 3 is a diagram showing a detailed circuit configuration of light source device 10a. Light source device 10a includes a control unit 24 that includes a microcomputer 25, a digital-to-analog converter (hereinafter referred to as "DAC") 26, analog-to-digital converters (hereinafter referred to as "ADC") 27 and 28, a subtractor 29, and an amplifier 30. Light source device 10a also includes two switches SW1 and SW2 that switch signal transmission paths.

[0028] The DAC 26 receives a control value indicating a target light intensity of the laser beam output from the microcomputer 25, converts the control value into a control signal, which is a voltage signal, and outputs it to the subtractor 29. The subtractor 29 outputs different types of signals depending on the mode. First, in the setting mode, the subtractor 29 receives the control signal and the detection signal output from the photodetector 22 via the switch SW2, generates a difference signal indicating the difference between the control signal and the detection signal, and outputs the difference signal to the amplifier 30. On the other hand, in the operation mode, the control signal output from the DAC 26 is output directly to the amplifier 30. The amplifier 30 also has different functions depending on the mode. First, in the setting mode, the subtractor 29 adjusts and outputs a drive signal so that the difference signal output from the subtractor 29 is minimized. On the other hand, in the operation mode, the amplifier 30 amplifies the control signal output from the subtractor 29 by a predetermined amplification factor and outputs the drive signal. The drive signal output from the amplifier 30 is converted by the driver 23 into a drive current supplied to the light-emitting element 21. That is, by controlling the drive signal, the drive current output from the drive unit 23 to the light emitting element 21 can be controlled, and the amount of laser light output from the light emitting element 21 can be controlled.

[0029] The switch SW2 switches the output destination of the detection signal generated by the photodetector 22 between the subtractor 29 and the ADC 28. The ADC 28 converts the amount of laser light indicated by the detection signal into a digital value and inputs it to the microcomputer 25. The SW1 is provided on the signal transmission path between the output of the amplifier 30 and the input of the ADC 27, and turns on / off the input of the drive signal from the amplifier 30 to the ADC 27. The ADC 27 converts the drive value indicated by the drive signal into a digital value and inputs it to the microcomputer 25.

[0030] The light source device 10a having the above configuration is configured to operate by switching between two types of functions, a setting mode and an operation mode, under the control of the microcomputer 25. Fig. 4 is a circuit diagram showing the switching state of the light source device 10a when operating in the setting mode, and Fig. 5 is a circuit diagram showing the switching state of the light source device 10a when operating in the operation mode.

[0031] When the setting mode function is operating, the operation is as follows. That is, as shown in FIG. 4, switch SW1 is turned on, and switch SW2 is switched so as to set the transmission path of the detection signal to the subtractor 29 side. As a result, the detection signal from the photodetector 22 is fed back to the control signal output by the DAC 26, and the drive signal output by the amplifier 30 is input to the ADC 27. With this connection configuration, when the setting mode function is operating, the control unit 24 corrects (adjusts) the drive signal so as to reduce the difference between the detection signal and the control signal. In other words, it performs APC control (Auto Power Control: automatic light amount control, first control) to reduce the drive signal when the light amount indicated by the detection signal is greater than the light amount indicated by the control signal, and to increase the drive signal when the light amount indicated by the detection signal is less than the light amount indicated by the control signal, and adjusts and outputs the drive signal based on the control signal and the detection signal. At this time, the control unit 24 executes APC control while changing the voltage value of the control signal within the range of laser light intensities that can be expected to be used in the confocal microscope unit 1, generates LUTs (Look Up Tables), which are data tables showing the correspondence between drive values ​​corresponding to the drive signals and the laser light intensities indicated by the voltage values ​​of the control signals, and stores the generated LUTs in an internal memory such as RAM in the microcomputer 25. Note that the drive values ​​corresponding to the drive currents set in the LUTs are values ​​obtained by converting the drive current values ​​to values ​​before amplification by the amplifier 30. Furthermore, the voltage value of the control signal is changed, for example, by changing the voltage value from 1% to 100% in 0.5% increments, with 100% being the voltage value corresponding to the maximum light intensity.

[0032] When the function in the operation mode is operating, the following operation is performed. That is, as shown in FIG. 5, switch SW1 is turned off, and switch SW2 is switched to set the transmission path of the detection signal to the ADC 28. As a result, the detection signal from the photodetector 22 is input to the ADC 28. With this connection configuration, when the function in the operation mode is operating, the control unit 24 inputs the control signal directly to the amplifier 30 and performs ACC control (Auto Current Control, second control), which outputs the control signal amplified by the amplifier 30 as a drive signal. When controlling the amount of laser light to a target amount of light, the control unit 24 reads LUTs from an internal memory such as RAM in the microcomputer 25 and identifies a drive value corresponding to the target amount of light. The control unit 24 then performs the ACC control using the control signal having the identified drive value. Additionally, when the function in the operation mode is operating, the control unit 24 monitors the amount of laser light indicated by the detection signal, and if the amount of light exceeds a predetermined reference value (e.g., 1.5 mW), controls the driver 23 to stop generating a drive current. This allows the laser output of the four light source devices 10a to 10d combined to be kept below four times the specified standard value (for example, 1.5mW x 4 = 4.5mW), allowing the confocal microscope unit 1 to be used safely even outside a laser-controlled area.

[0033] Next, the operation procedure of the functions of the light source device 10a of this embodiment, i.e., the control method of the confocal microscope unit of this embodiment, will be described. Fig. 6 is a flowchart showing the operation procedure of the functions of the setting mode of the light source device 10a, and Fig. 7 is a flowchart showing the operation procedure of the functions of the operation mode of the light source device 10a.

[0034] 6, when the operation of the setting mode in light source device 10a is started by inputting an instruction to microcomputer 25, control unit 24 sets the voltage value of the control signal to an initial value within a predetermined range (step S1). Then, control unit 24 executes APC control using the set control signal (step S2). In response, control unit 24 specifies a drive value corresponding to the drive signal output by control unit 24 (step S3). Next, control unit 24 records the correspondence between the specified drive value and the light amount indicated by the voltage value of the control signal at that time in the LUTs (step S4).

[0035] Thereafter, the control unit 24 determines whether or not the next voltage value of the control signal is within a predetermined range (step S5). If the determination result shows that the next voltage value is present (step S5; Yes), the voltage value of the control signal is set to the next value, and the processing of steps S1 to S4 is repeated. If the determination result shows that the next voltage value is not present (step S5; No), the control unit 24 stores the LUTs in which the correspondence relationships recorded by the processing up to that point are recorded in the microcomputer 25 (step S6). With the above processing, the operation of the setting mode is completed.

[0036] 7, when the operation of the operating mode in light source device 10a is started by inputting an instruction to microcomputer 25, control unit 24 sets a target light intensity of the laser light to be output (step S101). Next, control unit 24 reads out LUTs from microcomputer 25 (step S102). Then, control unit 24 refers to the LUTs and specifies a drive value corresponding to the set target light intensity (step S103). Furthermore, control unit 24 executes ACC control using a control signal having the specified drive value (step S104). In addition, control unit 24 specifies the light intensity of the laser light based on the detection signal output from photodetector 22 (step S105).

[0037] Then, the control unit 24 determines whether the identified light amount is equal to or less than a predetermined reference value (step S106). If the determination result shows that the light amount exceeds the reference value (step S106; No), the process proceeds to step S108. On the other hand, if the determination result shows that the light amount is equal to or less than the reference value (step S106; Yes), the control unit 24 determines whether to terminate the output of laser light corresponding to the scan on the sample M (step S107). If the determination result shows that the output of laser light is to continue (step S107; No), the target value of the light amount is changed to the next value corresponding to the scan, and the processes of steps S101 to S106 are repeated.

[0038] On the other hand, if it is determined in step S106 that the light amount exceeds the reference value (step S106; No), or if it is determined in step S107 that the output of the laser light is to be stopped (step S107; Yes), the control unit 24 stops the generation of the drive current by the drive unit 23 (step S108). The above processing completes the operation in the operation mode.

[0039] According to the confocal microscope unit 1 described above, the first excitation light output from the first subunit 6a is reflected by the dichroic mirror 9a and then scanned onto the sample M via the scan mirror 4 and the scan lens 7, and the first fluorescence generated from the sample M in response passes through the scan lens 7 and the scan mirror 4 and then reflects off the dichroic mirror 9a to enter the first subunit 6a, where its image is formed on the pinhole plate 12a in the first subunit 6a and detected by the photodetector 13a. In addition, the second excitation light output from the second subunit 6b is reflected by the dichroic mirror 9b in the second subunit 6b, passes through the dichroic mirror 9a in the first subunit 6a, and is then scanned onto the sample M via the scan mirror 4 and the scan lens 7. In response, the second fluorescence generated from the sample M passes through the scan lens 7 and the scan mirror 4, passes through the dichroic mirror 9a, reflects off the dichroic mirror 9b, and enters the second subunit 6b, where its image is formed on the pinhole plate 12b in the second subunit 6b and detected by the photodetector 13b. Similarly, the other second and third subunits 6c and 6d are configured so that the excitation light irradiated from their own units and the fluorescence generated in response to it are reflected by the dichroic mirror of their own units and transmitted through the dichroic mirror of the other units, thereby enabling independent detection of fluorescence generated in response to excitation light of two wavelengths.

[0040] In this embodiment, in the light source devices 10a to 10d, the setting mode function of the control unit 24 generates and stores in advance LUTs that indicate the correspondence between the drive current supplied to the light-emitting element 21 and the intensity of excitation light. The operation mode function of the control unit 24 outputs a control signal corresponding to a drive current corresponding to a target light intensity based on the LUTs to the driver 23, which supplies the drive current to the light-emitting element 21. Additionally, the operation mode function stops the drive current when the intensity of the excitation light exceeds a predetermined value. This allows the light-emitting element 21 to be driven quickly and accurately to change the intensity of the excitation light accordingly when a target light intensity is set so that the intensity of the excitation light changes in response to changes in the scanning speed of the excitation light on the sample M. Furthermore, the excitation light is stopped when the intensity of the excitation light exceeds a predetermined value, allowing the confocal microscope unit 1 to be used safely. As a result, the irradiation energy of the excitation light on the sample M can be uniformed with precision and safety, enabling highly safe confocal imaging.

[0041] 8 is a plan view showing an example of the scanning mode of the first excitation light on the sample M using the confocal microscope unit 1. As described above, an effective field of view range AR2 is set on the sample M inside the scanning range AR1 defined by the scan mirror 4. Within this scanning range AR1, two linear scans, one going back and forth along one direction, are repeated while shifting the scanning position in a direction perpendicular to the one direction, thereby scanning the entire scanning range AR1. At this time, in order to minimize the range of bleaching of the sample M due to the excitation light, the light source device 10a operates to irradiate the first excitation light only during scanning within the effective field of view range AR2 inside the scanning range AR1.

[0042] FIG. 9 shows the change over time in the amount of laser light output from the light source device 10a corresponding to the scanning pattern shown in FIG. 8 , and FIG. 10 shows the change over time in the amount of light in FIG. 9 corresponding to the sweep waveform of the scan mirror 4. The lower part of FIG. 10 shows the change over time in the amount of light, and the upper part of FIG. 10 shows the corresponding change over time in the sweep waveform W1 of the drive signal for the scan mirror 4 and the scanning speed V1 of the laser light. In this embodiment, a sine wave is used as the sweep waveform W1 of the drive signal for tilting the reflection angle of the scan mirror 4 in one direction. Therefore, if the amount of laser light output from the light source device 10a were constant, the irradiation energy of the laser light per unit area within the effective field of view AR2 on the sample M would be nonuniform, resulting in uneven fading. To prevent this, the light source device 10a intensity-modulates the LD light amount in accordance with the scanning speed of the scan mirror 4, thereby achieving uniform irradiation energy within the effective field of view AR2. That is, the light source device 10a operates to output laser light in one direction (forward) of the two-way (forward and backward) scanning within the effective field range AR2 of the sample M, and sets the amount of laser light during scanning so that the amount of laser light irradiated to the center of the effective field range AR2 is maximum and the amount of laser light irradiated from the center to the edge of the effective field range AR2 gradually decreases. Specifically, the light source device 10a sets the voltage value of the control signal to be set during operation in the operating mode so as to change in accordance with the waveform of the scanning speed V1, which is a differentiated waveform of the sweep waveform W1. This makes it possible to modulate the intensity of the LD light amount to achieve uniform laser light irradiation energy within the effective field range AR2 of the sample M.

[0043] Additionally, in the light source device 10a of this embodiment, the maximum light intensity of the laser light can be set according to an input value to the microcomputer 25 in order to set the average light intensity of the laser light suitable for observing the sample M. For example, in the light source device 10a, the modulation degree of the laser light intensity is kept constant, and the maximum light intensity can be set in the range of 1% to 100%. In this case, if the modulation degree is doubled, the light intensity of the laser light can be changed within a dynamic range of 100 times (200 times including modulation).

[0044] Furthermore, in this embodiment, the light source devices 10a to 10d have a switch SW2 that feeds back the detection signal from the photodetector 22 to the output of a control signal in the control unit 24 in the setting mode, and inputs the detection signal to the control unit 24 in the operation mode. With this configuration, the detection signal from the photodetector 22 can be output appropriately when the function in the setting mode is operating and when the function in the operation mode is operating. As a result, it is possible to achieve both uniform irradiation energy of the excitation light and improved safety.

[0045] In this embodiment, the control unit 24 performs APC control in the setting mode, increasing or decreasing the drive signal so as to minimize the difference between the detection signal and the control signal, and performs ACC control in the operation mode, outputting the drive signal based on the control signal. This configuration allows the relationship between the drive current and the excitation light intensity to be accurately stored during operation of the setting mode function, and also allows for faster drive of the light-emitting element during operation of the operation mode function. In particular, even if individual differences or changes over time in the light-emitting characteristics of the light-emitting element 21 occur, or changes in the environmental temperature occur, highly accurate control of the excitation light intensity is possible. As a result, the irradiation energy of the excitation light on the sample M can be made uniform with even greater precision.

[0046] Furthermore, in this embodiment, the drive unit 23 is configured by a voltage-controlled current source that generates a drive current based on a drive signal, which can simplify the configuration of the light source devices 10a to 10d.

[0047] Furthermore, in this embodiment, in the setting mode, the control unit 24 generates and stores LUTs that indicate the correspondence between drive values ​​corresponding to the drive current and the light intensity indicated by the control signal, and in the operation mode, the control unit 24 operates to perform ACC control using a control signal having a drive value corresponding to a target light intensity based on the LUTs. According to this configuration, the setting mode function of the control unit 24 generates and stores in advance LUTs that indicate the correspondence between drive values ​​for driving the light-emitting element 21 and the light intensity indicated by the control signal, and the operation mode function of the control unit 24 outputs, based on the LUTs, a control signal corresponding to a drive value corresponding to the target light intensity as a drive signal to the drive unit 23 that supplies a drive current to the light-emitting element 21. As a result, the irradiation energy of the excitation light on the sample M can be uniformed with high precision.

[0048] Various embodiments of the present disclosure have been described above, but the present disclosure is not limited to the above embodiments, and may be modified or applied to other things within the scope that does not change the gist of the claims.

[0049] In the confocal microscope unit 1 according to the above-described embodiment, the control unit 24 of the light source devices 10a to 10d may have the following function. That is, in the setting mode, the control unit 24 may generate and store LUTs that indicate the correspondence between the drive value corresponding to the drive signal and the light amount indicated by the detection signal output from the photodetector 22, and in the operation mode, perform ACC control based on the LUTs. Even with such a modification, the irradiation energy of the excitation light on the sample M can be uniformed with high precision.

[0050] 11 shows the circuit configuration of a light source device 110a according to a modified example. The light source device 110a does not have the switches SW1 and SW2 of the light source device 10a. Instead, the detection signal output from the photodetector 22 is input to the microcomputer via the ADC 28 as a digital value of the light intensity. When the setting mode function is active, the microcomputer 25 of the light source device 110a adjusts the control value in its firmware (FW) so that the detection signal output from the photodetector 22 becomes a desired value (desired light intensity), and outputs the control value to the DAC 26. The DAC 26 receives the adjusted control value output from the microcomputer 25, converts the control value into a control signal, which is a voltage signal, and outputs it to the amplifier 30. The amplifier 30 then generates a drive signal based on the input control signal and outputs it to the driver 23. The driver 23 generates a drive current to be supplied to the light-emitting element 21 based on the drive signal, and supplies the light-emitting element 21 with the drive current. The photodetector 22 again detects the emitted light, and the detection signal output from the photodetector 22 is again input to the microcomputer via the ADC 28 as a digital value of the light intensity. The microcomputer 25 performs APC control by repeating this process until the detection signal reaches a desired value, and records the control value when the desired value is reached in the LUTs along with the corresponding light intensity. Furthermore, when the function in the operation mode is operating, the microcomputer 25 performs ACC control using the LUTs and outputs the control value generated as a result of this control to the DAC 26. At the same time, when the function in the operation mode is operating, the microcomputer 25 controls the stopping of the drive current based on the digital value of the light intensity input from the ADC 28. Even with this modification, it is possible to precisely uniformize the irradiation energy of the excitation light on the sample M and achieve highly safe confocal imaging.

[0051] In the above embodiment, it is preferable that the light source unit has a switch that feeds back the detection signal from the photodetector to the output of a control signal in the control unit in the setting mode and inputs the signal to the control unit in the operation mode. In this case, the detection signal from the photodetector can be appropriately output when the function in the setting mode is operating and when the function in the operation mode is operating. As a result, it is possible to achieve both uniform irradiation energy of the excitation light and improved safety.

[0052] In the above embodiment, it is also preferable that the control unit, in the setting mode, executes automatic light intensity control, which increases or decreases the drive signal so as to reduce the difference between the detection signal and the control signal, and, in the operation mode, executes automatic current control, which outputs the drive signal based on the control signal. In this case, when the setting mode function is operating, the correspondence relationship between the drive current and the excitation light intensity can be accurately stored, and the light source can be driven at high speed when the operation mode function is operating. As a result, the excitation light irradiation energy on the sample can be made uniform with even greater precision.

[0053] Furthermore, in the above embodiment, it is preferable that the drive section has a voltage-controlled current source that generates a drive current based on the drive signal, which simplifies the configuration of the light source unit.

[0054] Furthermore, in the above embodiment, it is also preferable that in the setting mode, the control unit generates and stores a data table indicating the correspondence between drive values ​​corresponding to the drive current and the light intensity indicated by the control signal, and in the operation mode, executes the second control using a control signal having a drive value corresponding to a target light intensity based on the data table. According to this configuration, the setting mode function of the control unit generates and stores in advance a data table indicating the correspondence between drive values ​​for driving the light source and the light intensity indicated by the control signal, and the operation mode function of the control unit outputs a control signal corresponding to a drive value corresponding to the target light intensity as a drive signal to the driver that supplies drive current to the light source based on the data table. As a result, the irradiation energy of the excitation light on the sample can be uniformed with high precision.

[0055] Furthermore, in the above embodiment, it is also preferable that in the setting mode, the control unit generates and stores a data table indicating the correspondence between drive values ​​corresponding to the drive signal and the light intensity indicated by the detection signal, and in the operation mode, executes the second control using a control signal having a drive value corresponding to a target light intensity based on the data table. According to this configuration, the setting mode function of the control unit generates and stores in advance a data table indicating the correspondence between drive values ​​for driving the light source and the light intensity indicated by the detection signal of the excitation light, and the operation mode function of the control unit outputs a control signal corresponding to a drive value corresponding to the target light intensity as a drive signal to the driver that supplies drive current to the light source based on the data table. As a result, the irradiation energy of the excitation light on the sample can be accurately uniformed. [Industrial Applicability]

[0056] The embodiments are directed to a confocal microscope unit, a confocal microscope, and a control method for a confocal microscope unit, and are capable of uniformizing the irradiation energy of excitation light onto a sample with high precision. [Explanation of symbols]

[0057] 1...confocal microscope unit, 4...scan mirror, 10a, 10b, 10c, 10d, 110a...light source device (light source unit), 21...light emitting element (light source), 22...photodetector, 23...drive unit, 24...control unit, 50...microscope, A...confocal microscope, P1...connection port, SW1, SW2...switch, M...sample.

Claims

1. A confocal microscope unit that constitutes a confocal microscope by being attached to a connection port of a microscope having a microscope optical system, a light source unit including a light source that outputs excitation light, a photodetector that detects the excitation light output from the light source and outputs a detection signal, a driver that supplies a drive current to the light source, and a controller that outputs a drive signal to the driver that controls the drive current in response to a control signal that indicates a target light amount; a scan mirror that scans the excitation light output from the light source unit on a sample; a housing configured to be attachable to the connection port and to which the scan mirror and the light source unit are fixed; Equipped with The control unit has two types of functions: a setting mode and an operation mode. In the setting mode, a first control is executed to adjust and output the drive signal based on the control signal and the detection signal, and the first control is executed while changing the value of the control signal, thereby generating and storing a data table showing a correspondence relationship between the drive current and the light intensity of the excitation light; In the operation mode, a second control is executed to output the control signal as the drive signal, the data table is read, and based on the data table, the second control is executed using the control signal corresponding to the drive current corresponding to a target light amount, and a control is executed to stop the drive current when the light amount indicated by the detection signal exceeds a predetermined value; the target light intensity is set so as to change the light intensity of the excitation light in accordance with a change in the scanning speed of the excitation light on the sample. Confocal microscope unit.

2. the light source unit has a switch that feeds back the detection signal from the photodetector to the output of the control signal in the control unit in the setting mode, and inputs the detection signal to the control unit in the operation mode. The confocal microscope unit according to claim 1 .

3. The control unit executes automatic light amount control in the setting mode by increasing or decreasing the drive signal so as to reduce the difference between the detection signal and the control signal, and executes automatic current control in the operation mode by outputting the drive signal based on the control signal.

3. A confocal microscope unit according to claim 1 or 2.

4. the drive unit includes a voltage-controlled current source that generates the drive current based on the drive signal; The confocal microscope unit according to any one of claims 1 to 3.

5. In the setting mode, the control unit generates and stores a data table indicating a correspondence relationship between a drive value corresponding to the drive current and a light amount indicated by the control signal; In the operation mode, the second control is performed using the control signal having the drive value corresponding to a target light amount based on the data table. The confocal microscope unit according to any one of claims 1 to 4.

6. In the setting mode, the control unit generates and stores a data table indicating a correspondence relationship between a drive value corresponding to the drive signal and a light amount indicated by the detection signal; In the operation mode, the second control is performed using the control signal having the drive value corresponding to a target light amount based on the data table. The confocal microscope unit according to any one of claims 1 to 4.

7. A confocal microscope unit according to any one of claims 1 to 6, a microscope having a connection port to which the microscope optical system and the confocal microscope unit are attached; A confocal microscope comprising:

8. a control method using a confocal microscope unit that constitutes a confocal microscope by being attached to a connection port of a microscope having a microscope optical system, the confocal microscope unit including: a light source that outputs excitation light; a photodetector that detects the excitation light output from the light source and outputs a detection signal; a driver that supplies a drive current to the light source; and a controller that outputs a drive signal to the driver in response to a control signal indicating a target light amount to control the drive current; a scan mirror that scans a sample with the excitation light output from the light source unit; and a housing that is configured to be attachable to the connection port and to which the scan mirror and the light source unit are fixed, a step of executing a first control for adjusting and outputting the drive signal based on the control signal and the detection signal, and executing the first control while changing the value of the control signal, thereby generating and storing a data table showing a correspondence relationship between the drive current and the light intensity of the excitation light; a step of executing a second control for outputting the control signal as the drive signal, reading the data table, executing the second control using the control signal corresponding to the drive current corresponding to a target light amount based on the data table, and executing a control for stopping the drive current when the light amount indicated by the detection signal exceeds a predetermined value; Equipped with the target light intensity is set so as to change the light intensity of the excitation light in accordance with a change in the scanning speed of the excitation light on the sample. How to control a confocal microscope unit.

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