WO2018083772A1 - Microscope system - Google Patents

Abstract

A microscope system provided with: a first intensity modulation unit for modulating the intensity of first light at a frequency f1, the first light for exciting a first fluorescent substance included in a specimen; a second intensity modulation unit for modulating the intensity of second light at a frequency f2 different from the frequency f1, the second light for causing stimulated emission in the first fluorescent substance; a scanning unit for scanning the first light and the second light in the specimen; and a detection unit for detecting fluorescence from the specimen; the scanning unit having a resonant scanner having a resonance mirror, and the detection unit receiving fluorescence from the specimen and detecting a frequency f1 + f2 component.

Description

Microscope system

The present invention relates to a microscope system.

A microscope is known that excites a fluorescent substance with two photons, irradiates a laser beam that induces stimulated emission, acquires fluorescence attenuated by stimulated emission, and constructs an image (see, for example, Non-Patent Document 1). ). In a microscope, it is necessary to acquire an image at high speed.
Lu Wei et.al., Biomedical Optics Express 1465-1475, vol. 3, No. 6, 1 June 2012

In the first aspect of the present invention, the microscope system includes a first intensity modulation unit that modulates the intensity of the first light that excites the first fluorescent substance included in the sample at a frequency f1, A second intensity modulating unit that modulates the intensity of the second light that causes stimulated emission in the fluorescent substance at a frequency f2 different from the frequency f1, and a scanning unit that scans the first light and the second light on the sample; A detection unit that detects fluorescence from the sample, the scanning unit includes a resonant scanner having a resonance mirror, and the detection unit receives the fluorescence from the sample and detects a component of frequency f1 + f2.

In the second aspect of the present invention, the microscope system includes a first light that irradiates the specimen with first light that excites a fluorescent substance contained in the specimen and causes the detection unit to receive fluorescence from the specimen. An observation method, and intensity-modulating the first light with a frequency f1 and irradiating the specimen, and intensity-modulating the second light that causes stimulated emission in the fluorescent material with a frequency f2 different from the frequency f1 The second observation method can be selected so that the sample is irradiated, the fluorescence from the sample is received by the detection unit, and the frequency f1 + f2 component or the frequency f1-f2 component is detected.

The above summary of the invention does not enumerate all the features of the present invention. A sub-combination of these feature groups can also be an invention.

1 is a diagram illustrating a configuration of a microscope system 10 according to the present embodiment. It is a conceptual diagram which shows the relationship between the wavelength of pump light, probe light, and a detection wavelength area | region. It is a conceptual diagram explaining the time change of the fluorescence intensity. It is a conceptual diagram explaining the demodulation frequency in lock-in detection. It is a conceptual diagram explaining the scanning speed and the detection speed. An example of another scanning unit 151 is shown. Still another example of the scanning unit 156 is shown. It is a figure which shows the structure of the other microscope system. 3 is an example of a GUI screen 300 used in the microscope system 12. 3 is a flowchart showing an example of the operation of the microscope system 12. It is a flowchart of the operation | movement which selects the range of attenuation | damping fluorescence observation based on confocal observation. 5 is a flowchart of an operation of automatically selecting whether to perform confocal observation or attenuated fluorescence observation in the microscope system 12. 13 shows a GUI screen 350 used for the operation shown in the flowchart of FIG. It is a figure which shows the structure of other microscope system. It is a figure which shows the structure of the other microscope system 16, The relationship between the excitation / fluorescence spectrum of each fluorescent substance, pump light, probe light, and wavelength in the detection wavelength region is shown. It is a figure which shows the structure of the other microscope system 18. FIG. The wavelength relationship between the excitation / fluorescence spectrum, pump light, probe light, and detection wavelength region of each fluorescent material is shown. The relationship between the absorption / fluorescence spectrum and the detection wavelength region when the Stokes shift is small is shown. The relationship between fluorescence and excitation light that can be observed in each of the first detection wavelength region and the second detection wavelength region is shown. (A) shows time waveforms of fluorescence C ′ and fluorescence D ′, and (b) expresses fluorescence C ′ and fluorescence D ′ in a phasor display. The GUI screen 360 used for the two-color observation of FIGS. 15 to 21 is shown. It is a figure which shows the structure of the other microscope system. It is a figure which shows the structure of the other microscope system 22. FIG. It is a figure which shows the structure of the further microscope system 24. FIG. It is a figure which shows the structure of other microscope system 26. FIG. An example of another light source 101 is shown. The state of the time difference when the optical delay stage 700 is translated is shown. The decaying fluorescence signal reflects the fluorescence lifetime.

Hereinafter, the present invention will be described through embodiments of the invention. However, the following embodiments do not limit the invention according to the claims. In addition, not all the combinations of features described in the embodiments are essential for the solving means of the invention.

FIG. 1 is a diagram illustrating a configuration of a microscope system 10 which is an example of a microscope system according to the present embodiment. The microscope system 10 irradiates the sample with the intensity-modulated pump light and the probe light, thereby describing a fluorescence signal attenuated by stimulated emission generated from the fluorescent substance contained in the sample (hereinafter referred to as attenuated fluorescence, attenuated fluorescence signal, etc.). ) Lock-in detection. Thereby, the detection time can be shortened while improving the spatial resolution. Hereinafter, the attenuated fluorescence may be collectively referred to as fluorescence. Hereinafter, a microscope that acquires attenuated fluorescence is referred to as an attenuated fluorescence microscope. In FIG. 1, the xyz axis is shown for explanation.

The microscope system 10 includes a light source 100 that outputs pump light and probe light, an illumination optical system 140 that illuminates the specimen 186 with the pump light and probe light, an observation optical system 160 that observes light emitted from the specimen 186, A detection unit 136 that detects light via the observation optical system 160 is provided. The microscope system 10 further includes a stage 180 that supports the specimen 186. The microscope system 10 further includes a control unit 130 that controls the entire microscope system 10, an input unit 220 that transmits and receives signals to and from the control unit 130, a display unit 224, and a storage unit 226.

The illumination optical system 140 includes an acoustooptic tunable filter 114 (hereinafter also referred to as AOTF), an acoustooptic element 124 (hereinafter also referred to as AOM), dichroic mirrors 141 and 144, mirrors 142 and 143, and a scanning unit 150. A lens pair 173, a dichroic mirror 162, and an objective lens 164.
The observation optical system 160 includes an objective lens 164, a dichroic mirror 162, an optical filter 166, and a lens pair 172.

The specimen 186 has an observation object 184 and a slide glass 182 on which the observation object 184 is placed. The observation object 184 is a biological cell, for example. The observation object 184 contains a fluorescent material.

The light source 100 includes a laser light source 102 for pump light, a laser light source 104 for probe light, and a dichroic mirror 106 that combines the pump light and the probe light. The laser light sources 102 and 104 are, for example, continuous oscillation systems and output laser beams having different wavelengths. The pump light excites the fluorescent material to generate fluorescence. The probe light attenuates fluorescence by inducing stimulated emission in the fluorescent material. The wavelength of the pump light is shorter than the wavelength of the probe light. For example, the pump light is 532 nm and the amber probe light is 640 nm. The wavelengths of the pump light and the probe light are appropriately set according to the absorption band (absorption spectrum) and the fluorescence band (fluorescence spectrum) of the fluorescent substance. The wavelengths of the pump light and the probe light may be set automatically, or the input unit 220 may accept input from the user.

The AOTF 114 is arranged on the optical path of the laser beam synthesized coaxially by the dichroic mirror 106. The mirrors 142 and 143 constitute an optical path that does not pass through the AOM 124.

AOTF 114 functions as a diffraction grating for light. First-order diffracted light generated by the AOTF 114 is guided to the dichroic mirror 141. By controlling the voltage of the driver 112 applied to the AOTF 114, generation of the first-order diffracted light can be controlled for each wavelength. For each wavelength of light, the first-order diffracted light can always be generated (ON state, that is, the intensity is maximum), or not always generated (OFF state, that is, the intensity is minimum). It is also possible to modulate the light intensity. For example, when a constant voltage value is applied from the driver 112, the light intensity becomes a constant value according to the voltage value. For example, if the voltage value applied from the driver 112 is zero in time, the light intensity is also zero. For example, when the voltage waveform of the driver 112 is a sine wave, the intensity of light is modulated into a sine wave. In this embodiment, based on the oscillation from the oscillator 132, the AOTF 114 modulates the intensity of the pump light at the frequency f1, and the probe light is turned on (not modulated). The advantage of AOTF 114 is that the intensity of light having different wavelengths can be controlled independently. The AOTF 114 and the driver 112 constitute the first intensity modulation unit 110.

In this embodiment, the voltage of the driver 112 applied to the AOTF 114 is controlled so that the pump light is modulated at a predetermined frequency in the AOTF 114. Two acoustic frequencies corresponding to the wavelengths of the pump light and the probe light are given to the AOTF 114. Thereby, diffracted light of pump light and probe light is generated from the AOTF 114. Here, in order to modulate the intensity of only the pump light, the amplitude of the electrical signal of the acoustic frequency corresponding to the pump light wavelength is modulated at the modulation frequency f1. Thereby, the intensity of the pump light transmitted through the AOTF 114 is modulated by f1. On the other hand, since the amplitude of the electrical signal having an acoustic frequency corresponding to the probe light wavelength is not modulated, the intensity of the probe light is not modulated.

The dichroic mirror 141 reflects the pump light modulated by the AOTF 114 while transmitting the probe light. As a result, the probe light is guided to the AOM 124. By controlling the voltage of the driver 122 applied to the AOM 124, generation of the first-order diffracted light can be controlled. The first-order diffracted light can always be generated (ON state, that is, the state where the intensity is maximum), or the first-order diffracted light can always be generated (OFF state, that is, the state where the intensity is minimum). Can also be modulated. For example, when a constant voltage value is applied from the driver 122, the light intensity becomes a constant value according to the voltage value. For example, if the voltage value applied from the driver 122 is zero in time, the light intensity is also zero. For example, when the voltage waveform of the driver 122 is a sine wave, the light intensity is modulated into a sine wave. In the present embodiment, based on the oscillation from the oscillator 132, the intensity of the probe light is modulated by the AOM 124 at a frequency f2 different from the frequency f1. An advantage of the AOM 124 is that intensity modulation can be performed at a relatively high frequency of several tens of MHz. The AOM 124 and the driver 122 constitute a second intensity modulation unit 120.

On the other hand, after the pump light is reflected by the dichroic mirror 141, it is reflected by the mirrors 142 and 143 and enters the dichroic mirror 144. The dichroic mirror 144 coaxially combines the pump light and the probe light whose intensity is modulated respectively.

The scanning unit 150 is disposed at a position substantially conjugate with the pupil plane of the objective lens 164. For this reason, it is desirable that a lens pair 173 be installed between the scanning unit 150 and the dichroic mirror 162. An example of the scanning unit 150 is a galvano scanner, which includes a pair of galvanometer mirrors that can rotate in directions orthogonal to each other. The spot position of the laser beam on the specimen 186 is scanned in the xy direction by changing the angles of these galvanometer mirrors. Another example of the scanning unit 150 is a resonant scanner (resonance type scanner). The resonant scanner has a resonant mirror (resonant mirror) that operates by resonance. The resonant scanner includes, for example, a main scanning resonant mirror and a sub scanning galvanometer mirror. By using a resonant scanner, it is possible to scan at higher speed.

The laser beam output from the scanning unit 150 passes through the dichroic mirror 162 and is guided to the objective lens 164. The objective lens 164 focuses the laser beam on the sample 186.

Fluorescence generated from the fluorescent material of the sample 186 passes through the objective lens 164, is reflected by the dichroic mirror 162, and the pump light and the probe light are removed by the optical filter 166. The fluorescence is incident on the light receiving unit 174 installed at a position substantially conjugate with the objective lens pupil plane by the lens pair 172. Note that the dichroic mirror 162 may be disposed closer to the light source than the lens pair 173 and the scanning unit 150.

The detection unit 136 includes a light receiving unit 174 and a lock-in amplifier 134. The light receiving unit 174 is disposed at a position substantially conjugate with the pupil plane of the objective lens 164. An example of the light receiving unit 174 is a photomultiplier tube. The light receiving unit 174 outputs an electrical signal corresponding to the intensity of the received fluorescence by photoelectric conversion. The output of the light receiving unit 174 is input to the lock-in amplifier 134 to detect lock-in. The lock-in detection will be described later.

The input unit 220, the display unit 224, the storage unit 226, and the control unit 130 may be, for example, a PC. The input unit 220 receives input from the user to the control unit 130, and is, for example, a keyboard, a touch panel, a mouse, or the like. The display unit 224 is, for example, a display that displays a GUI, a detection result, and an observation image. The storage unit 226 stores a program for controlling the microscope system 10, parameters, and the like, detection results, observation images, and the like.

The control unit 130 includes a frequency control unit 229, a scanner control unit 228, and an image generation unit 222. The frequency control unit 229 controls the oscillation frequency generated by the oscillator 132 based on an input from the user or automatically based on the fluorescent material. The scanner control unit 228 controls the scanning unit 150. The image generation unit 222 generates an image based on the detection result of the detection unit 136 and displays the image on the display unit 224.

FIG. 2 is a conceptual diagram showing the relationship between the pump light, the probe light, and the wavelength in the detection wavelength region. In FIG. 2, a broken line indicates an absorption band of a specific fluorescent material, and a solid line indicates a fluorescent band of the fluorescent material. The wavelength of the pump light is preferably set to be included in the absorption band, and the probe light is preferably set to a wavelength longer than the intensity peak of the fluorescent band. Thereby, the wavelength region including the intensity peak of the fluorescent band can be set as a detection wavelength region which is a wavelength region in which the light receiving unit 174 detects fluorescence.

FIG. 3 is a conceptual diagram for explaining temporal changes in fluorescence intensity. FIG. 4 is a conceptual diagram illustrating a demodulation frequency in lock-in detection.

The pump light and the probe light are intensity-modulated at frequencies f1 and f2 that are temporally different from each other. If the time waveforms are I _Pump and I _Probe , they are expressed as follows.

Here, I1 and I2 are the intensity of pump light and probe light, and m and n are modulation contrasts. Since stimulated emission is proportional to the product of I _Pump and I _Probe , the fluorescence attenuated by stimulated emission (decayed fluorescence signal) I _RF is also proportional to the product of I _Pump and I _Probe and can be expressed as follows.

The time waveform of equation (1.3) is shown in FIG. 3 (normalized with the maximum value). Since the fluorescence signal acquired by the light receiving unit 174 includes a plurality of frequencies, the time waveform is as shown in FIG. Among these, since the fluorescence excited by the pump light is generated at the frequency f1 and the fluorescence excited by the probe light is generated at the frequency f2, the pump light is detected by detecting the component that fluctuates at f1-f2 or f1 + f2 from the equation (1.3). Only the attenuated fluorescence signal can be detected by removing the fluorescence excited by the single light and the probe light alone. Therefore, the detection unit 136 detects only a signal synchronized with a desired frequency by performing demodulation using a lock-in detection technique.

FIG. 4 shows time waveforms of the difference frequency (f1-f2) and the sum frequency (f1 + f2) included in the equation (1.3). Since the sum frequency is higher than the difference frequency, the cycle is also shortened. In the lock-in detection, since the period of this demodulation frequency determines the minimum integration time, the shorter the period, the faster the detection becomes possible.

Therefore, in this embodiment, demodulation is performed at the sum frequency. More specifically, the sum demodulation frequency is input from the oscillator 132 to the lock-in amplifier 134. The lock-in amplifier 134 extracts a signal synchronized with the demodulation frequency. While the scanning unit 150 scans the sample 186, the lock-in amplifier 134 performs lock-in detection for each pixel, and stores it in the storage unit 226 in association with the position information of the pixel. The image generation unit 222 reads the detection result associated with the position information from the storage unit 226, generates an observation image of attenuated fluorescence, and displays it on the display unit 224.

Here, since the stimulated emission phenomenon is a non-linear phenomenon, the signal generation region of the attenuated fluorescence is limited to a local region where the intensity of the condensed spot of the pump light and the probe light is high. Thereby, the spatial resolution can be improved.

FIG. 5 is a conceptual diagram for explaining the scanning speed and detection speed of the scanning unit 150. In the galvano scanner, the time required for scanning in the main scanning (in the x direction in the figure) is longer than that in the resonant scanner. Therefore, as shown in FIG. It can be considered that the position of the beam is almost unchanged. However, since the time required for scanning in the main scan (x direction in the figure) in the resonant scanner is shorter than that in the galvano scanner, the time required for detection by demodulation of the difference frequency is shown in FIG. As described above, there is a possibility that the position of the beam is greatly changed and it is difficult to obtain an accurate image. However, when demodulating at the sum frequency, the time required for detection is shortened because the demodulation frequency is high, and even if a resonant scanner is used, the position of the beam does not change during the predetermined time required for signal detection at a predetermined position. You can think about it. Therefore, it is possible to obtain an accurate image while detecting at high speed using a resonant scanner.

FIG. 6 shows an example of another scanning unit 151. The scanning unit 151 includes a resonant scanner 152, a galvano scanner 153, and a pair of mirrors 154 and 155. The pair of mirrors 154 and 155 are provided so as to be movable in the directions of the arrows in the figure, and based on these positions, it is selected which of the resonant scanner 152 and the galvano scanner 153 is used.

FIG. 6 shows a state where the resonant scanner 152 is selected. In this case, the mirror 154 is disposed on the optical path of the light emitted from the dichroic mirror 144, and the mirror 155 is disposed on the optical path of the light emitted from the resonant scanner 152. Thereby, the light reflected by the mirror 154 enters the resonant scanner 152. The light deflected in the predetermined direction by the resonant scanner 152 is reflected by the mirror 155, passes through the dichroic mirror 162, and enters the objective lens 164.

On the other hand, when the galvano scanner 153 is selected, the mirror 154 is retracted from the optical path of the light emitted from the dichroic mirror 144 and the mirror 155 is retracted from between the galvano scanner 153 and the dichroic mirror 162. As a result, light enters the galvano scanner 153, and light deflected by the galvano scanner 153 passes through the dichroic mirror 162 and enters the objective lens 164.

According to the scanning unit 151, the resonant scanner 152 and the galvano scanner 153 can be used properly according to the application. The means for moving the position of the pair of mirrors 154 and 155 includes, for example, a linear motor, but is not limited thereto, and each of them is arranged on a corresponding turret, and the mirrors 154 and 155 move by rotation of the turret. May be. By providing a pair of dichroic mirrors instead of the pair of mirrors 154 and 155, the resonant scanner 152 is used for light having a wavelength that reflects the pair of dichroic mirrors, and the wavelength that passes through the pair of dichroic mirrors. The galvano scanner 153 can be used with respect to the light. The positions of the resonant scanner 152 and the galvano scanner 153 may be opposite to those in FIG.

FIG. 7 shows another example of the scanning unit 156. In FIG. 7, the same components as those of FIG.

The scanning unit 156 includes a mirror 157 in which they are integrated instead of the pair of mirrors 154 and 155 of the scanning unit 151. The mirror 157 is provided to be movable in a direction perpendicular to the paper surface. Here, the state of FIG. 7 corresponds to the state of FIG. 6, and shows a state in which the resonant scanner 152 is used when light is reflected by the mirror 157. On the other hand, the mirror 157 moves in the direction perpendicular to the paper surface from the state of FIG. As a result, the galvano scanner 153 is used.

FIG. 8 is a diagram showing the configuration of another microscope system 12. Similar to the microscope system 10, the microscope system 12 can be used as an attenuated fluorescence microscope, and can also be used as a confocal microscope. In the microscope system 12, the same components as those in the microscope system 10 are denoted by the same reference numerals and description thereof is omitted.

In the microscope system 12, a dichroic mirror 402 that reflects fluorescence and transmits pump light and probe light is disposed on an optical path between the dichroic mirror 144 and the scanning unit 150. Furthermore, the optical filter 404, the lens 406, and the light-receiving part 410 which the light reflected by the dichroic mirror 402 injects are provided. The optical filter 404 and the light receiving unit 410 may have the same configuration as the optical filter 166 and the light receiving unit 174 of the microscope system 10. The microscope system 12 further includes a pinhole 408 as an example of an opening member having an opening. The pinhole 408 is arranged at a position conjugate with the specimen. The lens 406 collects light in the pinhole 408. The light receiving unit 410 is installed in the vicinity of the pinhole 408. Or it is good also as a structure by which a light-receiving part is installed in a pinhole and a substantially conjugate position with a lens not shown.

With the above configuration, the fluorescence from the sample 186 passes through the scanning unit 150, is reflected by the dichroic mirror 402, and is received by the light receiving unit 410 via the optical filter 404, the lens 406, and the pinhole 408. Thereby, even if the observation position of the sample 186 is changed by the scanning unit 150, the scanning unit 150 descans and the spot position in the pinhole 408 remains unchanged. The pinhole 408 has a variable hole size, and details will be described later. With the above configuration, it can be said that the pinhole 408 is provided between the light receiving unit 410 and the dichroic mirror 402. It can also be said that the pinhole 408 is provided between the light receiving unit 410 and the scanning unit 150. Furthermore, it can be said that the pinhole 408 is disposed between the light receiving unit 410 and the objective lens 164.

The microscope system 12 further includes a wavelength control unit 230 that controls the wavelength of light from the laser light sources 102 and 104.

FIG. 9 is an example of a GUI screen 300 used in the microscope system 12. The GUI screen 300 is displayed on the display unit 224 and accepts input from the user using the input unit 220.

A check box 302 is an input field for determining whether or not to acquire an image for confocal observation. The check box 304 is an input field for designating that the pump light is modulated in the confocal observation, and the check box 306 is an input field for designating that the pump light is not modulated.

The input column 308 is an input column for the modulation frequency of the pump light, and the designated modulation frequency is indicated by a thick vertical line together with a numerical scale in units of MHz. The input field 310 is an input field for specifying the size of the pinhole. “OPEN” in the input field 310 indicates that the size of the hole is the maximum. Further, the size of the designated hole is indicated by a vertical thick line along with a scale of “1” as an Airy size. Here, Airy size is the size of a diffraction-limited light spot determined by the wavelength and the numerical aperture, and is a value obtained by standardizing the pinhole diameter.

Further, the check box 312 is an input field for determining whether or not to acquire a time-lapse image for confocal observation. An input field 314 is an input field for time lapse time intervals.

Check box 316 is an input field for determining whether or not to acquire an image of attenuated fluorescence observation. The input column 318 is an input column for the modulation frequency of the pump light, and the designated modulation frequency is indicated by a thick vertical line along with a numerical scale in units of MHz. The input column 320 is an input column for the modulation frequency of the probe light, and the designated modulation frequency is indicated by a vertical thick line together with a numerical scale in units of MHz.

The input field 322 is an input field for the size of the pinhole 408 in the attenuated fluorescence observation, and has the same configuration as the input field 310. A check box 324 and an input field 326 are input fields related to time lapse in attenuated fluorescence observation, and have the same configuration as the check box 312 and the input field 314.

On the GUI screen 300, a confocal observation image 330 and an attenuated fluorescence observation image 332 are displayed side by side. Instead of this, they may be displayed in an overlapping manner. Further, by linking each other, an observation image 332 of attenuated fluorescence may be displayed when a target region of the confocal observation image 330 is clicked. Furthermore, when image acquisition by confocal observation time lapse is designated, time lapse images 334 are displayed side by side in time order. Similarly, when image acquisition by time lapse of attenuated fluorescence observation is designated, time lapse images 335 are displayed side by side in time order.

FIG. 10 is a flowchart showing an example of the operation (S10) of the microscope system 12.

In the flowchart S10, the control unit 130 determines whether to acquire an image of attenuated fluorescence observation based on the input of the check box 316 of the GUI screen 300 (S100). When the determination in step S100 is Yes, the control unit 130 sets the modulation frequencies of the pump light and the probe light for attenuated fluorescence observation based on the input in the input fields 318 and 320 (S102).

The control unit 130 sets the diameter of the pinhole 408 (S104). During the attenuated fluorescence observation, the pinhole 408 is opened by default, that is, “OPEN” is set by default in the input field 322 of FIG. When the user changes the value of the input field 322 from the default, the size of the pinhole 408 is set based on the changed value. In the decay fluorescence observation, by opening the pinhole 408, it is possible to detect the fluorescence whose imaging relation is disturbed due to scattering or the like despite being generated from the focal plane which is the pinhole conjugate plane. More photons can be detected, and as a result, the signal-to-noise ratio can be improved.

Based on the above settings, an image of attenuated fluorescence observation is acquired (S106). In this case, the demodulation frequency may be a difference frequency or a sum frequency, but is preferably a sum frequency when a resonant scanner is used. The method for acquiring the image of attenuated fluorescence observation is the same as that described in the microscope system 10, and the description thereof is omitted.

After step S106 or when the determination in step S100 is No, whether or not to acquire the confocal observation image is determined based on the input of the check box 302 (S108). When it is determined that a confocal observation image is acquired (S108: Yes), it is determined whether or not the pump light used for confocal observation is modulated based on the check boxes 304 and 306 (S112) and is modulated. In this case (S112: Yes), the modulation frequency input in the input field 308 is set (S114).

After step S114 or when pump light is not modulated in step S112 (S112: No), the diameter of the pinhole 408 is set based on the input to the input field 310 from the user (S116). The diameter of the pinhole in confocal observation is preferably smaller than the diameter of the pinhole in attenuated fluorescence observation.

The confocal observation image is acquired based on the above settings (S118). More specifically, the pump light is turned on or intensity-modulated, the probe light is turned off, the sample 186 is scanned by the scanning unit 150, and the fluorescence is detected by the detection unit 136 pixel by pixel. The detection result is stored in the storage unit 226 in association with the position information. When the intensity of the pump light is modulated, lock-in detection is performed at the modulation frequency in the lock-in amplifier. Note that when the pump light is turned on (when intensity modulation is not performed), a lock-in amplifier is not necessary, and thus it is desirable to directly input the output of the light receiving unit 410 to the image generation unit 222.

The image generation unit 222 reads out the detection result associated with the position information from the storage unit 226, generates the confocal observation image 330 and the attenuated fluorescence observation image 332, and displays them on the display unit 224 (S120).

Furthermore, when the acquisition of the time-lapse image for confocal observation is received in the check box 312, the microscope system 12 performs confocal observation at the time interval set in the input field 314 and generates each observation image. Similarly, when the acquisition of the time-lapse image of the attenuated fluorescence observation is received by the check box 324, the microscope system 12 performs the attenuated fluorescence observation at the time interval set in the input field 326 and generates each observation image.

In addition, it is good also as a structure which acquires the image of the wide visual field of the sample 186 by confocal observation, and designates the one part area | region among the images of confocal observation, and acquires the image of attenuation fluorescence observation. In this case, an image may be acquired by confocal observation, and a range suitable for attenuated fluorescence observation may be automatically selected.

FIG. 11 is a flowchart of an operation (S30) for selecting a range of attenuated fluorescence observation based on confocal observation. First, an image is acquired by confocal observation (S300). In this case, steps S112 to S118 in the operation (S10) of FIG. 10 are executed. Next, it is determined whether to automatically select the range of attenuated fluorescence observation based on the input from the user (S302).

In the case of automatic selection (S302: Yes), confocal images are subjected to image processing analysis and a range suitable for attenuated fluorescence observation is selected. For example, differential filtering is performed on the image, and a region where many peaks occur is selected.

On the other hand, if automatic selection is not performed (S302: No), an area for attenuated fluorescence observation is set based on designation from the user (S308). In this case, region designation may be received on the confocal observation image 330 of FIG.

For the region set in step S304 or S308, attenuated fluorescence observation is executed to obtain an observation image (S306). In this case, steps S102 to S106 in the operation (S10) of FIG. 10 are executed.

FIG. 12 is a flowchart of an operation (S20) for automatically selecting whether confocal observation or attenuated fluorescence observation is performed in the microscope system 12, and FIG. 13 shows a GUI screen 350 used in the operation.

The storage unit 226 associates the name of the fluorescent substance with the observation method, for example, whether confocal observation is preferable or attenuated fluorescence observation, and the wavelength of pump light in the case of confocal observation or attenuated fluorescence observation. In this case, the wavelengths of the pump light and the probe light are stored. On the GUI screen 350, the name 353 of the fluorescent substance stored in the storage unit 226 is displayed together with the check box 352.

The fluorescent material is selected by checking the check box 352 by the user (S200). The control unit 130 determines whether confocal observation is preferable or attenuated fluorescence observation is preferable with reference to the storage unit 226 according to the selected fluorescent substance (S202). When it is determined that confocal observation is preferable, the pump light box 355 is colored on the GUI screen 350, and the control unit 130 refers to the storage unit 226 to determine the wavelength of the pump light corresponding to the fluorescent substance. Is displayed in the display field 354 (S204). In this case, the probe light box 357 is white, and the wavelength display column 356 is grayed out. If it is determined that the attenuated fluorescence observation is preferable, the pump light box 355 is colored on the GUI screen 350, and the control unit 130 refers to the storage unit 226 to determine the wavelength of the pump light corresponding to the fluorescent material. The probe light box 357 is colored, and the wavelength of the probe light corresponding to the fluorescent material is determined and displayed in the display field 356 (S204). In either case, the display field 358 shows the relationship among the fluorescent material absorption band, the fluorescent band, the wavelength of the light source, and the detection wavelength region (S206).

When it is determined that the attenuated fluorescence observation is preferable, the wavelength control unit 230 sets the wavelength of the light from the laser light sources 102 and 104 based on the user's execution instruction. Further, the frequency control unit 229 sets the modulation frequency in the oscillator 132. In this case, the input of the modulation frequency may be received on the GUI screen 300 of FIG. 9, or the modulation frequency is associated with the fluorescent material and stored in the storage unit 226, and the storage unit 226 is selected along with the selection of the fluorescent material. The control unit 130 may automatically determine the modulation frequency by referring to FIG. Based on the above settings, an attenuated fluorescence observation image is acquired as in steps S102 to S106 of FIG.

On the other hand, when it is determined that confocal observation is preferable, the wavelength control unit 230 sets the wavelength of the light from the laser light source 102 based on an instruction to be executed by the user. Based on the above settings, confocal observation images are acquired as in steps S112 to S118 of FIG.

FIG. 14 is a diagram showing the configuration of still another microscope system 14. In the microscope system 14, the same components as those of the microscope systems 10 and 12 are denoted by the same reference numerals, and description thereof is omitted.

The microscope system 14 corresponds to a combination of the microscope system 10 and the microscope system 12. That is, a dichroic mirror 162 is disposed on the optical path between the objective lens 164 and the scanning unit 150, and an optical filter 166, a lens pair 172, and a light receiving unit 174 on which fluorescence reflected by the dichroic mirror 162 is incident are provided. Further, a dichroic mirror 402 is disposed on the optical path between the scanning unit 150 and the dichroic mirror 144, and includes an optical filter 404, a lens 406, a pinhole 408, and a light receiving unit 410 on which fluorescence reflected by the dichroic mirror 402 is incident. .

When the light receiving unit 174 receives fluorescence, the dichroic mirror 162 is advanced on the optical path between the objective lens 164 and the scanning unit 150, and the dichroic mirror 402 is moved between the scanning unit 150 and the dichroic mirror 144. Evacuate from the street. When the light reflected by the dichroic mirror 162 is received by the light receiving unit 174, a brighter signal can be detected because there are few optical elements through which the fluorescence passes. Therefore, it is preferable to use for attenuated fluorescence observation.

When fluorescence is received by the light receiving unit 410, the dichroic mirror 162 is retracted from the optical path between the objective lens 164 and the scanning unit 150, and the dichroic mirror 402 is moved between the scanning unit 150 and the dichroic mirror 144. Advance on the street. When the fluorescence reflected by the dichroic mirror 402 is received by the light receiving unit 410, it is descanned by the scanning unit 150. Therefore, it is preferably used for confocal observation using the pinhole 408.

15 is a diagram showing the configuration of still another microscope system 16, and FIG. 16 shows the relationship between the excitation / fluorescence spectrum, pump light, probe light, and wavelength of the detection wavelength region of each fluorescent substance. The microscope system 16 is used for attenuated fluorescence observation with multicolor fluorescence. For example, the microscope system 16 is used when there are two types of fluorescent materials, the pump light is common, and the probe light is different. In the microscope system 16, the same components as those in the microscope system 12 are denoted by the same reference numerals and description thereof is omitted.

The microscope system 16 includes a laser light source 500 for the second probe light in addition to the laser light source 104 for the first probe light, and a dichroic mirror 502 that combines the second probe light and the first probe light. And further. The dichroic mirror 106 combines the pump light with the first and second probe lights. As shown in FIG. 16, the wavelength of the pump light is in the common part of the absorption spectrum C of the first fluorescent material and the absorption spectrum D of the second fluorescent material, and excites both fluorescent materials. The wavelength of the first probe light is in the fluorescence spectrum A of the first fluorescent material, and induces stimulated emission in the first fluorescent material. The wavelength of the second probe light is in the fluorescence spectrum B of the second fluorescent material, and induces stimulated emission in the second fluorescent material. By using a fluorescent material having a large Stokes shift (wavelength difference between the absorption spectrum and the fluorescence spectrum) and making the pump light common, the number of light sources can be reduced and the apparatus configuration can be simplified. Note that the specimen 186 contains first and second fluorescent substances.

A drive voltage corresponding to each light is applied from the driver 112 to the AOTF 114, the pump light is turned on, the intensity of the first probe light is modulated at the frequency f2, and the intensity of the second probe light is modulated at the frequency f3. . Of the light diffracted by the AOTF 114, the pump light passes through the dichroic mirror 141, and the first and second probe lights are reflected by the dichroic mirror 141. The pump light is intensity-modulated by the AOM 124 at the frequency f1. On the other hand, the first and second probe lights are reflected by the mirrors 142 and 143 so as to bypass the AOM 124 and are combined with the pump light by the dichroic mirror 144. The pump light, the first probe light, and the second probe light pass through the dichroic mirror 402, pass through the scanning unit 150, and are collected on the sample 186 by the objective lens 164. The frequencies f1, f2, and f3 are set to be different from each other.

The pump light _{I Pump,} when the first probe light _{I Probe1,} the second probe light and _{I probe2,} each time the waveform is expressed as follows.

Note that I1, I2, and I3 are the light intensities of the pump light, the first probe light, and the second probe light, respectively.

The microscope system 16 includes a dichroic mirror 402 between the dichroic mirror 144 and the scanning unit 150. The dichroic mirror 402 includes a wavelength region including a wavelength region (first detection wavelength region) for detecting fluorescence from the first fluorescent material and a wavelength region (second detection wavelength region) for detecting fluorescence from the second fluorescent material. Reflect.

A dichroic mirror 412 is further arranged on the optical path of the light reflected by the dichroic mirror 402. The dichroic mirror 412 transmits the wavelength region including the first detection wavelength region and reflects the wavelength region including the second detection wavelength region.

Fluorescence from the first fluorescent material that has passed through the dichroic mirror 412 is cut in the wavelength region other than the first detection wavelength region by the optical filter 404 and is received by the light receiving unit 410 through the lens 406. The light-receiving signal photoelectrically converted by the light-receiving unit 410 is lock-in detected by the lock-in amplifier 134. The light receiving unit 410 and the lock-in amplifier 134 constitute a first detection unit 136.

On the other hand, the fluorescence from the second fluorescent material reflected by the dichroic mirror 412 is cut by the optical filter 414 in the wavelength region other than the second detection wavelength region, and detected by the light receiving unit 420 through the lens 416. The light receiving signal photoelectrically converted by the light receiving unit 420 is lock-in detected by the lock-in amplifier 135. The light receiving unit 420 and the lock-in amplifier 135 constitute a second detection unit 137.

When the signal received by the light receiving unit 410 and the signal received by the light receiving unit 420 are respectively I _RF1 and I _RF2 ,

Therefore, it is desirable to demodulate with f1 + f2 in the lock-in amplifier 134 and f1 + f3 with the lock-in amplifier 135, respectively.

As shown in FIG. 16, in general multicolor observation, since fluorescence generated from the first fluorescent material is also detected in the second detection wavelength region, crosstalk of fluorescence becomes a problem. However, the attenuated fluorescence signal from the first fluorescent material mixed in the second detection wavelength region is generated at the frequency f1 + f2, whereas the attenuated fluorescence signal from the second fluorescent material desired to be acquired in the second detection wavelength region. Occurs at frequency f1 + f3. Therefore, the crosstalk of the fluorescence can be suppressed by setting the demodulation frequency in the second detection wavelength region to f1 + f3. Thus, by setting the modulation frequencies of the two probe lights to different values, the crosstalk of the fluorescence signal can be suppressed, so that two-color simultaneous observation becomes possible. The dichroic mirror 412 may reflect the fluorescence in the first detection wavelength region and transmit the fluorescence in the second detection wavelength region.

FIG. 17 is a diagram showing the configuration of still another microscope system 18. FIG. 18 shows the relationship between the excitation / fluorescence spectrum, pump light, probe light, and wavelength of the detection wavelength region of each fluorescent substance. Similarly to the microscope system 16, the microscope system 18 is also used for attenuated fluorescence observation with multicolor fluorescence. For example, the microscope system 18 is used when there are two types of fluorescent materials, the probe light is common, and the pump light is different. In the microscope system 18, the same components as those in the microscope system 16 are denoted by the same reference numerals and description thereof is omitted.

The microscope system 18 includes, in addition to the first pump light laser light source 102, a second pump light laser light source 504, and a dichroic mirror 506 that combines the second pump light and the probe light. The dichroic mirror 106 combines the first pump light with the second pump light and the probe light.

As shown in FIG. 18, the wavelength of the first pump light is within the absorption spectrum H of the first fluorescent material, and excites the first fluorescent material. The wavelength of the second pump light is in the absorption spectrum G of the second fluorescent material, and excites the second fluorescent material. The wavelength of the probe light is at the intersection of the fluorescence spectrum E of the first fluorescent material and the fluorescence spectrum F of the second fluorescent material, and induces stimulated emission in both fluorescent materials. By using a fluorescent material having a large Stokes shift and sharing the probe light, the number of light sources can be reduced and the apparatus configuration can be simplified. Note that the specimen 186 contains first and second fluorescent substances.

A driving voltage corresponding to each light is applied from the driver 112 to the AOTF 114, the probe light is turned on, the intensity of the first pump light is modulated at the frequency f1, and the intensity of the second pump light is modulated at the frequency f4. . Of the light diffracted by the AOTF 114, the probe light passes through the dichroic mirror 141, and the first and second pump lights are reflected by the dichroic mirror 141. The probe light is intensity-modulated by the AOM 124 at the frequency f2. On the other hand, the first and second pump lights are reflected by the mirrors 142 and 143 so as to bypass the AOM 124 and are combined with the pump light by the dichroic mirror 144. The first pump light, the second pump light, and the probe light pass through the dichroic mirror 402, pass through the scanning unit 150, and are collected on the sample 186 by the objective lens 164. The frequencies f1, f2, and f4 are set to be different from each other.

A first pump light _{I Pump1,} a second pump light _{I Pump2,} when the probe light and _{I Probe,} each time the waveform is represented as follows.

Note that I1, I2, and I3 are the light intensities of the first pump light, the second pump light, and the probe light, respectively.

The microscope system 18 includes a dichroic mirror 402 between the dichroic mirror 144 and the scanning unit 150. The dichroic mirror 402 includes a wavelength region including a wavelength region (first detection wavelength region) for detecting fluorescence from the first fluorescent material and a wavelength region (second detection wavelength region) for detecting fluorescence from the second fluorescent material. Reflect.

A dichroic mirror 412 is further arranged on the optical path of the light reflected by the dichroic mirror 402. The dichroic mirror 412 transmits the wavelength region including the first detection wavelength region and reflects the wavelength region including the second detection wavelength region.

Fluorescence from the first fluorescent material that has passed through the dichroic mirror 412 is cut in the wavelength region other than the first detection wavelength region by the optical filter 404 and is received by the light receiving unit 410 through the lens 406. The light-receiving signal photoelectrically converted by the light-receiving unit 410 is lock-in detected by the lock-in amplifier 134.

On the other hand, the fluorescence from the second fluorescent material reflected by the dichroic mirror 412 is cut by the optical filter 414 in the wavelength region other than the second detection wavelength region, and detected by the light receiving unit 420 through the lens 416. The light receiving signal photoelectrically converted by the light receiving unit 420 is lock-in detected by the lock-in amplifier 135.

When the signal received by the light receiving unit 410 and the signal received by the light receiving unit 420 are respectively I _RF1 and I _RF2 ,

Therefore, it is desirable to demodulate at f1 + f2 in the lock-in amplifier 134 and at f4 + f2 in the lock-in amplifier 135, respectively.

Also in FIG. 18, the crosstalk of fluorescence becomes a problem. However, the attenuated fluorescence signal from the first fluorescent material mixed in the second detection wavelength region is generated at the frequency f1 + f2, whereas the attenuated fluorescence signal from the second fluorescent material desired to be acquired in the second detection wavelength region. Occurs at frequency f4 + f2. Therefore, by setting the demodulation frequency in the second detection wavelength region to f4 + f2, fluorescence crosstalk can be suppressed. Thus, by setting the modulation frequencies of the two pump lights to different values, the crosstalk of the fluorescence signal can be suppressed, so that two colors can be observed simultaneously.

A preferred mode for realizing simultaneous observation when excitation crosstalk becomes a problem in multicolor observation due to a small Stokes shift or the like will be described. FIG. 19 shows the relationship between the absorption / fluorescence spectrum and the detection wavelength region when the Stokes shift is small.

In the example of FIG. 19, the wavelength of the first pump light is located in the absorption spectrum K of the first fluorescent material, and the wavelength of the second pump light is located in the absorption spectrum L of the second fluorescent material. The probe light is located in the fluorescence spectrum I of the first fluorescent material and the fluorescence spectrum J of the second fluorescent material in order to induce stimulated emission. Further, the first detection wavelength region is set to include the peak of the fluorescence spectrum I of the first fluorescent material, and the second wavelength region is set to include the peak of the fluorescence spectrum J of the second fluorescent material.

FIG. 20 shows the relationship between fluorescence and fluorescent substances that can be observed in each of the first detection wavelength region and the second detection wavelength region. In each detection wavelength region, four types of signals may be observed.

In the first detection wavelength region, four signals of fluorescence A, B, C, and D may be detected. Among these, the signal to be detected is the fluorescence A from the first fluorescent material excited by the first pump light. Therefore, it is desirable to remove other fluorescence. Fluorescence B and D can be removed by setting the first detection wavelength region so that the fluorescence spectrum from the second fluorescent substance deviates from the first detection wavelength region. The fluorescence C can be removed by utilizing the difference in demodulation frequency.

In the second detection wavelength region, there is a possibility that four signals of fluorescence A ′, B ′, C ′, and D ′ are detected. Among these, the signal to be detected is the fluorescence D ′ from the second fluorescent material excited by the second pump light. Therefore, it is desirable to remove other fluorescence. Fluorescence A ′ and B ′ can be removed by utilizing the difference in demodulation frequency. This will be described in detail below.

The time waveforms of the first pump light, the second pump light, and the probe light are expressed by equations (2.6)-(2.8). When the time waveform of the fluorescence A ′ generated from the first fluorescent material by the first pump light and the fluorescence B ′ generated from the second fluorescent material by the first pump light is I _RF1 ,

On the other hand, if the time waveform of fluorescence D ′, which is the desired signal light, is I _RF2 ,

Therefore, the influence of the fluorescence A ′ and B ′ can be removed by demodulating with f4 + f2.

However, in the second detection wavelength region, the fluorescence C ′ from the first fluorescent material excited by the second pump light has the same frequency as the fluorescence D ′, which is the desired signal light, and therefore cannot be separated by frequency. . In this case, it is desirable that the second pump light does not excite the first fluorescent material. If this is difficult, it is desirable to reduce the contamination of the fluorescence C ′ by utilizing the difference in fluorescence lifetime between the first fluorescent material and the second fluorescent material. The following relationship holds among the fluorescence lifetime τ, the demodulation frequency f, and the phase θ.

FIG. 21 (a) shows the time waveforms of fluorescence C ′ and fluorescence D ′ detected at the demodulation frequency f = f4 + f2. If the fluorescence lifetimes of the first fluorescent material and the second fluorescent material are τ1 and τ2, respectively, the phases are θ1 and θ2. Therefore, in lock-in detection, the influence of the fluorescence C ′ can be reduced by setting the phase of the signal to be detected to the fluorescence D ′. FIG. 21B represents the signal intensities of fluorescence C ′ and fluorescence D ′ detected in lock-in by phasor display. By selecting θ2 as the phase of lock-in detection, the magnitude of fluorescence C ′ can be reduced by cos (θ1−θ2). In addition, when the first pump light excites the second fluorescent material and the fluorescence of the second fluorescent material is mixed into the first detection wavelength region, the phase of the first fluorescent material is adjusted in the first detection region. You may detect lock-in. The detection based on the phase may be applied to the case where the pump light is common and there are two types of probe light (for example, described in FIG. 16).

Note that (i) the fluorescence from the second fluorescent material by the first pump light is not mixed in the first detection wavelength region, and (ii) the second pump light by the second pump light is in the second detection wavelength region. When the fluorescence from one fluorescent material is not mixed, the number of light receiving units may be one. When the above conditions (i) and (ii) are not satisfied, as shown in FIG. 17, it is desirable to separate the fluorescence band by the dichroic mirror 412 and acquire each fluorescence.

FIG. 22 shows a GUI screen 360 used for the two-color observation of FIGS. 15 to 21. The check boxes 362 and 364 accept the selection of the fluorescent material. The wavelengths stored in the storage unit 226 in association with the selected fluorescent substance are read, and the wavelengths are displayed in the display columns 366 to 372, respectively. Further, the display column 374 displays the relationship between the excitation / fluorescence spectrum of the selected fluorescent substance, the pump light, the probe light, and the detection wavelength region. Instead of automatic setting, the user can arbitrarily select a light source wavelength or a detection wavelength region.

FIG. 23 is a diagram showing the configuration of still another microscope system 20. In the microscope system 20, the same components as those of the microscope system 10 and the like are denoted by the same reference numerals and description thereof is omitted.

In the microscope system 20, an intensity modulation unit 550 having a driver 552 and an AOTF 554 is disposed. A single AOTF 554 modulates the intensity of both the pump light and the probe light. In this case, in the AOTF 554, the pump light is f1 and the probe light is intensity-modulated by f2, and the generated fluorescence is demodulated by the lock-in amplifier 134 at the demodulation frequency of f1 + f2. Thereby, an observation image of attenuated fluorescence can be acquired with a simple configuration.

FIG. 24 is a diagram showing the configuration of still another microscope system 22. In the microscope system 22, the same components as those in the microscope system 10 are denoted by the same reference numerals, and the description thereof is omitted.

The microscope system 22 includes a laser light source 504 for the second pump light and a dichroic mirror 506 that multiplexes the second pump light with the probe light in addition to the laser light source 102 for the first pump light. The dichroic mirror 106 combines the first pump light with the second pump light and the probe light. Further, similarly to the microscope system 20, an intensity modulation unit 550 is disposed. A single AOTF 554 modulates the intensity of all of the two types of pump light and probe light. In this case, in the AOTF 554, the intensity of the first pump light is f1, the second pump light is f4, and the probe light is f2 respectively. Similarly to the microscope system 16, the fluorescence corresponding to the first pump light is received by the light receiving unit 410 and demodulated at the demodulation frequency of f1 + f2 by the lock-in amplifier 134. On the other hand, the fluorescence corresponding to the second pump light is received by the light receiving unit 420 and demodulated by the lock-in amplifier 135 at the demodulation frequency of f4 + f2. Thereby, a multicolor observation image of attenuated fluorescence can be acquired with a simple configuration.

FIG. 25 is a diagram showing the configuration of still another microscope system 24. In the microscope system 24, the same components as those of the microscope system 10 and the like are denoted by the same reference numerals and description thereof is omitted.

In the microscope system 24, an intensity modulation unit 560 is provided for the laser light source 102 for pump light, and an intensity modulation unit 570 is provided for the laser light source 104 for probe light. The intensity modulation unit 560 includes a driver 562 and an AOM 564, and modulates the intensity of the pump light with f1. The intensity modulation unit 570 includes a driver 572 and an AOM 574, and modulates the intensity of the probe light with f2. Further, the intensity-modulated pump light and probe light are combined by a mirror 452 and a dichroic mirror 450. The fluorescence received by the light receiving unit 410 is demodulated by the lock-in amplifier 134 at a demodulation frequency of f1 + f2. Thereby, it is possible to demodulate at a high modulation frequency, and therefore it is possible to shorten the time for acquiring an observation image of attenuated fluorescence.

FIG. 26 is a diagram showing the configuration of still another microscope system 26. In the microscope system 26, the same components as those of the microscope system 10 and the like are denoted by the same reference numerals and description thereof is omitted.

In the microscope system 26, an intensity modulator 560 is provided for the first laser light source 102 for pump light, an intensity modulator 570 is provided for the laser light source 104 for probe light, and the second pump light source. An intensity modulator 580 is provided for the laser light source 504. The intensity modulator 560 modulates the intensity of the first pump light with f1, and the intensity modulator 570 modulates the intensity of the probe light with f2. Further, the intensity modulation unit 580 includes a driver 582 and an AOM 584, and intensity-modulates the second pump light with f4. The intensity-modulated first pump light, second pump light, and probe light are combined by mirrors 458 and 460 and dichroic mirrors 454 and 456. Similarly to the microscope system 16, the fluorescence corresponding to the first pump light is received by the light receiving unit 410 and demodulated at the demodulation frequency of f1 + f2 by the lock-in amplifier 134. On the other hand, the fluorescence corresponding to the second pump light is received by the light receiving unit 420 and demodulated by the lock-in amplifier 135 at the demodulation frequency of f4 + f2. Thereby, even in multicolor observation, it is possible to demodulate at a high modulation frequency, and accordingly, it is possible to shorten the time for acquiring an observation image of attenuated fluorescence.

In the microscope systems 12, 14, 16, 18, 20, 22, 24, and 26, the scanning unit 151 in FIG. 6 or the scanning unit 156 in FIG. 7 may be used instead of the scanning unit 150.

In the microscope system 10 or the like, the probe light that is turned on by the AOTF 114 is modulated by the AOM 124. However, the probe light may be modulated by f1 by the AOTF 114, and the pump light may be modulated by f2 by the AOM 124.

In the microscope systems 10 to 26, intensity modulation may be performed using a mechanical shutter such as a chopper instead of the AOM 124 or the like. Instead, intensity modulation may be performed by switching the polarization direction at high speed using an EOM (electro-optical element) and a polarizer.

Although a continuous oscillation method is used as the laser light source 102 and the like, a pulse laser may be used. Since the pulse laser has a higher peak intensity, the stimulated emission is more efficiently generated and the attenuated fluorescence signal is also efficiently generated. The pulse repetition frequency is preferably determined in consideration of the fluorescence lifetime and damage due to the peak power of the pulse. On the other hand, the CW laser has the advantage that the price is lower. Further, although a photomultiplier tube is used as the light receiving unit 174 and the like, an avalanche photodiode (APD) may be used.

FIG. 27 shows an example of another light source 101. In the light source 101, the laser light sources 104 and 102 are pulse lasers. Fluorescence lifetime is measured using these pulse lasers. In the light source 101, the light output from the laser light source 104 for probe light is reflected by the mirror 470 and is incident on the optical delay stage 700. The optical delay stage 700 is movable in the direction of the arrow and includes a mirror 471 and a mirror 472. By translating the optical delay stage in the direction of the arrow, the optical path length of the probe light changes, and a time difference can be given to the optical pulses of the pump light and the probe light. The light reflected by the mirror 470 is reflected by the mirror 471 and the mirror 472 and guided to the mirror 473 in parallel with the light introduced from the mirror 470. The light reflected from the mirror 473 is combined with the pump light by the dichroic mirror 106. The subsequent steps are the same as in FIG.

FIG. 28 shows the time difference between the pump light pulse and the probe light pulse and the state of the time waveform of fluorescence when the optical delay stage 700 is translated. (A)-(c) shows the relationship between the amount of movement dz of the optical delay stage 700 and the time difference between pulses. When the movement amount dz is small, the time difference is small, and when the movement amount dz is large, the time difference is large. (D) shows a time waveform of fluorescence generated by the pump light. It is generally known that fluorescence decreases exponentially in this way with lifetime. In order to attenuate fluorescence by stimulated emission, the time difference between the probe light and the pump light needs to be shorter than the fluorescence emission duration. Also, the shorter the time difference, the more the fluorescence decreases, so the attenuated fluorescence signal also increases. Therefore, the fluorescence lifetime can be measured by adding a time difference by the optical delay stage 700 and acquiring the attenuated fluorescence signal at each time difference. This situation is shown in FIG. The attenuated fluorescence signal shows how the fluorescence lifetime is reflected. In imaging, fluorescence lifetime imaging can be performed by acquiring attenuated fluorescence images at a plurality of time differences.

In the above embodiment, one-photon excitation is used for excitation, but multiphoton excitation such as two-photon excitation or three-photon excitation may be used.

In the above embodiment, the pinhole 408 or the like may be narrowed down in the configuration in which fluorescence is detected by descanning during attenuated fluorescence observation. As a result, the point spread function of the imaging system also contributes to the improvement of the optical resolution, so that the resolution can be further improved. In the case of observing bright fluorescence with a sufficient amount of light, it is desirable that the pinhole 408 or the like be narrowed down. On the other hand, when observing dark fluorescence where the amount of light is not sufficiently secured, it is desirable to open the pinhole 408 and the like. In addition, since the microscope systems 14 to 26 shown in FIGS. 15 to 26 have the descanning optical system and the pinholes 408 and 418 are arranged, they can be used as a confocal microscope.

Also, when observing a wide field of view, the spot deviation in the in-plane direction of the pump light and the probe light becomes a problem due to the chromatic aberration of magnification. In this case, it is desirable to make the beam diameter of the pump light thinner (smaller) than the beam diameter of the probe light. As a result, the spot of the pump light spreads in the in-plane direction and the optical axis direction, and beam overlap becomes easier. The reason for making the beam diameter of the pump light narrower is that the wavelength of the pump light is shorter than that of the probe light. This is because the spot diameter is smaller than that of light.

Also in the microscope systems 10, 20, 22, 24, and 26, similarly to the microscope system 14 and the like, the wavelength controller 230 may be provided to control the wavelength of the light from the laser light source.

The dichroic mirrors 162 and 402 transmit the irradiation light and reflect the fluorescence. Alternatively, the dichroic mirrors 162 and 402 may reflect the irradiation light and transmit the fluorescence. The dichroic mirrors such as the dichroic mirrors 162 and 402 are an example of a wavelength separation member that separates light of a predetermined wavelength from light of other wavelengths.

Although the lock-in amplifier has been described as a method for detecting a signal component of a specific frequency, other methods may be used. For example, a signal component having a specific frequency may be detected by performing a Fourier transform on the time signal. For example, a reference signal having a demodulation frequency and signal light may be multiplied by a frequency converter to extract only a direct current component. Note that the DC component here corresponds to a value obtained by converting a vibration component of a sine wave into DC.

Note that the polarization of the pump light and the probe light is preferably the same. For example, the same linearly polarized light and circularly polarized light are desirable. Alternatively, a configuration that visualizes the polarization characteristics of the specimen by acquiring attenuated fluorescence images for the case where the polarization of the pump light and the probe light is orthogonal linear polarization and parallel polarization, and comparing them. It is also good. This is because the efficiency of stimulated emission depends on the polarization.

10, 12, 14, 16, 18, 20, 22, 24, 26 Microscope system 100, 101 Light source 102, 104, 500, 504 Laser light source 106, 141, 144, 162, 402, 412, 450, 454, 456, 506 Dichroic mirror 110 First intensity modulator 112 Driver 114 Acousto-optic tunable filter 120 Second intensity modulator 122 Driver 124 Acousto-optic device 130 Controller 132 Oscillator 134, 135 Lock-in amplifier 136, 137 Detector 140 Illumination optical system 142 , 143, 154, 155, 157, 452, 458, 460 Mirror 150, 151, 156 Scan unit 152 Resonant scanner 153 Galvano scanner 160 Observation optical system 164 Objective lenses 166, 404, 414 Optical filters 406, 41 6 Lens 172, 173 Lens pair 174, 410, 420 Light receiving unit 180 Stage 182 Slide glass 184 Observation object 186 Sample 220 Input unit 222 Image generating unit 224 Display unit 226 Storage unit 228 Scanner control unit 229 Frequency control unit 230 Wavelength control unit 230 300, 350, 360 GUI screen 302, 304, 312, 316, 324, 352, 362, 364 Check box 354, 356, 358, 366, 374 Display field 308, 310, 314, 318, 320, 322, 326 Input field 330, 332 Observation image 334, 335 Time-lapse image 408, 418 Pinhole 550, 560, 570, 580 Intensity modulation unit 552, 562, 572, 582 Driver 554 AOTF
564, 574, 584 AOM

Claims (22)

1. A first intensity modulator that modulates the intensity of the first light that excites the first fluorescent substance contained in the sample at the frequency f1,
   A second intensity modulator that modulates the intensity of the second light that causes stimulated emission in the first fluorescent substance at a frequency f2 different from the frequency f1,
   A scanning unit that scans the first light and the second light on the specimen;
   A detection unit for detecting fluorescence from the specimen,
   The scanning unit includes a resonant scanner having a resonant mirror,
   The detector is
   A microscope system that receives fluorescence from the specimen and detects a component having a frequency of f1 + f2.
2. The detector is
   A light receiving portion for receiving fluorescence from the specimen;
   The microscope system according to claim 1, further comprising: a lock-in amplifier that detects lock-in of the fluorescence received by the light receiving unit at a frequency f1 + f2.
3. The microscope system according to claim 1, wherein the scanning unit includes a resonance mirror that scans in a main scanning direction and a galvanometer mirror that scans in a sub-scanning direction.
4. An objective lens is provided between the scanning unit and the sample,
   A first wavelength separation member is provided between the objective lens and the scanning unit;
   The microscope system according to any one of claims 1 to 3, wherein the detection unit detects fluorescence transmitted or reflected by the first wavelength separation member.
5. A second wavelength separation member is disposed between the light source and the scanning unit;
   The microscope system according to any one of claims 1 to 4, wherein the detection unit detects fluorescence transmitted or reflected by the second wavelength separation member.
6. An opening member having an opening is provided between the second wavelength separation member and the detection unit,
   The microscope system according to claim 5, wherein a size of the opening of the opening member is variable.
7. The microscope system according to any one of claims 1 to 6, wherein the scanning unit is configured so that the resonant scanner and the galvano scanner can be selected.
8. The microscope system according to any one of claims 1 to 7, wherein a beam diameter of the first light is smaller than a beam diameter of the second light.
9. A storage unit that stores information that associates the first fluorescent substance with the wavelength of the first light, and information that associates the first fluorescent substance with the wavelength of the second light;
   A reception unit that receives selection of the first fluorescent material; and a control unit,
   The controller is
   The wavelength of the first light and the second light corresponding to the first fluorescent material received by the receiving unit is determined with reference to the storage unit. The described microscope system.
10. The storage unit
    Information associating the first fluorescent substance, the wavelength of the first light, and the modulation frequency of the first light, the wavelength of the first fluorescent substance, the second light, and the second light And stores information that associates the modulation frequency of
    The controller is
    Referring to the storage unit, the modulation frequency of the first light and the first light, the wavelength of the second light, and the second corresponding to the first fluorescent material received by the reception unit The microscope system according to claim 9, wherein a modulation frequency of light is determined.
11. 11. The microscope system according to claim 1, wherein one of the first intensity modulation unit and the second intensity modulation unit includes an acoustic tunable filter, and the other includes an acoustooptic element.
12. The specimen includes a second fluorescent material of a different type from the first fluorescent material,
    The first light excites the second fluorescent material;
    The second intensity modulator modulates the intensity of the third light that causes stimulated emission in the second fluorescent material at a frequency f3 different from the frequency f1 and the frequency f2,
    The detector is
    The microscope system according to any one of claims 1 to 11, wherein fluorescence from the sample is received and a component of frequency f1 + f2 and a component of frequency f1 + f3 are detected.
13. The microscope system according to claim 12, wherein at least one of the frequency f1 + f2 component and the frequency f1 + f3 component is detected at a predetermined phase.
14. The specimen includes a second fluorescent material of a different type from the first fluorescent material,
    The first intensity modulator modulates the intensity of the fourth light that excites the second fluorescent substance at a frequency f4 different from the frequency f1 and the frequency f2,
    The second light causes stimulated emission in the second fluorescent material;
    The detector is
    The microscope system according to any one of claims 1 to 11, wherein fluorescence from the specimen is received and the component of the frequency f1 + f2 and the component of the frequency f4 + f2 are detected.
15. The microscope system according to claim 14, wherein at least one of the frequency f1 + f2 component and the frequency f4 + f2 component is detected at a predetermined phase.
16. A first observation method of irradiating the specimen with first light for exciting a fluorescent substance contained in the specimen, and receiving fluorescence from the specimen at a detection unit;
    The first light is intensity-modulated at a frequency f1 and irradiated to the specimen,
    Irradiating the specimen with intensity-modulated second light that causes stimulated emission in the fluorescent material at a frequency f2 different from the frequency f1,
    A microscope system configured to be able to select a second observation method in which the detection unit receives fluorescence from the specimen and detects a component of frequency f1 + f2 or a component of frequency f1-f2.
17. An opening member having an opening is provided between the detection unit and the sample,
    The microscope system according to claim 16, wherein a size of the opening of the opening member is variable.
18. Having an objective lens,
    The microscope system according to claim 17, wherein the opening member is provided between the detection unit and the objective lens.
19. A scanning unit between the objective lens and the detection unit;
    The microscope system according to claim 18, wherein the opening member is provided between the detection unit and the scanning unit.
20. A wavelength separation member between the scanning unit and the detection unit;
    The microscope system according to claim 19, wherein the opening member is provided between the detection unit and the wavelength separation member.
21. The microscope system according to any one of claims 17 to 20, wherein a size of the opening of the opening in the first observation method is smaller than a size of the opening in the second observation method.
22. The microscope system according to any one of claims 17 to 21, wherein the first observation method or the second observation method is selected based on a type of the fluorescent substance.

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