Laser radiation system and method for radiating laser light to observation sample

Abstract

To provide a method for radiating a laser light from a camera port to an arbitrary target position with time control in a simple manner in applications such as optogenetics for applying laser light to an observation sample of an optical microscope.SOLUTION: There is provided a system connectable to a camera port of a microscope, the system including: an imaging element capable of capturing an image of a sample; a laser light source; and a single movable mirror for adjusting the position of radiating a focused spot of laser light emitted from the light source onto an observation sample, the laser light intensity and the position of the movable mirror being controlled by a single digital-to-analog device. There is also provided a method for laser radiation using the system.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] Channelrhodopsin is a central molecular tool in optogenetics, which uses light to manipulate neural activity in living organisms, and its use has led to the elucidation of many aspects of neural activity. (Patent Document 1) The number of channelrhodopsin types that respond to different wavelengths is increasing, making it possible to conduct more complex research than before. By connecting a laser light source to an optical microscope and irradiating laser light at any position within the microscope's observation field, it is possible to apply optogenetic techniques by irradiating laser light only to genetically unique cells discovered through microscopic observation, and it is now possible to experimentally verify the complex relationship between genes and neural activity.

[0002] Conventional optical microscopes or fluorescence microscopes have been used only for observational purposes, such as diagnostic applications for observing stained slides made from organs surgically removed from patients with diseases such as cancer, or for time-lapse observation applications, such as observing cell proliferation over time. However, in recent years, optical microscopes have been used to stimulate cells, such as the optogenetics technique using channelrhodopsin mentioned above, diversifying the applications of optical microscopes. The method of irradiating cells with laser light is not limited to optogenetics research using channelrhodopsin. It is also used in research using photoswitching materials (Non-Patent Document 1), which change the color of fluorescent reagents in the irradiated area. Furthermore, there are applications such as a method of irradiating living organisms with laser light at a wavelength of 1.5 microns, which is absorbed by water, to locally heat them for a short period of time and facilitate gene transfer (Non-Patent Document 2). Therefore, there are various applications for the technique of irradiating laser light at any position within the observation field of an optical microscope.

[0003] Figure 1 shows a schematic diagram of a fluorescence microscope commonly used in biological research. In a fluorescence microscope system 20, illumination light beam 8 emitted from a lamp light source 1, such as a mercury lamp, is guided by a condenser lens 2 through a fluorescence filter cube 11 in a filter turret 15 to an objective lens 3, illuminating a cell sample 19 placed, for example, in a culture vessel 16 together with a cell culture medium 18. Within the fluorescence filter cube 11, an excitation filter 12 is placed to select a wavelength appropriate for illuminating the fluorescent reagent, and an emission filter 14 is placed to observe the fluorescent wavelength emitted by the fluorescent reagent. Furthermore, a dichroic mirror 13 with reflection / transmission wavelength characteristics is placed so that the illumination light is reflected toward the objective lens 3 and the fluorescent wavelength is transmitted toward the camera 6. The fluorescence microscope system 20 is equipped with a filter turret 15 that holds multiple fluorescence filter cubes, allowing easy selection of a filter cube suitable for observation.

[0004] The fluorescence microscope system 20 is equipped with an imaging lens 4 and a camera 6, and can acquire image information of the fluorescence emitted by the cell sample being observed according to the observation magnification of the objective lens 3. The fluorescence microscope system 20 is equipped with a camera port 5 so that cameras tailored to the observer's needs can be attached. In the fluorescence microscope system 20 shown in Figure 1, the microscope observation light 9 observed through the cover glass 17 is converted into an observation beam 9 of predetermined wavelength components by the dichroic mirror 13 and emission filter 14 of the fluorescence filter cube 11. The observation beam 9 is then converged by the imaging lens 4, reflected by the mirror 10, and imaged on the image sensor 7 in the camera 6 connected to the camera port 5. Almost all optical microscopes are equipped with one or more camera ports conforming to a standard known as a C-mount, allowing the attachment of cameras tailored to the observer's needs. The C-mount standard is a standard in which a 25.4 mm (1 inch) inner diameter, 0.794 mm pitch thread is formed at the connection point, and the imaging position is located 17.526 mm from the end of the thread.

[0005] By standardizing the camera port of an optical microscope, when an operator focuses the image using the eyepiece, the camera can capture a nearly in-focus image of the sample, greatly improving the ease of use for the operator. Furthermore, researchers can attach a camera to the microscope that is appropriate for their research or work. In other words, attaching a camera to the camera port is not a custom job or microscope modification job performed by a microscope manufacturer, but rather a job that individual researchers can do themselves.

[0006] One method for irradiating a sample under observation with an optical microscope or a fluorescence microscope with laser light at a desired position is the DMD (Digital Microscope Device) method, which uses a DMD element with a large number of small mirrors arranged in a DMD array. (Patent Document 2) This method allows laser light to be irradiated in any desired shape onto the sample under observation. Figure 2 shows the principle of light irradiation using the DMD method. This optical system irradiates laser light onto all mirrors in a device with a large number of mirrors, each approximately 10 to 15 microns in size, arranged in a number of rows (e.g., approximately 2,000 horizontally and approximately 1,000 vertically). Each mirror can be switched between a position where laser irradiation is performed and a position where laser irradiation is not performed. For example, in the laser light irradiation device 120 shown in Figure 2, mirror 121 is in the irradiation position, and mirror 121 has the property of reflecting laser light irradiated by laser beam 124 in the direction indicated by arrow 125 so that it can be combined with lens 123. On the other hand, mirror 122 is positioned so that the sample is not irradiated, and mirror 122 has the property of reflecting the laser light irradiated by laser beam 124 in the direction shown by arrow 126 so that it does not couple with lens 123, and irradiating light absorption band 127. Each mirror can be switched between an irradiating position and a non-irradiating position at high speed in response to a command from a controller, and can be switched in about 10 microseconds.

[0007] However, since the laser beam is irradiated onto a large number of arranged mirrors, the light irradiated onto the edge of each mirror becomes stray light. Specifically, even for a mirror at a position 122 where the sample is not irradiated, the laser light irradiated onto the edge 122e is not reflected in the direction of the optical absorption band 127, and a part of the light is reflected toward the lens 123, so that a small amount of light is irradiated onto the position on the sample where it is not intended to be irradiated.

[0008] Because there are so many mirrors, even if a laser with an optical output of 100 milliwatts is used as the laser light source, there are 2,000,000 mirrors, so the energy is dispersed to each mirror at 0.05 microwatts. For example, if the mirror is a square with sides of 13 microns and is irradiated onto a sample at an optical magnification of 20, 5 microwatts will be irradiated onto a square with sides of 0.65 microns, resulting in an irradiation power of approximately 0.11 microwatts per square micrometer, which is not enough to obtain the irradiation energy of 1 kilowatt per square millimeter, or 10 microwatts per square micrometer, required to switch the photoswitching material described in Non-Patent Document 1.

[0009] On the other hand, the method of irradiating a sample with laser light incident on the illumination port of a microscope achieves a focal spot size defined by the numerical aperture (NA) of the objective lens, and the amount of light from the laser light source is not dispersed, so the sample can be irradiated with the energy required to switch the photoswitchable material. However, this method does not allow the irradiation position to be adjusted to any desired location. Furthermore, installing equipment on the illumination port of a microscope requires modification of the microscope, making it impossible for the user to do it alone. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] Patent No. 6363060 specification [Patent Document 2] U.S. Patent No. 1,112,309 [Non-patent literature]

[0011] [Non-Patent Document 1] Daniel Thedie, Romain Berardozzi, Virgile Adam, and Dominique Bourgeois, "Photoswitching of Green mEos2 by Intense 561 nm Light Perturbs Efficient Green-to-Red Photoconversion in Localization Microscopy", J. Phys. Chem. Lett. 2017, 8, 18, 4424-4430 [Non-patent document 2] Tomonori Deguchi, Mariko Itoh, Hiroko Urawa, Tomohiro Matsumoto, Sohei Nakayama, Takashi Kawasaki, Takeshi Kitano, Shoji Oda, Hiroshi Mitani, Taku Takahashi, Takeshi Todo, Junichi Sato, Kiyotaka Okada, Kohei Hatta, Shunsuke Yuba, Yasuhiro Kamei, "Infrared laser-mediated local gene induction in medaka, zebrafish and Arabidopsis thaliana", Development Growth and differentiation, Vol. 51, pp. 769-775 (2009) Summary of the Invention [Problem to be solved by the invention]

[0012] The present invention has been made in consideration of the above points, and provides a laser irradiation system that can be easily connected to the camera port of a fluorescence microscope without the need to modify the microscope, and that can irradiate any position on an observation sample with high-speed control of the energy required to switch a photoswitching material, and a method for irradiating a laser beam onto an observation sample. [Means for solving the problem]

[0013] To solve this problem, the laser irradiation system and method of irradiating a specimen with laser light of the present invention are configured to include an image sensor connectable to a camera port of a microscope and capable of capturing images of the specimen, a light source, and a movable mirror for adjusting the position of the focal spot of the laser light emitted from the light source on the specimen. A plate-shaped mirror that reflects the laser light is placed between the camera port and the image sensor to position the microscope image at the wavelength of the laser. The focal spot of the laser light emitted from the light source is formed near the focal spot, and the movable mirror adjusts the position of the focal spot, thereby irradiating the specimen with the laser light emitted from the laser light source. Furthermore, the position adjustment of the movable mirror and the power adjustment of the light source are performed in conjunction with each other with high time resolution by controlling the position adjustment of the movable mirror and the power adjustment of the light source using a single digital-analog controller. [Effects of the Invention]

[0014] The laser irradiation system and method for irradiating a specimen with laser light of the present invention can irradiate a desired position within the field of view of a specimen observed with a microscope with laser light from a light source. Since stray light is not generated during irradiation, high-contrast laser irradiation is possible. Furthermore, since a laser spot with high irradiation light density can be formed on the specimen surface, color conversion of photoswitching materials and fluorescence recovery after photobleaching (FRAP) can be easily performed even under low microscope objective lens conditions.

[0015] The laser irradiation system and the method for irradiating a laser beam onto an observation sample of the present invention are systems that can be easily installed by users without modifying a general optical microscope. The system can also be installed on a stereomicroscope. Since the laser irradiation system of the present invention has an image sensor and can acquire images of the observation sample, even on a microscope with only one camera port, it is possible to specify the laser irradiation position from the image information acquired by the camera and irradiate the laser at any position.

[0016] Furthermore, in the laser irradiation system and the method for irradiating a laser beam onto an observation sample of the present invention, the size of the focused laser beam spot irradiated onto the sample can be adjusted by inserting an optical system that adjusts the collimation state between the light source and the movable mirror. Also, by controlling the light source and the angular position of the movable mirror in a time series manner using signals from a single digital-to-analog converter, it is possible to easily irradiate the laser beam at any timing. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 is a schematic diagram illustrating the configuration of a typical fluorescence microscope. [Figure 2] FIG. 1 is a schematic diagram illustrating the configuration of a conventional laser irradiation system. [Figure 3] 1 is a schematic diagram illustrating the configuration of a laser irradiation system according to the present invention. [Figure 4] FIG. 1 is a schematic diagram illustrating the configuration of a laser irradiation system according to the present invention when attached to a fluorescence microscope. [Figure 5] 1 is an explanatory diagram showing a laser irradiation position when laser irradiation is performed using the laser irradiation system of the present invention. FIG. [Figure 6] FIG. 1 is a schematic diagram illustrating the configuration of a laser irradiation system according to the present invention when attached to a stereomicroscope. [Figure 7] 1 is an explanatory diagram showing a laser irradiation position when laser irradiation is performed using the laser irradiation system of the present invention. FIG. [Figure 8] 1 is a schematic diagram illustrating the configuration of a laser irradiation system according to the present invention. [Figure 9]FIG. 1 is a schematic diagram illustrating the configuration of a laser irradiation system according to the present invention when attached to a fluorescence microscope. [Figure 10] 1 is an explanatory diagram showing a laser irradiation position when laser irradiation is performed using the laser irradiation system of the present invention. FIG. [Figure 11] FIG. 1 is a schematic diagram illustrating the configuration of a laser irradiation system according to the present invention when attached to a stereomicroscope. [Figure 12] 1 is an explanatory diagram showing a laser irradiation position when laser irradiation is performed using the laser irradiation system of the present invention. FIG. [Figure 13] FIG. 4 is a diagram showing an example of a time chart of a control signal of the laser irradiation system of the present invention. [Figure 14] 10A and 10B are explanatory diagrams illustrating a method for generating a control signal for the laser irradiation system of the present invention. [Figure 15] 10A and 10B are explanatory diagrams illustrating a method for generating a control signal for the laser irradiation system of the present invention. [Figure 16] 10A and 10B are explanatory diagrams illustrating a method for generating a control signal for the laser irradiation system of the present invention. [Figure 17] 10A and 10B are explanatory diagrams illustrating a method for generating a control signal for the laser irradiation system of the present invention. [Figure 18] FIG. 4 is a diagram showing an example of a time chart of a control signal of the laser irradiation system of the present invention. [Figure 19] FIG. 1 is a schematic diagram illustrating the configuration of a laser irradiation system according to the present invention when attached to a fluorescence microscope. [Figure 20] 1 is a schematic diagram illustrating the configuration of a laser irradiation system according to the present invention. [Figure 21] FIG. 1 is a schematic diagram illustrating the configuration of a laser irradiation system according to the present invention when attached to a fluorescence microscope. [Figure 22] 1 is an explanatory diagram showing a laser irradiation position when laser irradiation is performed using the laser irradiation system of the present invention. FIG. [Figure 23] FIG. 4 is a diagram showing an example of a time chart of a control signal of the laser irradiation system of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0018] FIG. 3 shows a schematic diagram of a laser irradiation system 60 according to a first embodiment of the present invention. This laser irradiation system has a camera port connection port 5a compatible with the C-mount standard. When connected to a C-mount camera port of a microscope, an image sensor 7 is positioned at a location where the microscope observation light beam 9 of the sample is focused. A dichroic mirror 48, which selectively reflects the laser wavelength of the laser light source 41, is positioned between the camera port connection port 5a and the image sensor 7. The dichroic mirror 48 is a plate-shaped mirror approximately 1 mm in diameter. Although oblique placement of a plate in the focusing optical system generates astigmatism, the focal length of the imaging lens of an optical microscope is approximately 200 mm or 180 mm. Therefore, even if a plate-shaped dichroic mirror 48 made of glass with a refractive index of approximately 1.5 and a thickness of 2 mm or less (preferably approximately 1 mm) is placed in front of the image sensor 7 at a 45-degree angle, the image captured by the image sensor 7 can be captured without the influence of astigmatism. Furthermore, because astigmatism only affects the optical path that passes through glass materials, by arranging the reflective surface of dichroic mirror 48 on the light source 41 side, astigmatism does not affect the laser light emitted from laser light source 41. By using a cube-shaped dichroic mirror for dichroic mirror 48, it is possible to reduce astigmatism in the image captured by image sensor 7, but a plate-shaped dichroic mirror is cheaper than a cube-shaped dichroic mirror. By disposing the dichroic mirror 48 between the camera connection port 5a and the image pickup device 7, an image plane 37 is formed for the wavelength reflected by the dichroic mirror.

[0019] Laser light having a wavelength of, for example, 635 nm emitted from a laser light source 41 is collimated by a condenser lens 42, reflected by a movable mirror 45, and then formed into a focused spot 47 on an imaging plane 37 by a scan lens 46. Here, a quarter-wave plate 44 and a polarizing spectroscopic element 43 are disposed between the lens 42 and the movable mirror element 45, and the operation of the laser light source 41 can be stabilized by adjusting the angle of the quarter-wave plate 44 so that reflected light from the sample does not enter the light source 41.

[0020] The light source 41 uses a semiconductor laser element that emits laser light with a wavelength of approximately 635 nm, and is configured to emit light in response to a current supplied from a semiconductor laser driver board 81 via wiring 83. The current supplied from the semiconductor laser driver board 81 is configured to be controlled by a digital-to-analog converter (DAQ) 80 via analog signal supply wiring 82.

[0021] The movable mirror element 45 is connected to a mirror drive board 84 via a cable 87, and the two-dimensional angle of the movable mirror portion 45a of the movable mirror element 45 is controlled by a voltage signal supplied from a digital-to-analog converter (DAQ) 80 via analog signal supply wiring 85, 86. Here, the digital-to-analog converter (DAQ) 80 is connected to a computer to which the image sensor 7 is connected. The digital-to-analog converter (DAQ) 80 can also be disposed within the computer to which the image sensor 7 is connected. Furthermore, it is desirable to shorten the lengths of the cables 83, 87 by arranging the semiconductor laser driver board 81 and the mirror drive board 84 near the laser light source 41 and the movable mirror 45 and attaching them to the chassis of the laser irradiation system 60, for example.

[0022] A schematic configuration of a system 21 in which a laser illumination system 60 of the present invention is connected to a fluorescence microscope is shown in Figure 4. Because the laser illumination system 60 is connected to the camera port 5 of the microscope, images of wavelength components transmitted through the dichroic mirror 13, emission filter 14, and mirror 48 of the sample image magnified by the objective lens and imaging lens can be acquired by the image sensor 7 in the laser illumination system 60. Furthermore, the laser light emitted from the laser light source 41, which forms a focused spot 47 on the imaging plane 37, reflects off the spectral mirror 48, is substantially collimated by the imaging lens, then passes through the emission filter 14 and dichroic mirror 13, and is irradiated by the objective lens 3 onto sample positions corresponding to each focused spot 47. Here, if image observation using an imaging element is not required when the laser light source 41 is emitting light, the filter cube 11 can be retracted from the optical path within the filter turret 15, so that the laser light emitted from the laser light source 41 can be irradiated onto the sample even if the emission filter 14 and dichroic mirror 13 have wavelength characteristics that do not transmit the wavelength of the laser light source 41.

[0023] By using an F-θ lens as the scan lens 46 and disposing a movable mirror 45a at the back focus position of the scan lens 46, the position of the focused spot 47 of the laser light emitted from the laser light source 41 on the imaging plane 37 can be controlled by controlling the angle of the movable mirror 45a. Furthermore, since the focused spot 47 on the imaging plane 37 is reduced and projected onto the sample surface by the imaging lens 4 and the objective lens 3, the position of the focused spot 40 of the laser light emitted from the laser light source 41 on the cell 19 in the microscope observation sample shown in FIG. 5 can be adjusted by controlling the angle of the movable mirror 45a. The signal to be provided for control is a numerical value determined from the relationship between the position information of the image captured by the image sensor 7 and the angle information of the movable mirror 45.

[0024] In the example shown in Figure 5, the angle of the movable mirror 45a, i.e., the position of the focused spot 40 of light emitted from the laser light source 41, which is controlled by a signal from a mirror drive board 84, which is controlled by a voltage signal supplied from a digital-to-analog converter (DAQ) 80 via analog signal supply wiring 85, 86, is made to coincide with approximately the center of the cell 19.

[0025] The light emitted from the light source 41 is input to the semiconductor laser driver board 81 via an analog signal supply wiring 82 by a digital-to-analog converter (DAQ) 80, and the light is emitted in accordance with the current supplied via wiring 83. Therefore, it is possible to coordinate and control the timing of the control signals for the light source 41 and the movable mirror 45 output from the digital-to-analog converter (DAQ) 80.

[0026] FIG. 6 shows a schematic configuration of a system 22 in which a laser irradiation system 60 of the present invention is connected to a stereomicroscope. In the stereomicroscope system 22, an F-θ lens is used for the scan lens 46, and a movable mirror 45a is disposed at the back focus position of the scan lens 46. By controlling the angle of the movable mirror 45a, the position of the focused spot 47 of the laser light emitted from the laser light source 41 on the image forming plane 37 can be controlled. Furthermore, the focused spot 47 on the image forming plane 37 is enlarged or reduced and projected onto the sample surface by the imaging lens 4 and the objective lens 3. Therefore, the position of the focused spot 40 of the laser light emitted from the laser light source 41 on the microscope observation sample 30 shown in FIG. 7 can be adjusted by controlling the angle of the movable mirror 45a.

[0027] A schematic configuration diagram of a laser irradiation system 61 as a second embodiment of the present invention is shown in Fig. 8. In the laser irradiation system 61 of the present invention, a pair of lenses consisting of lenses 31 and 32 is inserted in the optical path of the laser light emitted from the light source 41 in the laser irradiation system 60 shown in Fig. 3, that is, the parallel light portion incident on the movable mirror 45, specifically, between the movable mirror 45 and the quarter-wave plate 44, and by moving the position of at least one of the lenses 31 or 32 and adjusting the distance between the lenses 31 and 32, an optical system is formed in which the laser light incident on the movable mirror 45 can be adjusted to divergent light, convergent light, or even parallel light.

[0028] FIG. 9 shows a system 23 in which a laser irradiation system 61 is attached to a fluorescence microscope. In the fluorescence microscope system 23 connected to the laser irradiation system 61, the laser light emitted from the light source 41 and incident on the movable mirror 45 can be adjusted to divergent light, convergent light, or even parallel light, thereby making it possible to adjust the position of the focused spot 47 formed near the image plane 37 to be closer to or farther away from the spectral mirror 48. When the focused spot 47 is located on the image plane 37, the focused spot 40 on the sample is in focus, so that by moving the position of the focused spot 47 formed near the image plane 37 closer to or farther away from the spectral mirror 48, the focused spot 40 on the sample becomes larger. When there is no room for space in the size of the movable part 45a of the movable mirror 45, if the laser light emitted from the light source 41 and incident on the movable mirror 45 is made divergent, it may go beyond the mirror 45a. Therefore, it is desirable to adjust the distance between the lenses 31 and 32 to make the size of the focused spot 40 on the sample focused or to increase the diameter, and to adjust the distance between the lenses 31 and 32 to make the laser light emitted from the light source 41 and incident on the movable mirror 45 parallel or convergent.

[0029] 10, the size of the focused spot 40 on the sample of the laser light emitted from the laser 41 is adjusted by adjusting the distance between the lenses 31 and 32 and the distance between the lenses 33 and 34. Then, the angle of the movable mirror 45 is adjusted so that the laser light is irradiated onto the entire cell 19, and the position of the focused spot 40 is adjusted to the position of the cell 19. Furthermore, the timing of the emission of the lasers 41 is also adjusted so that they are synchronized.

[0030] FIG. 11 shows a schematic configuration of a system 24 in which a laser irradiation system 61 of the present invention is connected to a stereomicroscope. In the stereomicroscope system 24 as well, the position of the focused spot 40 of the laser light emitted from the laser light source 41 on the microscope observation sample 30, the irradiation timing, and the size of the focused spot 40 are adjustable.

[0031] FIG. 12 shows an example in which the position and size of the focused spot 40 of the laser light emitted from the laser light source 41 on the microscope observation sample 30 are slightly larger than those in the example shown in FIG.

[0032] Here, we will explain the control signals for the light source 41 and the movable mirror 45 output from the digital-to-analog converter (DAQ) 80. The amount of light emitted by the light source 41 and the timing of light emission are controlled by a time-series signal 82t output from the digital-to-analog converter (DAQ) 80 and input to the semiconductor laser driver board 81. The angle of movable mirror 45 is controlled by time-series signals 85t, 86t that are output from digital-to-analog converter (DAQ) 80 and input to mirror drive board 84. Fig. 13 shows an example of time-series signals 82t, 85t, and 86t that are output from digital-to-analog converter (DAQ) 80. In this example, laser irradiation is performed from timing t1 to t2, and a method for generating the signals shown in Fig. 13 will be described using Figs. 14 to 17 below.

[0033] In the laser irradiation system of the present invention, the movable mirror 45 is a mirror device (model number: A8L2.2-5000AL-TINY48.4-A / W / TP) from Mirrorcle Technologies, which can be driven two-dimensionally in the X and Y directions, and the mirror drive board 84 is an analog control board (model number: DR-11-055-00) from Mirrorcle Technologies. In this case, both the X and Y axes have a characteristic in which the angle changes linearly from -4 degrees to +4 degrees in response to an input voltage signal from -10 V to +10 V. Furthermore, by using a scan lens from Thorlabs or an equivalent lens as the scan lens 46, the position of the spot 40 can be adjusted to any position on the microscope observation sample according to the voltage value input to the mirror drive board 84. The relationship between the voltage value that adjusts the position of the focal spot 40 of the laser light generated from the laser light source 41 and the position on the microscope observation sample observed by the image sensor 7 is the relationship between the position of the focal spot 47 formed on the imaging surface 37 and the position of each pixel on the image sensor 7, so the voltage value (VX1, VY1) to be input to the mirror drive board 84 can be determined from the pixel position information of the pixel coordinates (PX1, PY1) of the focal spot 40 shown in Figure 14.

[0034] Furthermore, when a semiconductor laser is used as the light source 41, as shown in FIG. 15, the laser light intensity increases almost linearly with the current Iop injected into the semiconductor laser light source above the oscillation threshold current Ith. While the characteristics shown in FIG. 15 vary depending on the individual semiconductor laser, by acquiring the characteristics of the semiconductor laser used in the device and specifying the required output power LP1, the current IP1 required to emit that power can be calculated as shown in FIG. 15. In the present invention, for example, by using a semiconductor laser drive board from Analog Technology, it is possible to output a current that responds linearly to an analog voltage signal. Furthermore, since Analog Technology's semiconductor laser drive board has a response on the order of MHz, applying a voltage in millisecond increments supplies the semiconductor laser with a current value corresponding to the input voltage without any time delay. Therefore, when supplying a current IP1 to the light source 41, it is sufficient to supply a current VP1 corresponding to the current IP1 to the semiconductor laser driver board 81.

[0035] FIG. 16 shows a flow 100 for generating the time-series signals 82t, 85t, and 86t shown in FIG. 13 in a state where the relationships shown in FIGS. 14 and 15 are obtained. The input data for creating the time series signals 82t, 85t, and 86t are the coordinates (PX1, PY1) of the irradiation target position of the laser spot 40 in the image captured by the imaging element 7, which are components of the input value table 101, the laser irradiation time Tillum, and the irradiation light amount of the laser spot 40: LP1. From this input table 101, first, the output calibrator 102 of the light source 41 converts the irradiation light amount LP1 of the laser spot 40 into the target light amount, an example of which is shown in Fig. 15, to obtain the current corresponding to the output LP1. Furthermore, the voltage value VL1 to be input to the semiconductor laser drive board is obtained.

[0036] Next, from the input table 101, the data output calibrator 103 of the mirror 45 determines the voltage value (VX1, VY1) to be input to the mirror drive board 84 from the coordinates (PX1, PY1) of the target position where the laser spot 40 is to be irradiated.

[0037] At this stage, the numerical values ​​of each signal value during laser irradiation have been obtained for the time series signals 82t, 85t, and 86t, but information on the transition state from the system's initial state to the irradiation state and the transition state from the end of irradiation back to the system's initial state has not been obtained.

[0038] Next, the contents of the voltage signal generator 106 that forms the time stream data will be described with reference to FIG. There are various ways to send a trigger signal to start irradiation, such as clicking a specified button on the program screen, inputting an external voltage trigger signal, or pressing a handheld trigger switch, but because there is a possibility that vibrations may be applied to the microscope when the program starts, it is desirable to provide a certain amount of time (Tmargin1 in Figure 17) at the beginning of the time series signals 82t, 85t, and 86t during which the voltage does not change. This time can be as short as a few milliseconds, but it depends on the situation.

[0039] The time it takes for the movable mirror to move to the designated position can be estimated from the frequency characteristics of the movable mirror, or it can be measured. When using a mirror device (model number: A8L2.2-5000AL-TINY48.4-A / W / TP) from Mirror Technologies, which can move two-dimensionally in the X and Y directions, the drive frequency is 120 Hz, so the time it takes for the mirror to move to the target position (Tmove in Figure 17) is set to 10 milliseconds. Because the mirror may vibrate due to the impact of stopping when it reaches the target position, a settling time (Tsettle in Figure 17) of, for example, 2 milliseconds is set. After Tsettle has elapsed, the movable mirror is stopped at the designated position, so the laser is emitted for a predetermined time (Tillum) from time t1 to t2. After the laser is emitted (after time t2), the present invention uses the following method to return the mirror to its initial state. First, after the laser irradiation is completed, a time of, for example, 1 millisecond is set as Tmargin2, and the mirror stops at that position after the laser irradiation is completed, and then moves to the initial position. The time for the mirror to move is set as the Tmove time described above, and then a fixed settling time (Tmargin3) of 10 milliseconds is set, and the time series signal is terminated.

[0040] As described above, the time series signals 82t, 85t, and 86t are generated by the voltage signal generator 106 taking into consideration the frequency characteristics of the device being used, thereby enabling stable laser irradiation at the target position.

[0041] Next, an example of a laser irradiation method using the laser irradiation system of the present invention will be described, in which a laser spot is irradiated onto two different points on a sample. It is also possible to make the time series signals 82t, 85t, and 86t output from the digital-to-analog converter (DAQ) 80 have the shape shown in Figure 18, and after the laser light source 41 emits light from time t1 to t2, 85t and 86t have different voltage values, and then emit light from the laser light source 41 from time t3 to t4.

[0042] FIG. 19 shows a system 25 in which a laser irradiation system 60 and a camera 116 are attached to a fluorescence microscope equipped with multiple camera ports 5 and 115. Depending on the state of the sample, it may be necessary to use a high-sensitivity camera when selecting the laser irradiation position. The camera 116 shown in FIG. 13 is assumed to be a high-sensitivity camera. Here, if the positions of the image sensor 7 in the laser irradiation system 60 and the camera 116 are calibrated in advance, even if the target irradiation position of the laser beam is specified in the image captured by the camera 116, the position of the image sensor 7 corresponding to that position can be estimated, and the voltage signal to be input to the movable mirror 45 can be obtained from that position, allowing laser irradiation to be performed.

[0043] Here, the positions of the image sensor 7 and the camera 116 can be calibrated by fixing them to the camera ports 5 and 115, respectively, taking images of the same sample, and comparing the images. Therefore, when determining the voltage signal to be input to the movable mirror 45 for the laser irradiation target position specified in the image taken by the camera 116, there is no need to take a new image using the image sensor 7. The laser irradiation system of the present invention has an internal imaging element, which simplifies the alignment process even when the laser irradiation position is specified from an image taken by a camera not in the laser irradiation system and laser light is irradiated at that position.

[0044] A schematic configuration diagram of a laser irradiation system 62 according to a third embodiment of the present invention is shown in Fig. 20. The laser irradiation system 62 of the present invention has a configuration in which an optical path of a second laser beam emitted from a light source 51 is added to the laser irradiation system 60 shown in Fig. 3. Laser light having a wavelength of, for example, 450 nm emitted from a laser light source 51 is collimated by a condenser lens 52, reflected by a mirror 58, and then aligned with the light path of the light source 41 by a dichroic mirror 59. The light is then reflected by a movable mirror 45 and focused onto an imaging plane 37 by a scan lens 46 to form a focused spot 47. A quarter-wave plate 54 and a polarizing spectroscopic element 53 are disposed between the lens 52 and the movable mirror element 45, and the angle of the quarter-wave plate 54 is adjusted to prevent light reflected from the sample from entering the light source 51, thereby stabilizing the operation of the laser light source 51. The dichroic mirror 59 transmits the laser light emitted from the light source 41 and reflects the laser light emitted from the light source 51. The laser light source 41 is assumed to have a wavelength of, for example, 1.5 microns.

[0045] The light source 51 uses a semiconductor laser element that emits laser light with a wavelength of approximately 450 nm, and is configured to emit light in response to a current supplied from a semiconductor laser driver board 91 via wiring 93. The current supplied from the semiconductor laser driver board 91 is configured to be controlled by a digital-to-analog converter (DAQ) 80 via analog signal supply wiring 92.

[0046] An example of a configuration in which the laser irradiation system 62 is connected to a fluorescence microscope is shown in Fig. 21. As in the example of the configuration shown in Fig. 4, the spot 47 formed on the imaging plane 37 is reduced and projected onto the sample by the imaging lens 4 and the objective lens 3. In FIG. 22, a focused spot 40 of laser light emitted from a light source 41 and a focused spot 50 of laser light emitted from a light source 51 are shown on a cell 19 . Figure 23 shows examples of time series signals 82t, 85t, 86t, and 92t output from digital-to-analog converter (DAQ) 80 when laser light emitted from light source 41 is irradiated onto cell 19 as spot 40, and immediately thereafter laser light emitted from light source 51 is irradiated onto cell 19 as spot 50.

[0047] Because the light emission wavelengths of light source 41 and light source 51 are different, the signal input to movable mirror 45 to form spot position 47 on imaging plane 37 by scan lens 46 differs slightly when forming spot 40 from light source 41 and when forming spot 50 from light source 51. Therefore, in laser irradiation system 62 of the present invention, it is not easy to simultaneously form spots from light source 41 and light source 51 at the same position on the sample at any position. However, as shown in Fig. 23, it is possible to make light source 51 emit light from time t3 to t4 by adjusting voltage signals 85t and 86t input to movable mirror 45 immediately after light source 41 emits light from time t1 to t2.

[0048] When a 1.5 micron laser light source that is absorbed by water and heats the irradiated area is used as light source 41, and a 450 nm wavelength light source that provides optical stimulation is used as light source 51, and irradiation is performed as shown in Figure 23, optical stimulation can be performed immediately after localized heating. [Industrial Applicability]

[0049] The laser irradiation system and method of irradiating laser light onto an observation sample of the present invention can be used in many areas, such as optogenetic techniques using channelrhodopsin, color conversion of photoswitching materials in which the color of a fluorescent reagent changes in the area where light is irradiated, and a technique for facilitating gene introduction by irradiating a living organism with laser light of a wavelength of 1.5 microns, which is absorbed by water, to cause localized heating for a short period of time. [Explanation of symbols]

[0050] 1... illumination light source, 2, 3, 4, 31, 32, 33, 34, 42, 52, 114... lens, 5, 115... camera port, 6, 116... camera, 7, 117... imaging element, 8... illumination light, 9... fluorescence light, 10, 48, 49, 58, 110... mirror, 11... fluorescence cube, 12... excite filter, 13... dichroic mirror, 14... emission filter, 15... fluorescence turret, 16... observation container, 17... cover glass, 18... culture medium, 19... cell, 20... fluorescence microscope, 21, 23, 25... fluorescence microscope connected to the laser irradiation device of the present invention, 22, 24... stereomicroscope connected to the laser irradiation device of the present invention, 30... sample, 37... image plane, 40, 50... laser light irradiation spot, 41, 51... laser light source, 43, 53... ...QWP (wave plate), 44, 54...Polarization separation element, 45, 55...Scanning mirror, 46, 56...Scan lens, 47, 57...Laser light spot on the image plane, 60, 61...Laser irradiation system, 80...Digital-to-analog converter DAQ, 81, 91...Laser drive board, 82, 83, 85, 86, 87, 92, 93, 95, 96, 97...Signal cable, 84, 94...Mirror drive board, 100...Voltage signal application flow diagram, 101...Input data storage, 102, 103, 104, 105...Data calibration converter, 106...Voltage signal generator, 120...DMD type laser irradiation device, 121, 122...Micromirror, 123...Coupling lens, 124...Laser light beam, 125, 126...Laser reflected light, 127...Laser light absorption band

Claims

1. It can be attached to and detached from the microscope camera port. A laser irradiation system having an image pickup element capable of taking an image and a laser light source, A plate-shaped mirror that reflects light emitted from the laser light source is provided between the detachable part for the microscope camera port and the imaging element, At or near the imaging position formed by placing a plate-shaped mirror, a lens that forms a focused spot of laser light emitted from a laser light source; a movable mirror capable of adjusting the position of a focused spot of laser light emitted from a laser light source formed at or near the imaging position, The angular position of the laser light source and the movable mirror is A laser irradiation system characterized by being controlled in a time series manner by signals from the same digital-to-analog conversion device.

2. In the above laser irradiation system, The angle of the movable mirror can be adjusted based on the image information of the sample observed by the microscope. The laser irradiation system according to claim 1 .

3. In the above laser irradiation system, The thickness of the plate-shaped mirror is approximately 1 mm. The laser irradiation system according to claim 1 .

4. In the above laser irradiation system, The size of the irradiated area of ​​the laser light emitted from the laser light source on the microscope observation sample is 2. The laser irradiation system according to claim 1, wherein the collimation state can be adjusted by an optical system arranged between the laser light source and the movable mirror for adjusting the collimation state.

5. In the above laser irradiation system, 2. The laser irradiation system according to claim 1, wherein two laser light sources are provided, and the angle of the movable mirror is finely adjusted in position according to each light source.

6. It can be attached to and detached from the microscope camera port. A method for irradiating a specimen with laser light using a laser irradiation system having an image capturing element and a laser light source, comprising: The laser irradiation system includes: A plate-shaped mirror that reflects light emitted from a laser light source is disposed between the detachable part to the microscope camera port and the image sensor, and at or near the imaging position formed by the mirror, a lens that forms a focused spot of the laser light emitted from the laser light source; a movable mirror capable of adjusting the position of a focused spot of laser light emitted from a laser light source formed at or near the imaging position, The emission of the laser light source and the angular position of the movable mirror are A method for irradiating a laser beam onto an observation sample, characterized in that the laser beam is irradiated in a time-series manner by signals from the same digital-to-analog converter.

7. In the above-mentioned method for irradiating a specimen with laser light, 7. The method for irradiating a laser beam onto an observation sample according to claim 6, wherein the angle of the movable mirror is adjusted based on image information of the microscope observation sample.

8. In the above laser irradiation system, The thickness of the plate-shaped mirror is approximately 1 mm.

7. The method for irradiating a specimen with laser light according to claim 6.

9. In the above laser irradiation system, The size of the irradiated area of ​​the laser light emitted from the laser light source on the microscope observation sample is 7. The method for irradiating a specimen with laser light according to claim 6, wherein the collimation state can be adjusted by an optical system arranged between the laser light source and the movable mirror for adjusting the collimation state.

10. In the above laser irradiation system, 7. The method for irradiating a laser beam onto an observation sample according to claim 6, wherein two laser beam sources are provided, and the angle of the movable mirror is finely adjusted in position according to each of the laser beam sources.

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