Laser radiation system and method for radiating laser to observation sample

Abstract

To provide a method that enables laser light emitted from a plurality of light sources to be simultaneously radiated easily, in applications such as optogenetics in which laser light is radiated onto an observation sample of an optical microscope.SOLUTION: The present invention relates to a system connectable to a camera port of a microscope, including: an imaging sensor capable of capturing an image of a sample; two light sources; and two movable mirrors for adjusting positions at which the observation sample is irradiated with focused spots of laser light emitted from the light sources, and also relates to a method for laser irradiation using the system.SELECTED DRAWING: Figure 2

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 an eyepiece (not shown), the camera can capture a nearly in-focus image of the sample, significantly improving the operator's operability. Furthermore, researchers can attach a camera to the microscope 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 any position is the DMD (Digital Microscope Device) method, which uses a DMD element with a large number of small mirrors arranged in a single array to perform laser irradiation (Patent Document 2). While this method allows the laser light to be irradiated in any shape onto the sample under observation, it is limited to irradiating only one wavelength at a time. Therefore, it is not possible to simultaneously induce reactions of channelrhodopsin that respond to different wavelengths, or to simultaneously irradiate a sample with a 1.5-micron laser light and a laser light to which channelrhodopsin responds, thereby simultaneously performing local heating and optogenetic neural stimulation.

[0007] Furthermore, since the DMD-type laser irradiation device shown in Patent Document 2 is installed in the light source section of a fluorescence microscope, modifications to the microscope are required for installation, making it difficult for researchers who use the device to perform tasks such as removing the device, and making it difficult to perform tasks such as attaching a DMD-type laser irradiation device to other microscopes on a daily basis. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Patent No. 6363060 specification [Patent Document 2] U.S. Patent No. 1,112,309 [Non-patent literature]

[0009] [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]

[0010] The present invention has been made in consideration of the above points, and provides a laser irradiation system and a method for irradiating a specimen with laser light, which can be easily connected to the camera port of a fluorescence microscope without the need to modify the microscope, and which can irradiate any position on the specimen with laser light of two wavelengths. [Means for solving the problem]

[0011] In order to solve this problem, the laser irradiation system and the method for irradiating a laser beam onto an observation sample of the present invention are configured to include an image sensor that can be connected to a camera port of a microscope and can capture an image of the sample, two light sources, and two movable mirrors that adjust the irradiation positions of the focused spots of the laser beams emitted from the respective light sources on the observation sample. A mirror that reflects the laser beams is disposed between the camera port and the image sensor to position the imaging position of the microscope image at the laser wavelength, and focused spots of the laser beams emitted from the two light sources, whose positions can be adjusted by the movable mirrors corresponding to the respective light sources, are formed near the imaging position, thereby irradiating the laser beams emitted from the two laser light sources onto the sample. [Effects of the Invention]

[0012] In the laser irradiation system and method of irradiating a laser beam onto an observation sample of the present invention, laser beams from two light sources can be simultaneously irradiated onto the desired position within the field of view of the sample being observed under a microscope. This makes it possible to perform complex laser irradiation that cannot be achieved with conventional irradiation systems that use laser beams emitted from a single light source, such as optical stimulation by channelrhodopsin that reacts to different wavelengths, or optical stimulation by channelrhodopsin while locally heating by irradiating with a laser wavelength absorbed by water.

[0013] 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.

[0014] 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 two light sources and the angular positions of the two movable mirrors 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]

[0015] [Figure 1] FIG. 1 is a schematic diagram illustrating the configuration of a typical fluorescence microscope. [Figure 2] 1 is a schematic diagram illustrating the configuration of a laser irradiation system according to the present invention. [Figure 3] 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 4] 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 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. 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 20] 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 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. DETAILED DESCRIPTION OF THE INVENTION

[0016] FIG. 2 shows a schematic diagram of a laser illumination system 60 according to a first embodiment of the present invention. This laser illumination 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 where the microscope observation light beam 9 focuses on the sample. A dichroic mirror 48 is positioned between the camera port connection port 5a and the image sensor 7, selectively reflecting the laser wavelengths of the laser light source 41 and the laser light source 51. 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, images 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 sources 41 and 51. 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.

[0017] 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 passed through a dichroic mirror 49 that transmits light of the wavelength of the laser light source 41 via a scan lens 46, before forming a focused spot 47 on an imaging plane 37. 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 angle of the quarter-wave plate 44 is adjusted so that reflected light from the sample does not enter the light source 41, thereby stabilizing the operation of the laser light source 41.

[0018] 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, reflected by a movable mirror element 55, reflected by a scan lens 56, reflected by a dichroic mirror 49 that reflects the wavelength of the laser light source 51, and then forms a condensed spot 57 on an imaging plane 37. Here, a quarter-wave plate 54 and a polarizing spectroscopic element 53 are disposed between the lens 52 and the movable mirror 55, and the angle of the quarter-wave plate 54 is adjusted so that reflected light from the sample does not enter the light source 51, thereby stabilizing the operation of the laser light source 51.

[0019] 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 controlled by a digital-to-analog converter (DAQ) 80 via analog signal supply wiring 82. 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 controlled by a voltage signal supplied from the digital-to-analog converter (DAQ) 80 via analog signal supply wiring 92.

[0020] 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. The movable mirror element 55 is connected to a mirror drive board 94 via a cable 97, and the two-dimensional angle of the movable mirror portion 55a of the movable mirror element 55 is controlled by a voltage signal supplied from the digital-to-analog converter (DAQ) 80 via analog signal supply wiring 95, 96. 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 may also be disposed within the computer to which the image sensor 7 is connected. In addition, it is desirable to shorten the length of the cables 83, 93, 87, 97 by arranging the semiconductor laser driver boards 81, 82 and the mirror drive boards 84, 94 near the laser light sources 41, 51 and the movable mirrors 45, 55 and attaching them to the chassis of the laser irradiation system 60.

[0021] A schematic configuration of system 21 in which a laser illumination system 60 of the present invention is connected to a fluorescence microscope is shown in Figure 3. Because laser illumination system 60 is connected to camera port 5 of the microscope, images of wavelength components transmitted through 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 image sensor 7 within laser illumination system 60. Furthermore, laser light emitted from laser light sources 41 and 51, which form focused spots 47 and 57 on imaging plane 37, reflects off spectral mirror 48, is substantially collimated by the imaging lens, then transmits through emission filter 14 and dichroic mirror 13, and is irradiated by objective lens 3 onto sample positions corresponding to the focused spots 47 and 57. Here, if image observation using an imaging element is not required when the laser light sources 41 and 51 are 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 sources 41 and 51 can be irradiated onto the sample even if the emission filter 14 and dichroic mirror 13 have wavelength characteristics that do not transmit the wavelengths of the laser light sources 41 and 51.

[0022] 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, 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. 4 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.

[0023] By using an F-θ lens as the scan lens 56 and locating a movable mirror 55a at the back focus position of the scan lens 56, the position of the focused spot 57 of the laser light emitted from the laser light source 51 on the imaging plane 37 can be controlled by controlling the angle of the movable mirror 55a. Furthermore, since the focused spot 57 on the imaging plane 37 is reduced and projected onto the sample surface by the imaging lens 4 and the objective lens, the position of the focused spot 50 of the laser light emitted from the laser light source 51 on the cell 19 in the microscope observation sample shown in FIG. 4 can be adjusted by controlling the angle of the movable mirror 55a. Similarly, a signal to be provided for control is input as a value determined from the relationship between the position information of the image and the angle information of the movable mirror 55, based on the image information captured by the image sensor 7.

[0024] In the example shown in Figure 4, the angle of movable mirror 45a, i.e., the position of the focused spot 40 of light emitted from laser light source 41 controlled by a signal from mirror drive board 84 controlled by a voltage signal supplied from digital-to-analog converter (DAQ) 80 via analog signal supply wiring 85, 86, is made to coincide with the angle of movable mirror 55a, i.e., the position of the focused spot 50 of light emitted from laser light source 51 controlled by a signal from mirror drive board 94 controlled by a voltage signal supplied from digital-to-analog converter (DAQ) 80 via analog signal supply wiring 95, 96.

[0025] The light emitted from light source 41 is input by digital-to-analog converter (DAQ) 80 to semiconductor laser driver board 81 via analog signal supply wiring 82, causing light to be emitted in response to current supplied via wiring 83, and the light emitted from light source 51 is input by digital-to-analog converter (DAQ) 80 to semiconductor laser driver board 91 via analog signal supply wiring 92, causing light to be emitted in response to current supplied via wiring 93, so it is possible to coordinate and control the timing of control signals output from digital-to-analog converter (DAQ) 80 to light source 41, light source 51, movable mirror 45, and movable mirror 55. With laser irradiation system 60 of the present invention, as shown in Fig. 4, it is possible to simultaneously irradiate the same location with focused spot 40 of laser light emitted from laser light source 41 and focused spot 50 of laser light emitted from laser light source 51.

[0026] In the example shown in FIG. 4, the focused spot 40 and the focused spot 50 of the laser light emitted from the laser light source 51 are located at the same position. However, with the laser irradiation system 60 of the present invention, as shown in FIG. 5, it is also possible to simultaneously irradiate the focused spot 40 of the laser light emitted from the laser light source 41 and the focused spot 50 of the laser light emitted from the laser light source 51 at different positions.

[0027] 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.

[0028] Similarly, by using an F-θ lens as the scan lens 56 and arranging a movable mirror 55a at the back focus position of the scan lens 56, the position of the focused spot 57 of the laser light emitted from the laser light source 51 on the imaging plane 37 can be controlled by controlling the angle of the movable mirror 55a. Furthermore, the focused spot 57 on the imaging plane 37 is projected onto the sample surface in an enlarged or reduced manner by the imaging lens 4 and the objective lens 3, so that the position of the focused spot 50 of the laser light emitted from the laser light source 51 on the microscope observation sample 30 shown in FIG. 7 can be adjusted by controlling the angle of the movable mirror 55a.

[0029] 7 shows an example in which the positions of the laser beam focusing spot 40 and the laser beam focusing spot 50 are different, but since the positions of the laser beam focusing spot 40 and the laser beam focusing spot 50 can be controlled independently, they can also be made to coincide as shown in Fig. 4. Furthermore, since the respective light emission timings (timings of supply current) can also be controlled independently, the timings can also be adjusted arbitrarily.

[0030] 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. 2, 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. Similarly, a pair of lenses consisting of lenses 33 and 34 is inserted in the optical path of the laser light emitted from light source 51, that is, the parallel light portion that is incident on movable mirror 55, specifically, between movable mirror 55 and quarter-wave plate 54, and by moving the position of at least one of lens 33 or lens 34 and adjusting the distance between lens 33 and lens 34, an optical system can be formed in which the laser light that is incident on movable mirror 55 can be adjusted to divergent light, convergent light, or even parallel light.

[0031] 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.

[0032] In the fluorescence microscope system 23 connected to the laser irradiation system 61, the laser light emitted from the light source 51 and incident on the movable mirror 55 can be adjusted to divergent light, convergent light, or even parallel light, thereby making it possible to adjust the position of the focused spot 57 formed near the image plane 37 to be closer to or farther away from the spectral mirror 48. When the focused spot 57 is located on the image plane 37, the focused spot 50 on the sample is in focus, and therefore, by moving the position of the focused spot 57 formed near the image plane 37 closer to or farther away from the spectral mirror 48, the focused spot 50 on the sample becomes larger. When there is no room for space in the size of the movable part 55a of the movable mirror 55, if the laser light emitted from the light source 51 and incident on the movable mirror 55 is made divergent, it may go beyond the mirror 55a. Therefore, it is desirable to adjust the distance between the lenses 33 and 34 to make the size of the focused spot 50 on the sample focused or to increase the diameter, and to adjust the distance between the lenses 33 and 34 to make the laser light emitted from the light source 51 and incident on the movable mirror 55 parallel or convergent.

[0033] 10, the sizes of the focal spot 40 on the sample of the laser light emitted from laser 41 and the focal spot 50 on the sample of the laser light emitted from laser 51 are adjusted by adjusting the distance between lens 31 and lens 32 and the distance between lens 33 and lens 34. The angles of movable mirrors 45 and 55 are then adjusted so that the entire cell 19 is irradiated with the laser light, and the positions of the focal spots 40 and 50 are adjusted to the position of cell 19. Furthermore, the emission timings of lasers 41 and 51 are also adjusted so that they are synchronized. In the laser irradiation system of the present invention, laser light emitted from two light sources 41 and 51 can be simultaneously irradiated onto almost the entire surface of cell 19.

[0034] 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, the position, irradiation timing, and size of the focal spot 40 of the laser light emitted from the laser light source 41 on the microscope observation sample 30 are adjustable, and the position, irradiation timing, and size of the focal spot 50 of the laser light emitted from the laser light source 51 are adjustable.

[0035] 12 shows an example in which the position and size of a focused spot 40 of laser light emitted from a laser light source 41 on a microscope observation sample 30 is adjusted to a position different from that of a focused spot 50 of laser light emitted from a laser light source 51. In FIG. 12, an example is shown in which the focused spot 40 of laser light and the focused spot 50 of laser light are positioned at different positions, but because the position of the focused spot 40 of laser light and the position of the focused spot 50 of laser light can be controlled independently, they can also be made to coincide. Furthermore, because the respective light emission timings (timings of supply current) can also be controlled independently, the timings can also be adjusted arbitrarily. In the above-described laser irradiation system 61, a system has been shown in which both the position and size of the focused spot 40 of the laser light emitted from the laser light source 41 and the focused spot 50 of the laser light emitted from the laser light source 51 can be adjusted, but if the adjustment of the size of the laser spot is to be performed only on the spot of the laser light emitted from either of the light sources, it is sufficient to install a spot size adjustment mechanism on only one of them.

[0036] Here, we will explain the control signals for light source 41, light source 51, movable mirror 45, and movable mirror 55 that are output from the digital-to-analog converter (DAQ) 80. The amount of light emitted by light source 41 and the timing of light emission are controlled by a time-series signal 82t that is output from the digital-to-analog converter (DAQ) 80 and input to a semiconductor laser driver board 81. The amount of light emitted by light source 51 and the timing of light emission are controlled by a time-series signal 92t that is output from the digital-to-analog converter (DAQ) 80 and input to a semiconductor laser driver board 91. The angle of movable mirror 45 is controlled by time-series signals 85t, 86t output from digital-to-analog converter (DAQ) 80 and input to mirror drive board 84, and the angle of movable mirror 55 is controlled by time-series signals 95t, 96t 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, 86t, 92t, 95t, and 96t 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.

[0037] In the laser irradiation system of the present invention, the movable mirrors 45 and 55 are, for example, mirror devices (model number: A8L2.2-5000AL-TINY48.4-A / W / TP) from Mirror Technologies that can be driven two-dimensionally in the X and Y directions, and the mirror drive boards 84 and 94 are analog control boards (model number: DR-11-055-00) from Mirror Technologies. In this case, both the X and Y axes have a characteristic in which angles change linearly from -4 to +4 degrees in response to input voltage signals ranging from -10 to +10 V. Furthermore, by using scan lenses from Thorlabs or equivalent lenses as scan lenses 46 and 56, the positions of spots 40 and 50 can be adjusted to any position on the microscope observation sample according to the voltage value input to the mirror drive boards 84 and 94. The relationship between the voltage value for adjusting the position of the focused 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 focused spot 47 formed on the imaging plane 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 found from pixel position information of the pixel coordinates (PX1, PY1) of the focused spot 40 shown in Fig. 14. The voltage value (VX2, VY2) to be input to the mirror drive board 94 can also be found from pixel position information of the pixel coordinates (PX2, PY2) of the focused spot 50 shown in Fig. 14.

[0038] 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, the current IP1 required to emit that output can be calculated as shown in FIG. 15 once the required output power LP1 is specified. Similarly, the current for the light source 51 can be calculated from the required output power. In this 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 time on the order of MHz, applying a voltage in milliseconds ensures that the semiconductor laser is supplied 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.

[0039] FIG. 16 shows a flow 100 for generating the time-series signals 82t, 85t, 86t, 92t, 95t, and 96t 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, 86t, 92t, 95t, and 96t 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 is a component of the input value table 101, the coordinates (PX2, PY2) of the irradiation target position of the laser spot 50, the laser irradiation time Tilm, the irradiation light intensity of the laser spot 40: LP1, and the irradiation light intensity of the laser spot 50: LP2. 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.

[0040] 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. Then, from the input table 101, the output calibrator 104 of the light source 51 converts the irradiated light amount LP2 of the laser spot 50 into a target light amount, an example of which is shown in Figure 15, to an injection current to determine the current IP2 corresponding to the output LP2, and further determines the voltage value VL2 to be input to the semiconductor laser drive board. Furthermore, from the input table 101, the data output calibrator 105 of the mirror 55 determines the voltage value (VX2, VY2) to be input to the mirror drive board 94 from the coordinates (PX2, PY2) of the target position where the laser spot 50 is irradiated.

[0041] At this stage, the numerical values ​​of each signal during laser irradiation have been obtained for the time series signals 82t, 85t, 86t, 92t, 95t, and 96t, 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.

[0042] 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, 86t, 92t, 95t, and 96t during which the voltage does not change. This time can be as short as a few milliseconds, but it depends on the situation.

[0043] 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.

[0044] As described above, by generating the time series signals 82t, 85t, 86t, 92t, 95t, and 96t using the voltage signal generator 106 that takes into account the frequency characteristics of the device being used, stable laser irradiation can be performed at the target position.

[0045] Next, an example of a laser irradiation method using the laser irradiation system of the present invention will be described in which the two laser spots shown in FIG. 13 are linked but have different light emission times. The time series signals 82t, 85t, 86t, 92t, 95t, and 96t output from the digital-to-analog converter (DAQ) 80 are shaped as shown in FIG. 18, and the laser light source 41 emits light from time t1 to time t3. However, it is also possible for the laser light source 51 to emit light from time t2 to time t3, which is a certain time later than t1.

[0046] Furthermore, as another example of laser irradiation using the laser irradiation system of the present invention, time-series signals 82t, 85t, 86t, 92t, 95t, and 96t output from the digital-to-analog converter (DAQ) 80 are shown in FIG. In this example, laser light sources 41 and 51 each emit light twice. Since control signals 85t and 86t for movable mirror 45 and control signals 95t and 96t for movable mirror 55 have different values ​​during the first emission from time t1 to t2 and the second emission from time t3 to t4, laser light sources 41 and 51 each emit laser light to different positions during the two emissions.

[0047] Furthermore, as another example of laser irradiation using the laser irradiation system of the present invention, time series signals 82t, 85t, 86t, 92t, 95t, and 96t output from the digital-to-analog converter (DAQ) 80 are shown in FIG. This example is a combination of the example shown in Figure 18 in which light source 41 and light source 51 have different light emission times and the example shown in Figure 19 in which light source 41 and light source 51 each irradiate laser light onto different positions twice, and is an example in which light source 41 and light source 51 each irradiate laser light onto different positions twice and the light emission times are different.

[0048] In the laser irradiation method using the laser irradiation system of the present invention, as described above, the device that controls the emission of laser 41, the device that controls the irradiation position of laser 41 on the sample, and the device that controls the emission of laser 51, the device that controls the irradiation position of laser 51 on the sample are independent, so there are no constraints on their control. Although there are no constraints on them, the time-series signals 82t, 85t, 86t, 92t, 95t, and 96t that control them are all output from a digital-to-analog converter (DAQ) 80, enabling highly accurate time coordination. Therefore, even if the emission times of laser light sources 41 and 51, i.e., the time from t1 to t2 and the time from t3 to t4, are 1 millisecond in the signals shown in FIGS. 13 and 19, it is possible to accurately irradiate the target position with laser light.

[0049] FIG. 13 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 light is specified in the image captured by the camera 116, the position of the image sensor 7 corresponding to that position can be estimated. From that position, the voltage signals to be input to the movable mirrors 45 and 55 can be obtained, and laser irradiation can be performed.

[0050] 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 signals to be input to the movable mirrors 45 and 55 for the laser irradiation target position specified in the image taken by the camera 116, it is not necessary 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.

[0051] In the above explanation, the light source 41 and the light source 51 have different wavelengths. However, in the laser irradiation system and the method of irradiating a laser beam onto an observation sample of the present invention, by using a half mirror as the mirror 49, it is also possible to use light sources having the same wavelength as the light source 41 and the light source 51. [Industrial Applicability]

[0052] 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]

[0053] 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...Polarization separation element, 44, 54...1 / 4 wave plate, 45, 55...Moving 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 device, 102, 103, 104, 105...Data calibration converter, 106...Voltage signal generator

Claims

1. It can be attached to and detached from the microscope camera port. A laser irradiation device with an image observation function having an image pickup element capable of taking an image, a first laser light source, and a second laser light source, placing an image sensor at a first imaging position of the image of the microscope observation sample; a first mirror that reflects light emitted from the first and second laser light sources is disposed between the detachable part for the microscope camera port and the imaging element; At or near the second image position formed by disposing the first mirror, a first lens that forms a focused spot of the laser light emitted from the first laser light source; a second lens that forms a focused spot of the laser light emitted from the second laser light source; a first movable mirror capable of adjusting the position of an irradiation portion of the laser light emitted from the first laser light source on the microscope observation sample by adjusting the position of a focused spot of the laser light emitted from the first laser light source formed at or near the second imaging position; a second movable mirror capable of adjusting the position of an irradiation portion of the laser light emitted from the second laser light source on the microscope observation sample by adjusting the position of a focused spot of the laser light emitted from the second laser light source formed at or near the second imaging position; A laser irradiation system comprising:

2. In the above laser irradiation system, 2. The laser irradiation system according to claim 1, wherein the angle of the first movable mirror is adjusted based on image information of the sample under microscope observation.

3. In the above laser irradiation system, 2. The laser irradiation system according to claim 1, wherein the angle of the second movable mirror is adjusted based on image information of the sample under microscope observation.

4. In the above laser irradiation system, The amount of laser light emitted from the first laser light source is adjustable. The laser irradiation system according to claim 1 .

5. In the above laser irradiation system, The amount of laser light emitted from the second laser light source is adjustable. The laser irradiation system according to claim 1 .

6. In the above laser irradiation system, Emission of a first laser light source; Emission of a second laser light source; the angular position of the first movable mirror; The angular position of the second movable mirror is 2. The laser irradiation system according to claim 1, wherein the laser irradiation system is controlled by signals from the same digital-to-analog conversion device.

7. In the above laser irradiation system, Emission of a first laser light source; Emission of a second laser light source; the angular position of the first movable mirror; The angular position of the second movable mirror is 2. The laser irradiation system according to claim 1, wherein the laser irradiation system is controlled in a time-series manner by signals from the same digital-to-analog converter.

8. In the above laser irradiation system, The size of the irradiated area of ​​the laser light emitted from the first 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 first laser light source and the first movable mirror for adjusting the collimation state.

9. In the above laser irradiation system, The size of the irradiated area of ​​the laser light emitted from the second 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 second laser light source and the second movable mirror for adjusting the collimation state.

10. It can be attached to and detached from the microscope camera port. A method for irradiating a laser beam onto an observation sample using a laser irradiation device with an image observation function, the laser irradiation device having an image capturing element capable of capturing an image, a first laser light source, and a second laser light source, the method comprising: The laser irradiation device with image observation function has an image sensor disposed at a first image forming position of an image of a microscope observation sample, a first mirror that reflects light emitted from the first and second laser light sources, between the detachable part to the microscope camera port and the image pickup element; At or near the second image position formed by disposing the first mirror, a first lens that forms a focused spot of the laser light emitted from the first laser light source; a second lens that forms a focused spot of the laser light emitted from the second laser light source; a first movable mirror capable of adjusting the position of an irradiation portion of the laser light emitted from the first laser light source on the microscope observation sample by adjusting the position of a focused spot of the laser light emitted from the first laser light source formed at or near the second imaging position; a second movable mirror capable of adjusting the position of an irradiation portion of the laser light emitted from the second laser light source on the microscope observation sample by adjusting the position of a focused spot of the laser light emitted from the second laser light source formed at or near the second imaging position; and A method for irradiating a laser beam onto an observation sample, comprising adjusting the angle of at least one of a first movable mirror and a second movable mirror based on image information of the observation sample under a microscope.

11. In the above-mentioned method for irradiating a specimen with laser light, Emission of a first laser light source; Emission of a second laser light source; the angular position of the first movable mirror; The angular position of the second movable mirror is 11. The method for irradiating a specimen with laser light according to claim 10, wherein the laser light is controlled by a signal from the same digital-to-analog converter.

12. In the above-mentioned method for irradiating a specimen with laser light, Emission of a first laser light source; Emission of a second laser light source; the angular position of the first movable mirror; The angular position of the second movable mirror is 11. The method for irradiating a specimen with laser light according to claim 10, wherein the laser light is controlled in a time-series manner by signals from the same digital-to-analog converter.

13. In the above-mentioned method for irradiating a specimen with laser light, The size of the irradiated area of ​​the laser light emitted from the first laser light source on the microscope observation sample is 11. The method for irradiating a laser beam onto an observation sample according to claim 10, characterized in that the collimation state can be adjusted by an optical system arranged between the first laser light source and the first movable mirror for adjusting the collimation state.

14. In the above-mentioned method for irradiating a specimen with laser light, The size of the irradiated area of ​​the laser light emitted from the second laser light source on the microscope observation sample is 11. The method for irradiating a laser beam onto an observation sample according to claim 10, wherein the collimation state can be adjusted by an optical system arranged between the second laser light source and the second movable mirror for adjusting the collimation state.

Images