Laser scanning microscope

Abstract

To miniaturize a laser scanning microscope and to enable observation in water or the like.SOLUTION: This microscope has a light source for emitting a scanning laser beam, a condenser lens, a photodetector for detecting fluorescence or the like from a sample, and an optical scanning mirror for scanning the laser beam from the light source, and the light source, the condenser lens, the photodetector, and the optical scanning mirror are stored in an integrated casing.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a laser scanning microscope that scans a light beam from a light source and observes fluorescent and reflected light from a sample, and relates to, for example, a laser scanning microscope that can be made compact and that can be used for observation underwater, etc. [Background technology]

[0002] Laser scanning microscopes, which are currently used as indispensable tools in the semiconductor industry and the bioscience and life science fields, are fairly large-scale devices consisting of an optical microscope, a laser light source and optical scanning system, and a display. Their configuration is complex, combining numerous components, including individual combined light source and scanning units, a light detection unit, and a microscope unit (see, for example, Patent Documents 1 and 2).

[0003] In addition, devices using semiconductor lasers as laser light sources have been proposed and commercialized, but these simply replace the light source of conventional laser scanning microscopes with semiconductor lasers, and remain complex devices made up of a large number of components (see, for example, Patent Document 3). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2008-052146 [Patent Document 2] Japanese Patent Application Laid-Open No. 2014-081417 [Patent Document 3] Japanese Patent Application Laid-Open No. 2014-240936 Summary of the Invention [Problem to be solved by the invention]

[0005] The laser scanning microscopes disclosed in Patent Documents 1 to 3 are complex and large-scale, combining a large number of parts, and have the drawbacks of being expensive, difficult to use, and requiring complicated maintenance.

[0006] SUMMARY OF THE INVENTION An object of the present invention is to realize an ultra-compact laser scanning microscope, thereby eliminating the above-mentioned drawbacks and enabling the use of the laser scanning microscope outdoors, underwater, etc. [Means for solving the problem]

[0007] In one aspect, the laser scanning microscope has a light source, a focusing lens, a photodetector, and an optical scanning mirror that scans the laser beam from the light source, and the light source, the focusing lens, the photodetector, and the optical scanning mirror are housed in an integrated housing. [Effects of the Invention]

[0008] One aspect is that the microscope system can be carried indoors or outdoors, allowing for high-resolution observation of fluorescence distribution and fine surface morphology, making it possible to perform "in situ measurements" in various natural science fields, such as the semiconductor industry, inorganic and organic material measurement, and even bio-, agricultural, and life sciences. [Brief explanation of the drawings]

[0009] [Figure 1] 1 is a conceptual configuration diagram of a laser scanning microscope according to an embodiment of the present invention. [Figure 2] FIG. 1 is an image diagram of a commercialized laser scanning microscope according to an embodiment of the present invention. [Figure 3] 1 is a conceptual configuration diagram of a laser scanning microscope according to a first embodiment of the present invention. [Figure 4] FIG. 1 is a configuration diagram of a light source of a laser scanning microscope according to a first embodiment of the present invention. [Figure 5] FIG. 2 is a configuration diagram of a photodetector of the laser scanning microscope according to the first embodiment of the present invention. [Figure 6]FIG. 2 is a perspective view of the main part of the scanning mirror unit of the laser scanning microscope according to the first embodiment of the present invention. [Figure 7] FIG. 1 is a conceptual configuration diagram of a laser scanning microscope according to a second embodiment of the present invention. [Figure 8] FIG. 10 is a conceptual configuration diagram of a laser scanning microscope according to a third embodiment of the present invention. [Figure 9] FIG. 10 is a conceptual configuration diagram of a laser scanning microscope according to a fourth embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0010] An example of a laser scanning microscope according to an embodiment of the present invention will now be described with reference to Figures 1 and 2. Figure 1 is a conceptual diagram of a laser scanning microscope according to an embodiment of the present invention. The laser scanning microscope of the present invention comprises three sections: a light source / light detection section 1, a light scanning section 2, and a scanning optical system / objective lens section 3.

[0011] The main components of the light source and light detection unit 1 are a light source 11, a condenser lens 13, a filter 14, a reflecting mirror 18, and a photodetector 15. The main components of the optical scanning unit 2 are prism reflecting mirrors 22 and 23, and an optical scanning mirror 21, and the main components of the scanning optical system and objective lens unit 3 are a scanning optical system 4 and an objective lens system 5. Depending on the configuration, the prism reflecting mirrors 22 and 23 may be replaced with a filter that also functions as a half mirror.

[0012] The irradiation laser beam 19( figure The irradiation laser beam (shown by a white outline in the figure) passes through a focusing lens 13 for focusing the irradiation laser beam, is shaped into collimated light or focused light close to collimated light, travels straight through a filter 14, and then is directed downward by a prism reflecting mirror 22, reaches the optical scanning mirror 21, and is reflected upward by the optical scanning mirror 21. The optical scanning mirror 21 may be a small galvanometer mirror, but a MEMS (Micro Electro Mechanical Systems) mirror is usually used.

[0013] When the optical scanning mirror 21 is in a stationary position, the reflected irradiation laser beam 19 is redirected by the prism reflecting mirror 23 to the same direction as the beam direction immediately after being emitted from the light source 11, and passes through the scanning optical system 4 consisting of the pupil projection lens 41 and the imaging lens 42 to reach the objective lens 51. After this, the irradiation laser beam 19 is focused by the objective lens 51 into an extremely narrow range at a specific position on the measurement sample, and irradiates the focused position. This irradiated focused position is called the irradiation focus point. When the optical scanning mirror 21 is moving, the irradiation laser beam 19 is scanned within the scanning angle range 24 as shown in the figure, and the irradiation focus point on the measurement sample is scanned over the sample.

[0014] In FIG. 1, the scanning angle range 24 shows the case where the irradiating laser beam 19 is scanned within the plane of the paper, but of course, two-dimensional scanning is normally performed in which the irradiating laser beam 19 is also scanned in a direction perpendicular to the plane of the paper. Also, in FIG. 1, the scanning state of the irradiating laser beam 19 is shown only up to the point after it is reflected by the prism reflecting mirror 23, and the subsequent beam is omitted. At the irradiated focal point, the irradiated irradiating laser beam 19 generates fluorescence, reflected light, and scattered light from the sample. Scattered light includes scattered light with no wavelength change, which is simply scattered light, Raman scattered light, Brillouin scattered light, harmonic scattered light, etc., which have wavelength changes. These fluorescence, reflected light, and scattered light are called the return light 1 from the sample. 10 (shown by a solid line in Figure 1). In the figure, the return light from the sample 1 10 is the return light from the sample 1 10 The central axis of the

[0015] Return light from sample 1 10The reflected light travels in the opposite direction to the path of the irradiating laser beam 19, passing through the objective lens 51, the scanning optical system 4, the prism reflecting mirror 23, the optical scanning mirror 21, and the prism reflecting mirror 22, before reaching the filter 14. The filter 14 is set to pass the irradiating laser beam 19 and to reflect the returning light from the sample. Typically, a bandpass filter that transmits only the wavelength of the irradiating laser beam is used. In this case, the returning light 1, such as Raman scattered light, Brillouin scattered light, and harmonic scattered light, which have wavelength variations, is filtered out. 10 The filter 14 causes the light to travel along a path different from that of the irradiating laser beam 19, and the light passes through a reflecting mirror 18 and a condenser lens 17 for collecting the returned light from the sample, before reaching the photodetector 15.

[0016] Return light from sample 1 10 If the wavelength of the irradiating laser beam 17 is approximately the same as that of the irradiating laser beam 19, the filter 14 cannot be set to pass only the irradiating laser beam 19 and reflect the return light from the sample, which will result in optical loss, so a simple semi-transparent mirror is used as the filter 14. It is desirable that the shape and characteristics of the condenser lens 13 for focusing the irradiating laser beam and the condenser lens 17 for focusing the return light from the sample are the same.

[0017] Here, with reference to FIG. 1 , the integrated housing 6 of the laser scanning microscope will be described. Generally, a housing refers to a box that houses a machine or electrical device with some function. The housing 6 defined in this invention is a single box that houses the light source / light detection unit 1 and the optical scanning unit 2. Here, as shown in FIG. 1 , it is preferable that the interior of the box is not divided into a single space, but partitions may be present inside the box. Note that the term "integrated housing" refers to a "single-molded housing made of a single member," a "housing made of connected components made of a single member," or a "housing made of connected molded components made of different members." Furthermore, while a rectangular parallelepiped is most suitable for the shape of the box, it does not necessarily have to be a rectangular parallelepiped; it can also be a cylindrical box or a columnar box with an elliptical, triangular, hexagonal, or octagonal cross section. Note that the scanning optical system / objective lens unit 3 does not necessarily have to be housed in the housing 6 that houses the light source / light detection unit 1 and the optical scanning unit 2.

[0018] Furthermore, it is desirable that the interior of the housing 6 be airtight, isolating it from the outside. If the internal gas can be isolated from the external environment, except for the light beam and electrical signals, and conversely, gases and liquids from the external environment can be prevented from entering the housing, this would offer the advantage of allowing the laser scanning microscope to be used in any measurement environment, even underwater. In the figure, reference numerals 12 and 16 denote a base, and reference numerals 43 and 52 denote lens holders. The light source is not limited to a semiconductor laser; as long as it can produce a light beam, it can also be one that generates a nonlinear beam, such as a harmonic generated by a nonlinear crystal, or even a solid-state laser, which has recently become increasingly miniaturized.

[0019] FIG. 2 is an illustration of a commercialized laser scanning microscope according to an embodiment of the present invention, showing a pencil-type laser scanning confocal microscope as an example. The scanning optical system 4, excluding the objective lens system 5, of the scanning optical system / objective lens unit 3 is housed in a housing 7 along with the light source / light detection unit 1 and the optical scanning unit 2. In FIG. 1, the connection between the housing 6 and the scanning optical system / objective lens unit 3 is depicted as if there is no partition. However, even in this case, if the scanning optical system / objective lens unit 3 itself is designed to prevent the intrusion of gases and liquids from the outside, the interior of the housing 6 can be made airtight and isolated from the outside. Essentially, there is no problem as long as the interior of the housing is essentially airtight and isolated from the outside.

[0020] To make the laser scanning confocal microscope portable, it is desirable to house the light source and light detection unit 1, the optical scanning unit 2, and the scanning optical system and objective lens unit 3 in the same housing 7 as the drive circuit and communication unit 9. The drive circuit and communication unit 9 includes a power supply and drive circuit for driving the laser and scanning the irradiating laser beam, a circuit for forming an image using the returned light from the sample, and other components, and by communicating with the outside, it is possible to display the obtained results on an external monitor or the like.

[0021] Although a panel display can be used as the external monitor, the use of an eyeglass-type display makes the entire system more portable. Furthermore, if a laser scanning eyeglass-type display is used as the eyeglass-type display, it becomes possible to set the eyeglass-type display to scan the image in synchronization with the scanning of the irradiated focal point on the sample in the laser scanning confocal microscope, thereby simplifying the display circuitry.

[0022] In the example of FIG. 2, the objective lens system 5 is installed outside the housing 7, and is an example of a structure that allows for easy attachment and detachment. Furthermore, the central axis of the emitted irradiation laser beam coincides with the central axis of the irradiation laser beam 19 after passing through the objective lens of the objective lens system 5, resulting in a structure that corresponds to a pencil shape. To achieve a conveniently portable pencil shape, a clip portion 10 is provided, and the inclination between the central axis of the emitted irradiation laser beam 19 and the central axis of the irradiation laser beam 19 after passing through the objective lens 51 should be within 10°. Furthermore, by covering the objective lens system 5 with a transparent covering portion 8, direct measurement can be performed underwater or inside the body, eliminating the need for sampling the measurement target.

[0023] It is desirable that the overall size of a pencil-type laser scanning confocal microscope be as small as possible. If it is assumed that the microscope will be portable and will be used for measurements while held in the hand, the official size of an athletic baton (39mm diameter) that can be held freely even during exercise is a good reference. Therefore, if the cross section is circular, the overall size of a pencil-type laser scanning confocal microscope should be a maximum of about 40mm in diameter. Also, if the cross section is square, the length of the diagonal should be 39mm, and the length of one side should be 39mm / 2. 1 / 2 = 27.6 mm, so it is desirable that the length of one side of the entire pencil-type laser scanning confocal microscope be at most about 30 mm. Furthermore, when using the microscope by inserting it into the human body, the size of the endoscope is a reference. A lower gastrointestinal endoscope has a diameter of about 13 mm, so when using the microscope as an endoscope, it is desirable that the size of the entire pencil-type laser scanning confocal microscope be at most about 13 mm in diameter if the cross section is circular. Furthermore, when the cross section is square, the length of the diagonal is 13 mm, and the length of one side is 13 mm / 2 1 / 2 = 9.2 mm, it is desirable that the length of one side of the entire pencil-type laser scanning confocal microscope be at most about 10 mm.

[0024] Although the present invention is not limited to confocal laser scanning microscopes, when used in a confocal laser scanning microscope, it is desirable to form a confocal optical system by providing a pinhole member with a pinhole in the photodetector 15 so that the laser beam emission position of the light source 11 and the pinhole are at conjugate positions.

[0025] The light source 11 preferably includes multiple semiconductor lasers with different laser beam emission positions, and a confocal optical system is formed so that the beam emission positions corresponding to each semiconductor laser and the pinhole of the pinhole member are conjugate positions. In this case, the number of semiconductor lasers may be one or more, but typically three: a red semiconductor laser, a green semiconductor laser, and a blue semiconductor laser. In this case, the central axes of the laser beams of each semiconductor laser may be parallel to each other or may be non-parallel to each other. In this case, when multiple semiconductor lasers are used as the light source 11, the laser beams from each semiconductor laser may be multiplexed using a waveguide-type multiplexer.

[0026] The central axis of the laser beam from the semiconductor laser and the substrate of the optical scanning unit 2 may be parallel or tilted at an angle of ±10° or less. It is also desirable that the tip of the transparent coating 7 that covers the objective lens system 5 is located at the focal point of the light emitted by the final objective lens of the objective lens system 5. The light source / light detection unit 1 can also be used by adding only this light source / light detection unit 1 to another microscope, and in this case, the entire device based on that other microscope can be made smaller. [Example]

[0027] Next, a laser scanning microscope according to a first embodiment of the present invention will be described with reference to Figs. 3 to 6. Fig. 3 is a conceptual diagram of the laser scanning microscope according to the first embodiment of the present invention. The laser scanning microscope according to the first embodiment of the present invention comprises three parts: a light source, a condenser lens, a photodetector, and a scanning optical system / objective lens unit. The light source is provided in a housing 14 with a window 15, a base 12 provided in the housing 14, The laser scanning microscope includes a semiconductor laser 13 mounted on a base 12. The photodetector includes a housing 27 with a window 28, a base 22 mounted within the housing 27, a light-receiving element 23 mounted on the base 22, a filter member 25, and a pinhole member 26. The optical scanning unit includes an optical scanning mirror substrate 41, a scanning mirror 42 mounted on the optical scanning mirror substrate 41, and prism reflectors 43 and 44. The scanning optical system / objective lens unit includes a scanning optical system in which a pupil projection lens 61 and an imaging lens 62 are held by a lens holder 63, and an objective lens system in which an objective lens 71 is held by a lens holder 72. The housing 50 is airtight, isolating the interior from the outside. As a result, the internal gas is isolated from the external environment except for the light beam and electrical signals, and conversely, gases and liquids from the external environment can be prevented from entering the housing. This allows the laser scanning microscope to be used in a variety of measurement environments, including underwater.

[0028] 4A and 4B are explanatory diagrams of the light source of the laser scanning microscope of Example 1 of the present invention, with Fig. 4A being a plan view and Fig. 4B being a side view. The light source is an example in which three semiconductor lasers 131, 132, and 133 are arranged in parallel in the horizontal direction. The semiconductor lasers 131, 132, and 133 are installed on a pedestal 12 inside an airtight housing 14, and the emitted irradiation laser beams 191, 192, and 193 are guided to the outside through a window 15.

[0029] Components other than major components such as conductors for injecting current into the semiconductor lasers are omitted in Fig. 4. The three semiconductor lasers of the light source in Fig. 4 are an example in which the wavelengths of the emitted light are different, and for example, semiconductor laser 131 on the left side in the emission direction is a green semiconductor laser with an oscillation wavelength of 532 nm and an output of 20 mW, semiconductor laser 132 in the middle is a blue semiconductor laser with an oscillation wavelength of 488 nm and an output of 20 mW, and semiconductor laser 133 on the right side in the emission direction is a red semiconductor laser with an oscillation wavelength of 640 nm and an output of 20 mW, with the semiconductor lasers 131, 132, and 133 spaced apart by 150 μm.

[0030] The semiconductor lasers 131, 132, and 133 are aligned horizontally so that the beam emission positions 181, 182, and 183 are at the same height relative to the base 12. The emission directions of the irradiation laser beams 191, 192, and 193 do not necessarily need to be the same, taking into account factors such as chromatic aberration of the lenses. The condenser lens 16 converts the irradiation laser beams 191, 192, and 193 emitted from the three semiconductor lasers 131, 132, and 133 into collimated light or condensed light close to collimated light. While a single lens is shown here, a combination of multiple lenses may also be used. In the case of a single lens, it is necessary to collectively convert the irradiation laser beams 191, 192, and 193, which have different wavelengths and are emitted from different locations, into collimated light or condensed light close to collimated light. Therefore, a lens with a free-form surface or a toroidal surface is desirable.

[0031] Although Figure 4 shows a case where the light source consists of three semiconductor lasers 131, 132, and 133, a single semiconductor laser may be used for monochromatic sample observation, and the number of semiconductor lasers is arbitrary. Even when the light source does not consist of a semiconductor laser, the light beams from the semiconductor lasers may be multiplexed in a multiplexer to generate the irradiation laser beam. In this case, an optical waveguide multiplexer is suitable. In addition, the light source is not limited to semiconductor lasers, and any light source that can generate an optical beam may be used, such as a nonlinear beam generated by a nonlinear crystal to generate a nonlinear beam, such as a harmonic, as the irradiation laser beam. Although the light source in Figure 4 has a housing 14 and a window 15, these are not necessarily required if the microscope itself is isolated from the outside.

[0032] FIG. 5 shows an example of a photodetector according to the first embodiment of the present invention, with FIG. 5(a) being a plan view and FIG. 5(b) being a side view. The return light from the sample travels along the same optical path as the irradiation laser beams 191, 192, and 193 in the opposite direction, passes through a condenser lens 29, and arrives at the photodetector. Within the photodetector, the return light passes through a window 28, a pinhole member 26, and a filter member 25 before reaching a light-receiving element 23. In the case of FIG. 1, the photodetector is installed upside down on the ceiling opposite the light source, so that the return light from the sample due to the irradiation laser beam 191 reaches the light-receiving surface 243 as return light 313, the return light from the sample due to the irradiation laser beam 192 reaches the light-receiving surface 242, and the return light from the sample due to the irradiation laser beam 193 reaches the light-receiving surface 241.

[0033] The diameter of the pinholes 261, 262, 263 provided in the pinhole member 26 is, for example, 0.5 mmφ, and the intervals between the pinholes 261, 262, 263 are 150 μm, the same as the intervals between the semiconductor lasers 131, 132, 133.

[0034] As in FIG. 1, this is an example in which the photodetector is installed upside down on the ceiling opposite the light source, and beam emission positions 181, 182, and 183 of semiconductor lasers 131, 132, and 133 and pinhole 26 3,A confocal optical system is formed so that the laser beams 191, 192, and 193 are at conjugate positions. Note that, during actual measurements, the illumination and focusing points on the sample are also in a confocal relationship as conjugate positions. That is, in an example where three semiconductor lasers 131, 132, and 133 are arranged in parallel, the beam emission position 181 and the pinhole 263 are at conjugate positions, the beam emission position 182 and the pinhole 262 are at conjugate positions, and the beam emission position 183 and the pinhole 261 are at conjugate positions. When the beam emission positions 181, 182, and 183 from the semiconductor lasers 131, 132, and 133 are different, as in the example where three semiconductor lasers 131, 132, and 133 are arranged in parallel, the illumination and focusing points from which the return light is emitted are different for each of the illumination laser beams 191, 192, and 193. This results in a more efficient stray light removal than conventional coaxial confocal microscopes, and allows for the production of scanned images with good contrast. Although the beam emission positions 181, 182, and 183 of the semiconductor lasers 131, 132, and 133 are shown here to be different, if a light source other than a semiconductor laser is used to emit a light beam and the beam emission positions are set to be different, the efficiency of removing stray light will be better than that of conventional coaxial confocal microscopes, and a scanned image with good contrast will be obtained in the same way.

[0035] In order to make the beam emission positions 181, 182, 183 of the semiconductor lasers 131, 132, 133, the irradiation focusing points on the sample, and the pinholes 263, 262, 261 all conjugate positions, it is desirable that the shape characteristics of the focusing lens 29 for the return light from the sample be the same as those of the focusing lens 16 for the irradiation laser beams 191, 192, 193.

[0036] The wavelength-selective filters 251, 252, and 253 provided in the filter member 25 are different for each pinhole 261, 262, and 263, and the wavelength of the transmitted light is set to correspond to the irradiation wavelength. For example, when observing a fluorescence distribution, the irradiation wavelength is set to the excitation wavelength of the fluorescence, and the transmission wavelength of the wavelength-selective filter is set to a wavelength range whose peak is the fluorescence wavelength. For example, the transmission peak wavelength of the wavelength-selective filter 253 on the right side of the figure, facing the returned light, is set to 630 nm to detect the fluorescent substance Texas Red. The transmission peak wavelength of the wavelength-selective filter 252 in the center of the figure is set to 530 nm to detect the fluorescent substance Alexa Fluor 488. The transmission peak wavelength of the wavelength-selective filter 251 on the left side of the figure, facing the returned light, is set to 660 nm to detect the fluorescent substance APCT. When simply observing scattered light without wavelength change due to scattered light, the transmission wavelength of the wavelength-selective filter is set to match the wavelength of the irradiating laser beam.

[0037] In the example of FIG. 5 , a wavelength-selective filter 251 is located immediately after the pinhole 261 on the left side of the pinholes as viewed from the returning light, with the light-receiving surface 241 of the light-receiving element located behind it. A wavelength-selective filter 252 is located immediately after the central pinhole 262, with the light-receiving surface 242 of the light-receiving element located behind it. A wavelength-selective filter 253 is located immediately after the pinhole 263 on the right side of the pinhole as viewed from the returning light, with the light-receiving surface 243 of the light-receiving element located behind it. With the above configuration, the selected wavelengths of the wavelength-selective filters 253, 252, and 251 correspond to the irradiation wavelengths of the irradiation laser beams 191, 192, and 193, respectively. Note that while the selected wavelengths of the wavelength-selective filters 251, 252, and 253 are typically different, it is not essential that the selected wavelengths be different, as the same wavelength may be acceptable in some cases. The wavelength selection filters 251, 252, and 253 may be configured so that the entire wavelength selection filters 251, 252, and 253 can be replaced depending on the application.

[0038] The light receiving element 23 having the light receiving surfaces 241, 242, and 243 is preferably an array type light receiving element capable of receiving each return light individually, or an avalanche photodiode capable of improving sensitivity, and typically a silicon photodiode array is used.

[0039] Figure 5 shows three pinholes 261, 262, and 263 corresponding to the three semiconductor lasers 131, 132, and 133 in Figure 4. However, for monochromatic sample observation, only a single pinhole is required, corresponding to the case of a single semiconductor laser. Thus, any number of pinholes can be used to accommodate any number of semiconductor lasers. Even if the microscope is configured with the same number of pinholes as the number of semiconductor lasers, the actual number of semiconductor lasers and pinholes used may be fewer than the actual number. While the above discussion focuses on a laser scanning confocal optical microscope using pinholes, a simple laser scanning microscope using only wavelength-selective filters and photodetectors without pinholes can also be used, although the resolution of the sample observation will be reduced. The photodetector in Figure 5 includes a housing 27 and a window 28. However, these are not necessarily required if the microscope itself is isolated from the outside.

[0040] Next, the configuration of the optical scanning unit will be described with reference to Figure 3 again. Here, the central axis of the irradiation laser beam from the light source and the optical scanning mirror substrate 41 of the optical scanning mirror 42 are parallel, and this configuration makes it possible to reduce the size of the housing 50. If it is difficult to make the central axis of the irradiation laser beam from the light source parallel to the optical scanning mirror substrate 41 of the optical scanning mirror 42, it is sufficient that the inclination between the central axis of the irradiation laser beam and the optical scanning mirror substrate 41 of the optical scanning mirror 42 is ±10° or less.

[0041] 6 is a schematic perspective view of an example of an optical scanning mirror of the laser scanning microscope according to the first embodiment of the present invention. The optical scanning mirror 42 is a MEMS (Micro Electro Mechanical Systems) mirror having a reflecting portion 421, a rotating outer frame 423 that supports the reflecting portion 421 with a pair of first hinges 422 that serve as a first optical scanning rotation axis, and a non-rotating outer frame 425 that supports the rotating outer frame 423 with a pair of second hinges 424 that serve as a second optical scanning rotation axis and are provided in a direction perpendicular to the first hinges 422. The optical scanning mirror 42 is also provided with a magnetic field generator 426 that includes at least an AC magnetic field generator for driving the reflecting portion 421. The first hinge 422 and the second hinge 424 need to be determined so that the high-speed rotation becomes the natural rotation frequency of the reflecting portion 421. This natural rotation frequency is determined by the shape and mass of reflecting section 421, the spring constant of the rotating section, etc., and the thickness of first hinge 422 and second hinge 424 is generally 2 μm to 50 μm, with a typical thickness being 10 μm. It is desirable to use a hard magnetic thin film on the surface side of reflecting section 421 whose coercive force is equal to or greater than a predetermined value, particularly a hard magnetic thin film whose coercive force is such that the ratio of the magnetic field generated by magnetic field generating device 426, which includes at least an AC magnetic field generator, to the coercive force is 0.2 or less, i.e., a hard magnetic thin film whose coercive force is 5 (= 1 / 0.2) or more of the magnetic field generated by magnetic field generating device 426. The MEMS mirror is not particularly limited and may use electrostatic or piezoelectric elements in addition to those that use a magnetic field.

[0042] The scanning method of the MEMS mirror is raster scanning, and for example, the scanning angle is 36° maximum in the high-speed scanning direction and 26° maximum in the low-speed scanning direction.

[0043] Next, an example of the configuration of the scanning optical system / objective lens unit will be described with reference to Figure 3 again. The scanning optical system / objective lens unit is composed of a scanning optical system equipped with a pupil projection lens 61 and an imaging lens 62, and an objective lens system equipped with an objective lens 71. The pupil projection lens 61 and the imaging lens 62 are held by a lens holder 63, and the objective lens 71 is also held by a lens holder 72. For example, a dispersion-compensating doublet lens is used as the pupil projection lens 61 and the imaging lens 62 of the scanning optical system, and the magnification of the objective lens 71 is set to 20x.

[0044] The scanning optical system / objective lens unit has the function of irradiating the sample with the irradiation laser beam traveling from the optical scanning unit, and returning the return light from the sample to the optical scanning unit. The pupil projection lens 61, imaging lens 62, and objective lens 71 usually use doublet lenses or dispersion compensation lenses, but of course they can also be single lenses. The scanning optical system / objective lens unit can be connected either fixedly to the optical scanning unit or detachably. Furthermore, the optical scanning system and objective lens system can also be fixed or detachably. If the objective lens system is detachable and objective lenses 71 with different magnifications are provided, the magnification of the microscope can be changed.

[0045] The irradiation laser beam 19( figure The laser beam (shown by the white outline in the figure) passes through a focusing lens 16 for focusing the irradiation laser beam, is shaped into collimated light or focused light close to collimated light, travels straight through a filter 17, is then directed downward by a prism reflecting mirror 43, reaches an optical scanning mirror 42, and is reflected upward by the optical scanning mirror 42.

[0046] When the optical scanning mirror 42 is in a stationary position, the direction of the reflected irradiation laser beam is changed by the prism reflecting mirror 44 to the same direction as the beam direction immediately after being emitted from the semiconductor laser 13, and passes through a scanning optical system consisting of a pupil projection lens 61 and an imaging lens 62 to reach an objective lens 71. After this, the irradiation laser beam condensed by the objective lens 71 is condensed into an extremely narrow range at a specific position on the measurement sample, and irradiates the condensed position. When the optical scanning mirror 42 is in operation, the irradiation laser beam 19 scans within an angular range of a scanning angle 45, as shown in the figure, and the irradiated condensed point on the measurement sample is scanned over the sample.

[0047] Return light 31 from the sample (shown by a solid line in the figure) retraces the path of the irradiation laser beam 19 in reverse, passing through objective lens 71, scanning optical system, prism reflecting mirror 44, optical scanning mirror 42, and prism reflecting mirror 43, before reaching filter 17. Filter 17 uses a bandpass filter and is configured to pass the irradiation laser beam and reflect the return light from the sample. Raman scattered light, Brillouin scattered light, harmonic scattered light, and other light whose wavelengths are shifted by the bandpass filter are redirected by filter 17 to return along a path different from the path of the irradiation laser beam, passing through reflecting mirror 30, condenser lens 29, pinhole member 26, and filter member 25, before reaching light-receiving surface 24.

[0048] Here, the photodetector is placed upside down on the ceiling of the housing 50 on the opposite side from the light source. However, the installation method of the photodetector is not necessarily limited to the configuration shown in FIG. 3 . For example, the photodetector may be installed on the bottom of the housing 50 in the same manner as the light source, or on the side of the housing 50 at an angle of 90° from the light source. In this case, the orientation and installation position of the filter 17 and the reflecting mirror 30 must be optimized so that the return light from the sample returns to the photodetector. In the case of a confocal optical microscope, the laser beam emission position of each light source and the corresponding pinhole should be located at conjugate positions. [Example]

[0049] Next, a laser scanning microscope according to a second embodiment of the present invention will be described with reference to Fig. 7. Fig. 7 is a conceptual configuration diagram of the laser scanning microscope according to the second embodiment of the present invention. The basic structure of the laser scanning microscope according to the second embodiment of the present invention is the same as that of the laser scanning microscope according to the first embodiment of the present invention, except for a slight difference in the configuration of the light source and light detection unit. That is, the condenser lens 29 of the photodetector is omitted, and the condenser lens 16 of the light source is provided on the light scanning mirror 42 side of the filter 17.

[0050] The filter 17 is placed between the light source and the condenser lens 17 so that the irradiation laser beam and the return light from the sample are condensed by the same condenser lens 17. This configuration allows the number of condenser lenses to be reduced from two to one, thereby reducing the number of parts. However, if the space between the light source and the condenser lens 17 is small, the degree of freedom in design is reduced.

[0051] In Example 2, the photodetector is also placed upside down on the ceiling inside the housing 50 on the opposite side from the light source, but the installation method of the photodetector is not necessarily limited to the configuration shown in Figure 7, and it may be installed, for example, on the bottom of the housing 50 in the same way as the light source, or on the side of the housing 50 at an angle of 90 degrees from the light source. [Example]

[0052] Next, a laser scanning microscope according to a third embodiment of the present invention will be described with reference to FIG. 8. FIG. 8 is a conceptual diagram of the laser scanning microscope according to the third embodiment of the present invention. In the laser scanning microscope according to the third embodiment, the configuration of the optical scanning unit is changed so that the direction of the irradiation laser beam 19 after passing through the condenser lens 16 is directed upward by 90° from the direction of the beam immediately after being emitted from the semiconductor laser 13 when the optical scanning mirror 42 is in a stationary position. That is, the direction of the scanning optical system and objective lens unit is tilted by 90° compared to that of the first embodiment in order to direct the direction of the irradiation laser beam 19 toward the sample and the return light 31 from the sample upward by 90° from the direction of the beam immediately after being emitted from the semiconductor laser 13. Note that the structures of the light source, light detection unit, and scanning optical system and objective lens unit are the same.

[0053] Here, instead of the prism reflectors 43 and 44, a filter 46 is installed at a 45° angle relative to the direction of travel of the irradiating laser beam. In this case, the irradiating laser beam is incident perpendicularly on the optical scanning mirror 42 by the filter 46. The beam reflected by the optical scanning mirror 42 then passes through the filter 46 and travels upward. The return light from the sample travels in the opposite direction, from top to bottom, passes through the filter 46, is reflected by the optical scanning mirror 42, and is then reflected again by the filter 46, traveling in the opposite direction to the irradiating laser beam. Furthermore, when the optical scanning mirror 42 is operating, the irradiating laser beam 19 scans within a scanning angle range 47, as shown in the figure, and the focal point of the irradiation on the measurement sample is scanned across the sample. To allow the light beam to travel in this manner, the filter 46 must be a semitransparent mirror. Therefore, the irradiation laser beam 19 and a part of the return light 31 travel to the right in the figure, and each time the beam passes through and is reflected by the filter 46, the beam is split into two and the beam intensity is reduced by half, but the irradiation laser beam 19 can be made to travel upward.

[0054] As can be seen from the difference between the configurations in Fig. 3 and Fig. 8, the emission direction of the irradiation laser beam 19 can be changed by changing the optical system of the optical scanning unit. Note that a configuration is possible in which the emission direction of the irradiation laser beam is changed to any direction other than a direction that differs by 90° from the beam direction immediately after being emitted from the light source. Note that in the case of Example 3 as well, as in Example 2, the condenser lens 29 of the photodetector may be omitted, and the condenser lens 16 of the light source may be provided on the optical scanning mirror 42 side of the filter 17.

[0055] Next, a laser scanning microscope according to a fourth embodiment of the present invention will be described with reference to FIG. 9. FIG. 9 is a conceptual configuration diagram of the laser scanning microscope according to the fourth embodiment of the present invention. The laser scanning microscope according to the fourth embodiment of the present invention has the same basic structure as the laser scanning microscope according to the first embodiment of the present invention, except for the configuration of the housing that houses the light source, light detection unit, and light scanning unit. That is, the light source, light detection unit, and light scanning unit are housed in housing 52, and the light scanning unit is housed in housing 48 made of a different material or structure, and housing 52 and housing 48 are connected to form an integrated unit. Note that, in the case of the fourth embodiment as well, the condenser lens 29 of the light detector may be omitted, and the condenser lens 16 of the light source may be provided on the light scanning mirror 42 side of the filter 17, as in the second embodiment.

[0056] Here, the following notes are added to the embodiments of the present invention including Examples 1 to 4. (Appendix 1) a light source, a condenser lens, a photodetector, and an optical scanning mirror that scans the laser beam from the light source; The laser scanning microscope includes a housing in which the light source, the condenser lens, the photodetector, and the optical scanning mirror are integrated. (Appendix 2) 2. A laser scanning microscope according to claim 1, wherein the inside of the housing is airtight and isolated from the outside of the housing. (Appendix 3) 3. The laser scanning microscope according to claim 1, wherein the photodetector has a pinhole member with a pinhole formed therein, and a confocal optical system is formed so that the laser beam emission position of the light source and the pinhole are at conjugate positions. (Appendix 4) 3. A laser scanning microscope according to claim 1 or 2, wherein the light source is composed of a plurality of light sources each having a different laser beam emission position, and a confocal optical system is formed so that the laser beam emission position corresponding to each of the light sources and the pinhole of the pinhole member are at conjugate positions. (Appendix 5) 5. The laser scanning microscope according to claim 1, wherein the light source comprises three or more semiconductor lasers having different oscillation wavelengths. (Appendix 6) 5. The laser scanning microscope according to claim 4, wherein the central axes of the laser beams of the plurality of semiconductor lasers are parallel to each other. (Appendix 7) 5. The laser scanning microscope according to claim 4, wherein the central axes of the laser beams of the plurality of semiconductor lasers are non-parallel to each other. (Appendix 8) 8. The laser scanning microscope according to any one of claims 1 to 7, wherein the central axis of the laser beam from the light source and the substrate of the optical scanning mirror are parallel to each other or tilted at an angle of ±10° or less. (Appendix 9) 9. The laser scanning microscope according to any one of claims 1 to 8, wherein an optical scanning system / objective lens unit including a lens is connected to the laser beam emission end of the optical scanning mirror. (Appendix 10) 10. The laser scanning microscope according to claim 9, wherein the tip of the optical scanning system / objective lens unit is located at the position of the irradiation focal point of the final stage objective lens of the optical scanning system / objective lens unit. (Appendix 11) 11. The laser scanning microscope according to claim 1, further comprising a half mirror that bends the laser beam from the light source, reflected by the optical scanning mirror, in a vertical direction. [Explanation of symbols]

[0057] 1 Light source and light detector 11 light sources 12 pedestals 13 Condenser lens 14 filters 15 photodetectors 16 pedestals 17 condenser lens 18 reflector 19 Irradiation laser beam 1 10 Return light 2 Optical scanning unit 21 Optical scanning mirror 22,23 Prism reflector 24 scanning angle range 3. Scanning optical system. Objective lens section 4. Scanning optical system 41 pupil projection lens 42 Imaging Lens 43 Lens holder 5 Objective lens system 51 objective lenses 52 Lens Holder 6,7 Case 8 Transparent clothing section 9 Drive circuit and communication section 10 Clip part 11,12 Pedestal 13 Semiconductor laser 14. Case 15. Window 16 Condenser lens 17 filters 181, 182, 183 Beam emission positions 191, 192, 193 Irradiation laser beam 21,22 Pedestal 23 Photodetector 24,241,242,243 light receiving surface 25 Filter material 251, 252, 253 wavelength selection filters 26 Pinhole member 261, 262, 263 pinhole 27 Case 28 Windows 29 Condenser Lens 30 Reflector 31,311,312,313 Return light 41 Optical scanning mirror substrate 42 Optical scanning mirror 421 Reflector 422 First Hinge 423 rotation outer frame 424 Second Hinge 425 non-rotating outer frame 426 AC magnetic field generator 43,44 Prismatic reflector 45,47 scanning angle range 46 filters 48, 50, 51, 52 housing 61 Pupil projection lens 62 Imaging Lens 63 Lens holder 71 Objective Lens 72 Lens holder

Claims

1. a light source, a condenser lens, a photodetector, and an optical scanning mirror that scans the laser beam from the light source; The laser scanning microscope includes a housing in which the light source, the condenser lens, the photodetector, and the optical scanning mirror are integrated.

2. 2. The laser scanning microscope according to claim 1, wherein the inside of the housing is airtight and isolated from the outside of the housing.

3. 3. A laser scanning microscope according to claim 1, wherein the photodetector has a pinhole member with a pinhole formed therein, and a confocal optical system is formed so that the laser beam emission position of the light source and the pinhole are at conjugate positions.

4. A laser scanning microscope as described in claim 1 or claim 2, wherein the light source consists of a plurality of light sources each having a different laser beam emission position, and a confocal optical system is formed so that the laser beam emission position corresponding to each of the light sources and each pinhole of the pinhole member are at conjugate positions.

5. 3. The laser scanning microscope according to claim 1, wherein the central axis of the laser beam from the light source and the substrate of the optical scanning mirror are parallel to each other or tilted at an angle of ±10° or less.

Images