Microscope

Abstract

This microscope includes: a placement part on which an object is placed; an irradiation optical system for irradiating the object with coherent light; a first detection optical system for detecting signal light separated from first emission light from the object; an illumination optical system for irradiating the object with plane wave light; a second detection optical system for detecting second emission light emitted from the object due to the plane wave light; and a scanning part for scanning the coherent light and the second emission light.

Description

microscope

[0001] The present invention relates to a microscope.

[0002] By applying an imaging method to a microscope that utilizes nonlinear optical effects to detect scattered light, including the sample's nonlinear response to the intensity of the irradiated light, it is possible to obtain functional information about the sample based on molecular information, such as molecular vibrations contained in the sample. Known nonlinear optical effects that can be applied to microscopic imaging include multiphoton-excited fluorescence, second-harmonic generation, coherent anti-Stokes Raman scattering, and stimulated Raman scattering. When utilizing nonlinear optical effects, it is desirable for the irradiated light intensity to change rapidly over time, while it is necessary to prevent damage to the sample by the irradiated light. For this reason, the irradiated light is generally moved and scanned spatially relative to the sample.

[0003] Furthermore, cells are a sample with low visibility in bright-field observation. Quantitative phase imaging is known as a method for obtaining morphological information about cells. Non-Patent Document 1 discloses a microscope that irradiates cells with plane waves of second-harmonic light and mid-infrared light in a non-scanning manner, captures the transmitted light, and obtains morphological images using quantitative phase images and functional images using label-free mid-infrared photothermal images.

[0004] ACS Nano. 2022 Aug 23; 16(8): 11516-11544.

[0005] When imaging molecular information and morphology of cells, it is preferable to be able to align the images from the perspective of the usefulness of the imaging information. In the multimodal microscope disclosed in Non-Patent Document 1 that acquires quantitative phase images and mid-infrared photothermal images, there was concern that the positional relationship between the two captured images would be misaligned due to misalignment caused by differences between the quantitative phase imaging optical system and the mid-infrared photothermal optical system. When a user acquires morphological and functional images of living cells using the microscope system described in Non-Patent Document 1, the misalignment between the images makes it difficult to understand the morphological features and biological functions. The present invention aims to provide a microscope that ensures the positional relationship between the quantitative phase image (QPI) and the nonlinear optical image (NOI).

[0006] A microscope according to an embodiment of the present invention includes a mounting section on which an object is mounted, a scanning section that scans coherent light emitted from a first light source and containing a first wavelength, and a first objective lens that collects the coherent light scanned by the scanning section, and an illumination optical system that illuminates the object with the coherent light via the scanning section and the objective lens, in this order; a first detection optical system that includes a second objective lens that collects first emission light emitted from the object by the coherent light, on the opposite side of the mounting section from the first optical system, a first separation section that separates signal light from the first emission light, and a first detection section that detects the signal light; an illumination optical system that irradiates the object with the illumination light, the illumination light being emitted from a second light source and including a second wavelength different from the first wavelength, and a second separation unit that transmits either the first emitted light or the first emitted light and reflects the other; and a second detection optical system that includes a third separation unit that transmits either the second emitted light emitted from the object by the illumination light or the coherent light and reflects the other, the second detection optical system including a second detection unit that detects the second emitted light via the first objective lens, the scanning unit, and the second separation unit in this order.

[0007] According to the present invention, it is possible to provide a microscope in which the positional relationship between the quantitative phase image QPI and the nonlinear optical image NOI is guaranteed.

[0008] FIG. 1 is a diagram showing the configuration of a microscope 100 according to a first embodiment; FIG. 2 is a diagram showing the configuration of a first light source and a scanning unit according to the first embodiment; FIG. 3 is a diagram showing the configuration of a microscope 300 according to a second embodiment; FIG. 4 is a diagram showing the configuration of a first light source and a scanning unit according to the second embodiment.

[0009] First Embodiment A microscope 100 according to this embodiment is a multimodal microscope capable of outputting a fusion image in which the voxel spaces of the volume data of the acquired stimulated Raman scattering light image and quantitative phase image QPI match. The positional relationship of the volume data of the stimulated Raman scattering light image and quantitative phase image QPI match, and the image spaces of the two images match.

[0010] The illumination optical system and the first detection optical system for acquiring a stimulated Raman scattering light image, which will be described later, can be replaced with other optical elements for nonlinear optical imaging, such as optical elements that utilize multiphoton excitation fluorescence, second harmonic generation, or coherent anti-Stokes Raman scattering.

[0011] (Placement Unit) As shown in FIG. 1 , the microscope 100 includes a placement unit 161 on which the object 160 is placed. The placement unit 161 can be described as a positioning member that determines the position of the object 160 between a pair of first and second objective lenses, which are opposed to each other and sandwich the object 160, as described below. The placement unit 161 may be mechanically coupled to an adjustment mechanism 163. The adjustment mechanism 163 is configured with a multi-axis stage that can adjust the Cartesian coordinate system (X, Y, Z axes), the tilt angle θ, and the azimuth angle Φ. The control unit 135 is configured to acquire position information of the placement unit 161 via the adjustment mechanism 163 and adjust the relative position of the object 160 and the placement unit 161 via the adjustment mechanism 163. The control unit 135 can be described as adjusting the relative position of the object 160 and the focal point fs of the coherent light 1001. In other words, the mounting unit 161 mounts the object 160 at the focusing position of the first objective lens 153, and the object 160 at the focusing position of the second objective lens 170. Since the focusing point fs overlaps with the focusing position, the focusing position may be referred to as the focusing position fs.

[0012] (Object) The object 160 includes a biological sample, an organic composition, an inorganic composition, and a flexible, non-self-supporting sample that is amorphous and plastically deformable. Such a non-self-supporting object 160 is supported by a support member (not shown), and the posture and position of the object 160 relative to the optical system of the microscope are determined by the support member and the mounting portion 161. The support member is configured to be translucent to primary and secondary light for observing the object 160 and is composed of an optical member that is fluorescently inactive. Examples of optical members include a prepared preparation, a slide glass, etc.

[0013] 1, the microscope 100 includes an illumination optical system 140 and a first detection optical system 142 as a nonlinear optical observation system 143, and an illumination optical system 146 and a second detection optical system 148 as a quantitative phase observation system 149. The illumination optical system 140 and the second detection optical system 148, which are located on the same side of the mounting unit 161, share an optical path with multiple optical members. Similarly, the first detection optical system 142 and the illumination optical system 146, which are located on the same side of the mounting unit 161, share an optical path with multiple optical members.

[0014] 1 , the irradiation optical system 140 includes a scanning unit 130 that scans coherent light 1001 emitted from the first light source 110 and including a first wavelength λ1, and a first objective lens 153 that collects the coherent light 1001 scanned by the scanning unit 130. The irradiation optical system 140 irradiates the object 160 with the coherent light 1001 via the scanning unit 130 and the first objective lens 153 in this order. The coherent light 1001 includes excitation light 1011 with a wavelength λe1 and probe light 1021 with a wavelength λp1, both of which are synchronously emitted from the light source 110.

[0015] The irradiation optical system 140 includes a mirror 151 that changes the optical path of the coherent light 1011 by 90 degrees.

[0016] (First Light Source) The irradiation optical system 140 includes a first light source 110 that irradiates the object 160 with coherent light 1001 that generates a nonlinear optical effect. The first light source 110 includes an excitation light source 101 that emits an excitation light pulse train and a probe light source 102 that emits a probe light pulse train. The first light source 110 further includes an adjustment unit 103 that adjusts the time difference Δt between one of the excitation light 1011 and the probe light 1021 and the other, and a multiplexing unit 105 that multiplexes the one light and the other light whose time difference Δt has been adjusted. The multiplexing unit 105 is configured with a dichroic mirror. In other words, the irradiation optical system 140 includes the first light source 110 that is arranged to be optically coupled to the first objective lens 153.

[0017] As shown in Fig. 2, the first light source 110 includes an excitation light source 101 that emits excitation light 1011 in a direction toward the dichroic mirror 105. The excitation light 1011 is pulsed light from the viewpoint of the efficiency of the elementary processes of wave light of nonlinear optical light. The pulse width of the excitation light 1011 is on the order of femtoseconds to picoseconds. Furthermore, the pulse repetition frequency of the excitation light 1011 is set to 1 MHz or higher from the viewpoint of mitigating the effects of intensity fluctuations of the excitation light 1011.

[0018] As a laser that satisfies these pulse widths and repetition frequencies, a titanium sapphire laser that oscillates in the near-infrared band can be used as the excitation light source 101. When a titanium sapphire laser is used as the excitation light source 101, the excitation wavelength can be set to 700 nm or more and 1000 nm or less.

[0019] The first light source 110 includes a probe light source 102 that emits a probe light 1021. The probe light 1021 emitted from the probe light source 102 is guided to an optical delay system 103 that provides a predetermined delay time for synchronization with the pulse train of the excitation light 1011, and is then guided to a dichroic mirror 105 via a mirror 104. The probe light 1021 reflected by the dichroic mirror 105 is multiplexed with the excitation light 1011. The multiplexed excitation light 1011 and probe light 1021 are guided to a third separator 210 as coherent light 1001. The third separator 210 is composed of a dichroic mirror and has spectral transmission characteristics that transmit the coherent light 1001.

[0020] The pulse width of the probe light 1021 is on the order of femtoseconds to picoseconds, similar to that of the excitation light 1011. The wavelength of the probe light 1021 is different from that of the excitation light 1011. The wavelengths of the probe light wavelength λp1 and the excitation light wavelength λe1 are made different so as to ensure a Raman shift (wavenumber difference Δk) corresponding to molecular information of the target molecule of interest for inclusion in the object 160.

[0021] When the excitation light source 101 is a titanium sapphire laser, the wavelength of the probe light 1021 can be swept over a wavelength band from 1000 nm to 1100 nm. An optical fiber laser such as a Yb fiber laser or an Er fiber laser is used as the probe light source 102. The pulse repetition frequency of the probe light 1021 can be set to (1 / natural number), such as (1 / 2) or (1 / 3), times the pulse repetition frequency of the excitation light 1011. Alternatively, the pulse repetition frequencies of the excitation light 1011 and the probe light 1021 can be matched, and intensity modulation can be applied to either the excitation light 1011 or the probe light 1021. Alternatively, different intensity modulations can be applied to the excitation light 1011 and the probe light 1021, respectively.

[0022] The probe light 1021 passes through an optical delay system 103 and a mirror 104, is reflected by a dichroic mirror 105, and is coaxially combined with the excitation light 1011. When combined by the dichroic mirror 105, the optical path length of the probe light 1021 is adjusted using the optical delay system 103 so that the pulses of the excitation light 1011 and the probe light 1021 coincide in time.

[0023] (Scanning Unit) The irradiation optical system 140 includes a scanning unit 130 that scans the coherent light 1001 emitted from the first light source 110 and including the first wavelength λe1. The scanning unit 130 is in other words a scanning unit 130 that scans the excitation light 1011 and the probe light 1021 from the first light source 110 over the object 160. The scanning unit 130 will be described below.

[0024] In the scanning unit 130, the excitation light 1011 and the probe light 1021 are angularly displaced in two directions perpendicular to the traveling direction by the scanning mirrors 131 and 132. The scanning mirrors 131 and 132 may be galvanometer scanners, resonant scanners, or polygon scanners. The scanning mirrors 131 and 132 are arranged so that the midpoints on the optical paths between them are optically conjugate with the entrance pupil of the first objective lens 153. An aperture 133 that defines the beam diameters of the coherent light 1001 and the second emitted light 1092 is arranged between the scanning mirror 131 and the third separation unit 210. The scanning mirrors 131 and 132 are connected to the control unit 135 and are oscillated and scanned so that the normals of their reflecting surfaces are parallel to each other. The scanning mirror 131 and the scanning mirror 132 output information about the direction of their normals to the control unit 135 .

[0025] The irradiation optical system 140 includes a dichroic mirror as a second separation unit 210 between the first light source 110 and the scanning unit 130. The excitation light 1011 and the probe light 1021 are transmitted through the second separation unit 210. Note that, in the present specification, for the sake of simplicity, the reference symbols of optical elements shared by the nonlinear optical observation system 143 and the quantitative phase observation system 149 are shown in the drawings with an indicator line connecting only one element of the nonlinear optical observation system 143 or the quantitative phase observation system 149. Reference symbols of optical elements shared by the nonlinear optical observation system 143 and the quantitative phase observation system 149 include 172, 170, 153, 130, and 210.

[0026] Coherent light 1001 including excitation light 1011 and probe light 1021 that have been angularly displaced and scanned two-dimensionally is reflected by a mirror 151 and focused on an object 160 via a first objective lens 153 .

[0027] ((Nonlinear Optical Effect)) When the Raman shift due to molecular vibration contained in the object 160 matches the wavelength difference between the excitation light 1011 and the probe light 1021, the stimulated Raman scattering effect causes a slight decrease in the intensity of the excitation light 1011 and a slight increase in the intensity of the probe light 1021.

[0028] Hereinafter, the pump light 1011 modulated by the nonlinear optical effect will be referred to as signal light 1012. A modified form in which the probe light 1021 is used as the signal light 1012 may be employed.

[0029] ((First Detection Optical System)) The nonlinear optical observation system 153 includes a first detection optical system 142 on the opposite side of the mounting portion 161 from the irradiation optical system 150. The first detection optical system 142 includes a second objective lens 170 that collects first emission light 1002 emitted from the object 160 by the coherent light 1001, and a first separation unit 173 that separates signal light 1012 from the first emission light 1002. The first detection optical system 142 further includes a first detection unit 174 that detects the signal light 1012.

[0030] The signal light 1012 generated by the nonlinear optical effect travels as a parallel light with an angular displacement by the second objective lens 170. The signal light 1012 is wavelength-selectively guided by the second separation unit 172. The second separation unit has spectral transmission characteristics and spectral reflection characteristics that transmit either the illumination light 1091 or the first emitted light 1002 (described later) and reflect the other. The second separation unit 172 of this embodiment reflects the first emitted light 1002. The first separation unit 172 is configured with a dichroic mirror.

[0031] The first separating unit 173 reflects a probe light component emitted light 1022 of the first emitted light 1002 that corresponds to the wavelength component of the probe light 1021, and selectively transmits a signal light 1012 that corresponds to the wavelength component of the excitation light 1011. The signal light 1012 is detected by the first detecting unit 174. A photodiode is used as the photodetector employed in the first detecting unit 174. The first detecting unit 174 may also be referred to as a photodetector 174, a light-receiving element 174, or a light-receiving sensor 174.

[0032] The control unit 135 acquires an output signal including information about the light intensity of the signal light 1012 from the first detection unit 174. The computer 136 includes an image formation unit 136if that performs image reconstruction processing and forms an image based on the information about the light intensity of the signal light 1012 acquired via the control unit 135 and angular displacement information about the scanning mirrors 131 and 132. Some or all of the functions of the computer 136 may be located remotely. The image formation unit 136if can construct a three-dimensional image of the object 160 when the mounting unit 161 is scanned in the optical axis direction. In other words, the image formation unit 136if is a first image formation unit that acquires a nonlinear optical image NOI of the object 160 based on the first detection information ip from the first detection unit 174 and information if about the position of the optical axis center of the coherent light 1001.

[0033] The first detection unit 174 may be configured to perform lock-in detection of the signal light 1012 using, as a reference input, a signal synchronized with the pulse repetition frequency of the probe light 1021. The object 160 is illuminated with plane wave light 1092 obtained by shaping the illumination light 1081 into a plane wave by the plane wave forming unit 182, and therefore the plane wave light 1092 in this embodiment may be referred to as the illumination light 1081.

[0034] (Quantitative Phase Contrast Observation System) Next, quantitative phase contrast observation by the microscope 100 according to this embodiment will be described.

[0035] As shown in FIG. 1, the microscope 100 includes an illumination optical system 146 that illuminates the object 160 with illumination light 1091 , and a quantitative phase observation system 149 that detects second emitted light from the object 160 .

[0036] (Illumination Optical System) The quantitative phase observation system 149 includes an illumination optical system 146 that illuminates the object 160 with plane wave light 1091. The illumination optical system 146 includes a second light source 181 that emits second emission light 1081 and a plane wave forming unit 182 that illuminates the object 160 with the second emission light 1081 as plane wave light 1091. The second light source 181 may be referred to as an illumination light source 181. The illumination light 1081 emitted from the second light source 181 passes through the plane wave forming unit 182 and the first separating unit 172 and is focused by the second objective lens 170. The focusing point fs is adjusted by the mounting unit 161 so as to overlap with the object 160. The second light source 181 is optically coupled to the second objective lens 170 via the plane wave forming unit 182 and the second separating unit 172. The focal point fs forms an irradiation spot is on the irradiation surface.

[0037] (Second Light Source) The quantitative phase observation system 149 includes a second light source 181 that emits illumination light 1081 of a second wavelength λ2 for irradiating the plane wave light 1091. The second light source 181 may be a white LED, a lamp, or the like. The wavelength band of the illumination light 1081 is the visible light band. The second wavelength λ2 of the illumination light 1081 may be a monochromatic light source using a bandpass filter.

[0038] The second wavelength λ2 is different from the first wavelength λ1 contained in the coherent light 1001 including the pump light 1011 and the probe light 1021, and is 450 nm to 650 nm, which is shorter than the wavelengths of the pump light 1011 and the probe light 1021.

[0039] (Plane Wave Forming Unit) The illumination optical system 146 includes a plane wave forming unit 182 between the second light source 181 and the second objective lens 170, which converts the illumination light 1081 into plane wave light 1091 so that the plane wave light 1091 is illuminated on the object 160 via the second objective lens 170. The plane wave forming unit 182 employs optical elements such as a lens and a pinhole. The plane wave forming unit 182 of this embodiment is a lens arranged in a position optically conjugate with the focal position of the second objective lens 170 on the second light source 181 side. The lens arranged in such an optically conjugate position is replaced by a pinhole or a fiber end (not shown). The plane wave forming unit 182 of the microscope 100 may be an optical element constituting the second light source 181 that emits the plane wave light 1091.

[0040] (Second Detection Optical System) The quantitative phase observation system 149 includes a second detection optical system 149 that detects second emission light 1092 emitted from the object 160 by illumination with plane wave light 1091. The second detection optical system 149 includes a third separation unit 210 that transmits either the second emission light 1092 or the coherent light 1001 and reflects the other, and a second detection unit 187 that detects the second emission light 1092. The second detection unit 187 detects the second emission light 1092 via the first objective lens 153, the scanning unit 130, the third separation unit 210, the imaging lens 185, and the aperture 188, in this order.

[0041] The detection process of the second detection optical system 148 that constitutes the quantitative phase observation system 149 will be described in order. A portion of the plane wave light 1091 passes through the object 160, and the remaining portion of the plane wave light 1091 is diffracted and scattered by the object 160. Second outgoing light 1092, which is emitted from the object 160 on which the plane wave light 1091 is incident to the opposite side of the second objective lens 170, is collected by the first objective lens 153. The second outgoing light 1092 collected by the first objective lens 153 is guided to the scanning unit 130 via the mirror 151.

[0042] (Scanning unit) The second detection optical system 148 constituting the quantitative phase observation system 149 shares, with the irradiation optical system 140 constituting the nonlinear optical observation system 143, the scanning unit 130 that scans the optical axes of the second emitted light 1092 and the coherent light 1001 in a direction intersecting the optical axes. In the microscope 100, the optical axes of the second emitted light 1092 and the coherent light 1001 are anti-parallel to each other but coaxial.

[0043] The scanning unit 130 scans the optical axis center of the second emitted light 1092 in a direction intersecting the optical axis of the coherent light 1001. The scanning unit 130 synchronously scans the optical axis center of the coherent light 1001 and the optical axis center of the second emitted light 1092 in a direction intersecting the optical axis of the coherent light 1001.

[0044] (Third separation section) The second detection optical system 148 that constitutes the quantitative phase observation system 149 shares with the irradiation optical system 140 that constitutes the nonlinear optical observation system 143 a third separation section 210 that transmits either the second emitted light 1092 or the coherent light 1001 and reflects the other.

[0045] 2, the second emitted light 1092 incident on the scanning unit 130 is reflected by the scanning mirrors 131 and 132 and guided toward the aperture 133. A portion of the second emitted light 1092 passes through the aperture 133, thereby passing through the scanning unit 130, and is guided to the third separation unit 105. A dichroic mirror is employed for the third separation unit 133, similar to the second separation unit.

[0046] ((Imaging Lens and Aperture)) The second emitted light 1092, which is wavelength-selectively separated from the coherent light 1001 by the third separator 133, is imaged on the sensor surface of the first detector 187 via the imaging lens 185, as shown in Figures 1 and 2. The second detector 187 employs a photodiode or a single or arrayed light-receiving array element such as a CMOS image sensor.

[0047] An aperture 188 is provided in front of the first detection unit 187. The aperture 188 can adjust the spatial resolution and sensitivity of the quantitative phase by changing the size of the opening diameter. By reducing the size of the opening diameter, the spatial resolution increases and the sensitivity decreases.

[0048] The sensor surface of the second detecting unit 187 or the center of the aperture 188 is disposed at a position optically conjugate with the center of light emission of each of the excitation light source 101 and the probe light source 102 constituting the first light source 110. When the excitation light source 101 and the probe light source 102 are fiber light sources, the center of the emission end of each fiber is in a position conjugate with the sensor surface of the second detecting unit 187 or the center of the aperture 188.

[0049] The irradiation spot is in the sample surface of the object 160, onto which the coherent light 1001 from the nonlinear optical observation system 143 is irradiated, is changed by the scanning unit 130. At the irradiation time of each pulse light train of the excitation light 1011 and the probe light 1021 constituting the coherent light 1001, the second emitted light 1092 is detected by the second detection unit 187. The excitation light, excitation light pulse train, and excitation light source may be alternatively referred to as pump light, pump light pulse train, and pump light source, respectively. Furthermore, the probe light, probe light pulse train, and probe light source may be alternatively referred to as Stokes light, Stokes light pulse train, and Stokes light source, respectively.

[0050] (Acquisition of Bright-Field Image) The microscope 100 has a scanning unit 130 configured to perform point scanning of a common focal spot fs (focal spot) with the coherent light 1001 of the nonlinear optical observation system and the plane wave light 1091. The scanning unit 130, the adjustment mechanism 163, and the control unit 135 are configured to enable three-dimensional scanning of the focal spot fs on the object 160.

[0051] The microscope 100 can analyze the output signal of the first detection unit 187 acquired via the control unit 135, together with the angular displacement information of the primary information scanning unit 130 at the focal point fs, in the image forming unit 136if, and visualize it as a light intensity image to obtain a bright-field image as a morphological image.

[0052] The image forming unit 136if can be said to be a second image forming unit that acquires a morphological image of the object 160 based on the second detection information is from the second detection unit 187 and information regarding the position of the optical axis center of the illumination light 1081.

[0053] (Acquisition of Quantitative Phase Images) The microscope 100 can defocus the object 160 and the focal point fs of the plane wave light 1091 by relatively moving them in the optical axis direction of the plane wave light 1091, thereby acquiring multiple light intensity images of the object 160 with different defocus amounts. The mechanism for moving the mounting unit 161 in the optical axis direction of the plane wave light 1091 may use the same adjustment mechanism 163 for both nonlinear optical observation and quantitative phase observation, or may use another adjustment mechanism (not shown) that differs from the adjustment mechanism 163 in at least one of accuracy, pitch, and range. The image forming unit 136if can acquire a quantitative phase image of the object 160 by a known calculation method, including the intensity transport method, based on the multiple light intensity images with different defocus amounts.

[0054] The microscope 100 according to this embodiment is configured to acquire a signal derived from a common focal point fs by the scanning unit 130 shared by the nonlinear optical observation system 143 and the quantitative phase observation system 149 via the first detection unit 173 and the second detection unit 87. Therefore, the microscope 100 according to this embodiment is a microscope capable of acquiring a quantitative phase image QPI and a nonlinear optical image NOI whose positional relationship is guaranteed.

[0055] The image forming unit 136if may be referred to as an image processing unit that performs other signal processing on the two images, the nonlinear optical image NOI and the quantitative phase image QPI. The image processing unit, which is a modified version of the image forming unit 136if, performs at least one of the following processes: a mode change process that changes the display mode of the two images, an image fusion process that fuses the two images, and an image generation process that displays the two images so that they can be compared spatially or temporally.

[0056] The irradiation timings of the coherent light 1001 and the illumination light 1081 (plane wave light 1091) may be set to the same time so as to overlap, or may be set to different times.

[0057] Second Embodiment (Microscope 300) A microscope 300 according to this embodiment differs from the microscope 100 according to the first embodiment in that it is provided with a nonlinear optical observation system 153 that acquires a two-photon excitation fluorescence image. The microscope 300 according to this embodiment will be described with reference to Figures 3 and 4.

[0058] (Excitation Light Source) The microscope 300 includes an excitation light source 310 that emits excitation light 1041 for exciting two-photons in the object 160. The object 160 is excited by two-photon absorption and emits second emission light 1032 from the object 160, the second emission light 1032 including fluorescence 1052, which is a fluorescent component, and transmitted component light 1042 of the primary light.

[0059] The second emitted light 1032 emitted from the object 160 is detected by the first detection unit 174 as signal light, in the same manner as the first detection optical system according to the first embodiment, with the fluorescence 1052 wavelength-selectively received via the first separation unit 173.

[0060] Similar to the microscope 100, the microscope 300 according to this embodiment acquires two-photon excitation fluorescence images TPI and quantitative phase images QPI with the positional relationship maintained.

[0061] In addition, the nonlinear optical image NOI includes at least one of a distribution of molecular information of the object 160, a distribution of Raman scattering spectroscopic information, and a distribution of two-photon excitation fluorescence information, and the quantitative phase image QPI includes morphological information of the object 160.

[0062] The present invention is not limited to the above-described embodiments, and various modifications and variations can be made without departing from the spirit and scope of the present invention. Therefore, the following claims are appended to apprise the public of the scope of the present invention.

[0063] This application claims priority based on Japanese Patent Application No. 2024-088416, filed May 30, 2024, the entire contents of which are incorporated herein by reference.

[0064] 100 Microscope 101 Excitation light source 102 Probe light source 103 Optical delay system 104 Mirror 105 Dichroic mirror 110 First emission optical system (light source unit) 130 Scanning unit 131 Scanning mirror 132 Scanning mirror 133 Aperture 135 Control unit 136 Computer 149 Illumination optical system 151 Mirror 153 First objective lens 160 Sample 161 Mounting unit 170 Second objective lens 172 Second separation unit (dichroic mirror) 173 First separation unit (filter) 174 First detection unit 181 Second emission optical system (second light source) 185 Imaging lens 187 First detection unit 188 Aperture 210 Third separation unit (dichroic mirror) 300 Microscope 310 Excitation light source 473 Filter 1001 Coherent light 1011 Excitation light 1021 Probe light 1012 Signal light 1041 Excitation light 1042 Excitation wavelength component of first emitted light 1052 Signal light 1081 Illumination light 1091 Plane wave light 1092 Second emitted light

Claims

1. A mounting section on which an object is mounted; an illumination optical system including a scanning section optically coupled to a first light source that emits coherent light including a first wavelength and that scans the coherent light, and a first objective lens that collects the coherent light scanned by the scanning section, and that illuminates the object with the coherent light via the scanning section and the objective lens, in that order; a first detection optical system including a second objective lens on the opposite side of the mounting section from the illumination optical system that collects first emission light emitted from the object by the coherent light, a first separation section that separates signal light from the first emission light, and a first detection section that detects the signal light; and an illumination optical system that includes a second separation section that transmits either the illumination light emitted from a second light source that includes a second wavelength different from the first wavelength or the first emission light and reflects the other, and that illuminates the object with the illumination light. a microscope comprising: a second detection optical system including a third separation unit that transmits either the second emission light emitted from the object by the illumination light or the coherent light and reflects the other, and a second detection unit that detects the second emission light via the first objective lens, the scanning unit, and the second separation unit in this order.

2. The microscope of claim 1, further comprising a second light source optically coupled to the second objective lens.

3. The microscope of claim 1 or 2, further comprising the first light source optically coupled to the first objective lens.

4. A microscope according to claim 1 or 2, wherein the first light source comprises an excitation light source that emits an excitation light pulse train, a probe light source that emits a probe light pulse train, an adjustment unit that adjusts the time difference between one of the excitation light and the probe light relative to the other, and a combining unit that combines the one light and the other light whose time difference has been adjusted.

5. A microscope according to claim 1 or 2, wherein the first light source comprises an excitation light source that emits an excitation light pulse train.

6. A microscope according to claim 1 or 2, wherein the scanning unit scans the optical axis center of the coherent light and the optical axis center of the second emitted light in a direction intersecting the optical axis of the coherent light.

7. A microscope as described in claim 1 or 2, comprising a first image forming unit that acquires a nonlinear optical image of the object based on first detection information from the first detection unit and information regarding the position of the optical axis center of the coherent light.

8. A microscope as described in claim 7, further comprising a second image forming unit that acquires a quantitative phase image of the object based on second detection information from the second detection unit and information regarding the position of the optical axis center of the illumination light.

9. A microscope as described in claim 8, comprising an image processing unit that performs at least one of the following processes: a mode change process that changes the display mode of at least one of the nonlinear optical image and the quantitative phase image; an image fusion process that fuses the nonlinear optical image and the quantitative phase image; and an image generation process that displays the nonlinear optical image and the quantitative phase image in a manner that allows them to be compared spatially and temporally.

10. A microscope as described in claim 8, wherein the nonlinear optical image includes at least one of a distribution of molecular information of the object, a distribution of Raman scattering spectroscopic information, and a distribution of fluorescent information, and the quantitative phase image includes morphological information of the object.

11. A microscope according to claim 1 or 2, wherein the first detection unit detects the signal light from the object via the second objective lens and the first separation unit in this order.

12. A microscope according to claim 1 or 2, wherein the illumination optical system irradiates the illumination light onto the object via the second separation section and the second objective lens in this order.

13. A microscope according to claim 1 or 2, wherein the illumination light is applied to the object as plane wave light.

14. The microscope of claim 1, wherein the first wavelength is longer than the second wavelength.

15. The microscope according to claim 1 or 2, wherein the first light source comprises an excitation light source that emits excitation light containing the first wavelength as the coherent light, and a probe light source that emits probe light containing a third wavelength as coherent light having a wavelength that is longer than either the first wavelength or the second wavelength.

16. The microscope according to claim 1 or 2, further comprising a control unit for controlling the relative position between the object and the focal point of the coherent light.

17. The microscope according to claim 16, further comprising an adjustment mechanism that supports the mounting portion and adjusts the position of the mounting portion in the direction of the optical axis of the coherent light.

18. The microscope according to claim 17, wherein the control unit controls the position of the mounting unit via the adjustment mechanism.

19. The microscope of claim 1 or 2, wherein the scanning unit includes a pair of optical elements, a first optical element and a second optical element, sandwiched between them in a position optically conjugate with the entrance pupil of the first objective lens, the illumination light is irradiated onto the first optical element, the second optical element, and the object in that order, and the second emission light is irradiated onto the second optical element, the first optical element, and the second detection unit in that order.

20. An illumination optical system that irradiates the object with the coherent light, the illumination optical system including: a scanning unit optically coupled to a first light source that emits coherent light including a first wavelength, and that scans the coherent light; and a first objective lens that condenses the coherent light scanned by the scanning unit; a mounting unit that places the object at a condensing position of the first objective lens; a second objective lens that collects first emission light emitted from the object by the coherent light, a first separation unit that separates signal light from the first emission light, and a first detection unit that detects the signal light; and an illumination optical system that irradiates the object with the illumination light, the illumination optical system including: a second light source that emits illumination light including a second wavelength different from the first wavelength, and a second separation unit that transmits either the illumination light or the first emission light and reflects the other. a microscope comprising: a second detection optical system including a third separation unit that transmits either the second emission light emitted from the object by the illumination light or the coherent light and reflects the other, and a second detection unit that detects the second emission light via the first objective lens, the scanning unit, and the second separation unit in this order.

21. The microscope of claim 20, further comprising the first light source.

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