Microscope

Abstract

A microscope according to the present invention is configured such that a phase difference observation optical system and a nonlinear optical observation optical system are provided opposite each other with an object to be provided therebetween. The microscope comprises: a ring diaphragm that functions as a diaphragm for primary light for phase difference observation and transmits secondary light that includes nonlinear optical effects from the object; and a phase ring that is provided at a location that is optically conjugate with the ring diaphragm.

Description

microscope

[0001] The present invention relates to a microscope.

[0002] Phase contrast observation is known as a method for observing the morphology of samples such as cells, which have poor contrast when observed using bright-field observation, etc. In phase contrast observation, a donut-shaped aperture (ring aperture) that partially blocks the illumination is placed at the pupil position of the condenser, and a ring-shaped phase film (phase ring) that is conjugate with the aperture is placed at the pupil position of the objective lens.

[0003] There is also a known method of utilizing nonlinear optical effects to obtain the distribution of molecular vibrations contained in a sample by detecting scattered light that includes the sample's nonlinear response to the intensity of the irradiated light. Examples of methods that utilize linear optical effects include multiphoton excitation fluorescence, second harmonic generation, coherent anti-Stokes Raman scattering, and stimulated Raman scattering.

[0004] A microscope that combines these methods and has an optical system that shares part of the illumination and detection optical paths before and after the sample is disclosed, for example, in Japanese Patent Application Laid-Open No. 2019-35859.

[0005] Japanese Patent Application Laid-Open No. 2019-35859

[0006] If the optical path for phase contrast observation and an observation method utilizing nonlinear optical effects (hereinafter referred to as nonlinear optical observation) is shared before and after the sample, the aperture used in the former will block the illumination light or scattered light of the latter, resulting in a decrease in signal volume and efficiency. To solve this problem, Japanese Patent Application Laid-Open No. 2019-35859 proposes using a relay optical system to create a position conjugate with the pupil of the condenser, placing an aperture there, and branching the optical paths for phase contrast observation and nonlinear optical observation between the sample and the aperture. However, there was an issue that the optical system would become larger due to the relay optical system mentioned above.

[0007] That is, an object of the present invention is to provide a microscope equipped with a compact optical system that can simultaneously obtain a phase contrast image by a phase contrast method and a nonlinear optical image by a nonlinear optical method.

[0008] A microscope according to an embodiment of the present invention includes a mounting section on which an object is mounted; a first optical system including a first condensing lens that condenses first light emitted from a first light source and irradiates the object with first illumination light; a first detection section located on the opposite side of the mounting section from the first optical system and that detects first emission light, which includes a first wavelength and is emitted from the object due to a nonlinear optical effect caused by the first illumination light, via a second condensing lens that is arranged opposite the first condensing lens and the mounting section; a second optical system including a second condensing lens that is arranged opposite the first condensing lens and that irradiates the object with second illumination light, which is emitted from a second light source and includes a second wavelength different from the first wavelength; and a second detection section that detects second emission light, which is emitted from the object due to the second illumination light, via the first condensing lens. The first optical system includes a first separation unit that transmits either the first light or the second emitted light and reflects the other, and the second optical system includes a second separation unit that transmits either the second light or the first emitted light and reflects the other, and a first optical element that is arranged between the second separation unit and the second focusing lens and has a spectral transmittance at the first wavelength higher than the spectral transmittance at the second wavelength and attenuates a portion of the second light, and a second optical element that is arranged between the second detection unit and the first focusing lens and is arranged in a position optically conjugate with the first optical element.

[0009] According to the present invention, it is possible to provide a microscope that is equipped with a compact optical system that can simultaneously obtain a phase contrast image by a phase contrast method and a nonlinear optical image by a nonlinear optical method.

[0010] 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; and FIG. 4 is a diagram showing the configuration of a microscope 400 according to a third embodiment.

[0011] First Embodiment The microscope 100 according to this embodiment is a multimodal microscope capable of acquiring physical property images, including stimulated Raman scattering light, in a common region of interest of a common sample, so that an observer can utilize nonlinear optical observation methods in addition to morphological images obtained by phase contrast observation. The nonlinear optical detection method provided by the microscope of this embodiment can also be applied to nonlinear optical scattering other than stimulated Raman scattering. Nonlinear optical scattering other than stimulated Raman scattering light includes multiphoton excitation fluorescence, second-harmonic generation light, coherent anti-Stokes Raman scattering light, etc. FIG. 1 is a diagram showing a schematic configuration of the microscope 100 according to the first embodiment. Physical property images provided by the microscope 100 of this embodiment include molecular vibration spectrum images and interatomic bond distributions in an object, and examples of the object include organic matter and biological materials.

[0012] 1-1 Nonlinear Optical Light Detection System The first light source in the nonlinear optical detection system of the microscope 100 according to this embodiment will be described with reference to FIG.

[0013] The first light source 110 includes an excitation light source 101 that emits excitation light 1011 and a dichroic mirror 105 that selectively transmits the excitation light 1011 in a wavelength-selective manner. The excitation light 1011 is pulsed light to efficiently generate a nonlinear optical process. The pulse width of the excitation light 1011, which is pulsed light, is on the order of femtoseconds to picoseconds. The pulse repetition frequency may be 1 MHz or higher to mitigate the effects of fluctuations in the intensity of the excitation light 1011. When a titanium sapphire laser oscillating in the near-infrared band is used as the excitation light source 101, the wavelength of the excitation light 1011 is set between 700 nm and 1000 nm.

[0014] Next, the probe light source 102 included in the first light source 110 will be described. The probe light source 102 emits probe light 1021. The probe light 1021 is pulsed light, and its pulse width is on the order of femtoseconds to picoseconds. The wavelength of the probe light 1021 is set to be different from the wavelength of the excitation light 1011, and it is desirable that the difference be approximately the same as the wavelength corresponding to the Raman shift (wavenumber) of the molecular information of the target object. When a titanium sapphire laser is used as the excitation light source 101, a probe light source 102 capable of sweeping the wavelength of the probe light 1021 between 1000 nm and 1100 nm is used. Such a probe light source 102 includes an optical fiber laser such as a Yb fiber laser or an Er fiber laser. Furthermore, by setting the pulse repetition frequency of the probe light 1021 to half the pulse repetition frequency of the excitation light 1011, the nonlinear optical effect of the object can be used to modulate the signal light 1012 at the same frequency as the pulse repetition frequency of the probe light 1021. However, the pulse repetition frequencies of the excitation light 1011 and the probe light 1021 may be matched, and intensity modulation may be applied to the excitation light 1011 or the probe light 1021.

[0015] 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.

[0016] The scanning unit 130 for scanning the object 160 with the first irradiation light 1031c obtained by further converging the first light 1031 including the excitation light 1011 and the probe light 1021 from the first light source 110 using a converging lens will now be described.

[0017] The scanning unit 130 includes a pair of scanning mirrors 131 and 132 that are given angular displacements in two directions perpendicular to the directions of travel of the excitation light 1011 and the probe light 1021. A galvano scanner, a resonant scanner, or a polygon scanner is employed as the pair of scanning mirrors 131 and 132. The pair of scanning mirrors 131 and 132 are configured to be controlled by a control unit 135 and to output angular displacement information to the control unit 135.

[0018] A first optical system 190 relating to a nonlinear optical observation system of a microscope 100 according to this embodiment will be described with reference to FIG.

[0019] 1, the first light 1031 (excitation light 1011 and probe light 1021) to which the angular displacement has been given is reflected by the first separator 151 (dichroic mirror) and passes through the second optical member 152 (phase ring). The first light 1031 (excitation light 1011 and probe light 1021) that has entered the first condenser lens 153 via the second optical member 152 (phase ring) is condensed by the first condenser lens 152 toward the object 160 placed on the mounting unit 161.

[0020] By providing a movable stage coupled to the mounting unit 161, the object 160 can be moved in the optical axis direction of the first light 1031 (excitation light 1011 and probe light 1021) and in a direction intersecting the optical axis direction via the mounting unit 161. The mounting unit 161 and the control unit 135 are communicatively connected, and position information of the mounting unit 160 is output to the control unit 135, which can then move the mounting unit via a movable stage (not shown).

[0021] Next, the second optical system 195 related to the nonlinear optical observation system of the microscope 100 according to this embodiment will be described with reference to FIG.

[0022] When the Raman shift due to molecular vibrations contained in the object 160 matches the wavenumber 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.

[0023] Hereinafter, an example in which the emitted light 1012 (secondary light) of the first emitted light 1032 modulated by the stimulated Raman scattering effect and having the same wavelength as the excitation light 1011 is used as the signal light 1012 will be described as the microscope 100 of the first embodiment. On the other hand, the present invention also includes a modified embodiment (not shown) in which the emitted light 1022 (secondary light) of the first emitted light 1032 and having the same wavelength as the probe light 1021 is used as the signal light 1022.

[0024] The excitation light 1011 and the signal light 1012 of the first emitted light 1032 generated by the nonlinear optical effect travel as parallel light with an angular displacement by the second condenser lens 170. The signal light 1012 passes through the first optical member 171 (ring diaphragm) and is wavelength-selectively reflected by the second separator 172 (dichroic mirror).

[0025] The emitted light 1012 (secondary light) reflected by the second separating unit 172 (dichroic mirror) passes through a spectral filter 173 that attenuates the wavelength of the probe light 1021 and selectively transmits the wavelength of the excitation light 1011, and is guided to the first detecting unit 174. The emitted light 1012 is detected as signal light 1012 by the first detecting unit 174.

[0026] The first detector 174 outputs a detection signal to the controller 135. The angular displacement information of the scanning mirror pair 131 and the scanning mirror 132 and the detection signal are analyzed and imaged in the computer 136. By combining this with scanning in which the mounting unit 161 is moved in the optical axis direction by the controller 135, a three-dimensional physical property image of the object 160 can be acquired.

[0027] The output signal from the first detection unit 174 can also be subjected to high-sensitivity detection by locking in the signal light 1012 (emitted light 1012) using a lock-in amplifier (not shown) with the pulse repetition frequency signal of the probe light 1021 as a reference input.

[0028] The first detection unit 174 is located on the opposite side of the mounting unit 160 from the first optical system 190. The first detection unit 174 is configured to detect, via the second condenser lens 170, the first emitted light 1032 containing the first wavelength λ1 that is emitted from the object 160 due to the nonlinear optical effect of the first irradiation light 1031c. The second condenser lens 170 is disposed opposite the first condenser lens 153 with the mounting unit 160 interposed therebetween.

[0029] 1-2 Phase Difference Detection System The phase difference detection system of the microscope 100 according to this embodiment will be described with reference to FIG.

[0030] As shown in FIG. 1, the phase difference detection system includes a second optical system 195 including a plurality of optical elements on the side of the second light source 181 that emits second light 1081 of a second wavelength and the mounting portion 160.

[0031] The second optical system 195 includes a second separation unit 172 (dichroic mirror) that is optically coupled to the second light source 181 and that wavelength-selectively transmits the second light 1081. The second optical system 195 also includes a first optical member 171 (ring diaphragm) that partially blocks the luminous flux of the second light 1081 that has transmitted through the separation unit 172, and a second condenser lens that condenses the luminous flux of the second light 1081 and irradiates the object 160 with the luminous flux.

[0032] Here, a white LED or lamp having a plurality of emission wavelengths is adopted as the second light source 181, and the wavelength band of the second light 1081 can be a visible light band, but a monochromatic light source equipped with a wavelength selection filter may also be used. The wavelength of the second light 1081 is preferably different from that of the excitation light 1011 and the probe light 1021, and can be, for example, 450 to 650 nm, which is shorter than that of the excitation light 1011 and the probe light 1021.

[0033] Between the second light source 181 and the second separator 172 (dichroic mirror), an element such as a lens (not shown) is arranged so that the second light 1081 forms Koehler illumination through the second condenser lens 170. The first optical member 171 (ring diaphragm) is arranged at the pupil position of the second condenser lens 170, and partially blocks the luminous flux of the second light 1081 while transmitting the remaining luminous flux. The first optical member 171 (ring diaphragm) has a predetermined transmission area, but does not necessarily have to be ring-shaped.

[0034] Second illumination light 1081c, which is obtained by collecting the remaining light beam of second light 1081, is irradiated onto object 160, and a portion of second illumination light 1081c is diffracted by object 160. Second illumination light 1081c may also be referred to as illumination light 1081c or second illumination light 1081c. Second exit light 1082, which includes scattered light and diffracted light and corresponds to transmitted light that has passed through object 160, is guided to pass through first condenser lens 153. Second exit light 1082 guided via first condenser lens 153 passes through optical element 152 (phase ring). Second optical element 152 (phase ring) is optically conjugate with first optical element 171 (diaphragm ring) via first condenser lens 153 and second condenser lens 170. In order to obtain a conjugate relationship between the first optical element 171 (ring aperture) and the second optical element 152 (phase ring), a position adjustment mechanism may be provided on at least one of the first optical element 171 (ring aperture) and the second optical element 152 (phase ring).

[0035] The second optical member 152 (phase ring) includes a phase film and a neutral density filter having a shape corresponding to that of the first optical member 171 (ring diaphragm). The second emitted light 1082 that passes through the second optical member 152 is transmitted through the first separator 151 (dichroic mirror) and is imaged on the sensor surface of the second detector 186 via the imaging lens 185. The second detector 186 can obtain a phase contrast image corresponding to the refractive index distribution of the object 160. The phase contrast image may also be referred to as a morphological image.

[0036] 1-3 First Optical Member (Aperture Ring) Next, the first optical member 171 (aperture ring), which constitutes a feature of the microscope 100 of this embodiment, will be described with reference to FIG.

[0037] The first optical member 171 (ring diaphragm) is configured to have spectral transmittance characteristics such that it functions as a diaphragm to block a portion of the second light 1081, but does not function as a diaphragm to transmit the signal light 1012 of the first emitted light 1032. In this way, the first optical member 171 has an optical arrangement and spectral transmittance, which ensures signal strength for nonlinear optical observation and enables simultaneous observation with phase contrast observation.

[0038] The first optical member 171 (ring diaphragm) is made of a material having wavelength-selective transmittance in at least a part of the region through which the luminous flux of the second light 1081 passes. For example, when the second light 1081 is visible light and the signal light 1012 of the first emitted light 1032 is near-infrared light, the first optical member 171 (ring diaphragm) can be made of a material having a higher absorption coefficient for visible light than for near-infrared light.

[0039] In addition, the first optical member 171 can be configured to have a ring-shaped dielectric multilayer structure on a substrate made of transparent glass, and to have a higher reflectance for visible light than for near-infrared light.

[0040] Furthermore, the first optical member 171 (ring diaphragm) can be configured so that the transmittance of at least a portion of its area has a predetermined spectral transmittance, similar to the filter 173. That is, the spectral transmittance of a portion of the first optical member 171 (ring diaphragm) is configured to be higher for the excitation light 1011 and the second light 1081 than for the probe light 1021. With this configuration, the signal light 1012, which corresponds to the wavelength of the excitation light 1011 in the first emitted light 1032, can be detected by the first detecting unit 174. For example, the ring diaphragm can be configured by drilling a ring-shaped hole in a bandpass filter corresponding to the wavelength of the excitation light 1011.

[0041] The excitation light 1011 constituting the first light 1031 from the first light source 110 can be considered to have a first wavelength, the second light 1081 from the second light source 181 can be considered to have a second wavelength, and the probe light 1021 constituting the first light 1031 from the first light source 110 can be considered to have a third wavelength.

[0042] In this case, as described above, the first wavelength of the excitation light 1011 is longer than the second wavelength of the second light. In other words, the first light source 110 is configured to emit third light (probe light 1012) including a third wavelength that is different from both the first wavelength corresponding to the excitation light 1011 and the second wavelength corresponding to the second light 1081. The first wavelength can be set to be longer than the third wavelength.

[0043] The first separating unit 151 (dichroic mirror) is an element constituting the first optical system 190, and can be said to be configured to transmit either the first light 1031 or the second emitted light 1082 and reflect the other. In other words, the first separating unit 151 is disposed in a section on the optical path of the first light 1031 from the first light source 110 to the first condenser lens 153 and on the optical path of the second emitted light 1082 from the first optical system 190 to the second detecting unit 186.

[0044] Similarly, the second separating unit 172 (dichroic mirror) is an element constituting the second optical system 195, and can be said to be configured to transmit either the second light 1081 or the first emitted light 1032 and reflect the other. In other words, the second separating unit 172 is disposed in a section on the optical path of the second light 1081 from the second light source 181 to the second condenser lens 170 and on the optical path of the first emitted light 1032 from the second condenser lens 170 to the first detecting unit 174.

[0045] Second Embodiment A microscope 300 according to this embodiment will be described with reference to Fig. 3. The microscope 300 according to this embodiment differs from the microscope 100 according to the first embodiment in the position where the second optical member 152 (phase ring) is provided and in that the microscope 300 includes a relay optical system 301.

[0046] The relay optical system 301 is composed of a lens pair, and relays a position optically conjugate with the first optical member 171 (ring diaphragm) between the first separator 151 (dichroic mirror) and the imaging lens 185. The position optically conjugate with the first optical member 171 (ring diaphragm) corresponds to the position where the second optical member 152 (phase ring) in the first embodiment was provided.

[0047] In this embodiment, the second optical element 152 (phase ring) is positioned optically conjugate with the first optical element 171 (diaphragm ring) formed by the relay optical system 301. As a result, the excitation light 1011 and the probe light 1021 do not pass through the second optical element 152 (phase ring), thereby improving the design flexibility of the second optical element 152 (phase ring). For example, even if a material that absorbs the excitation light 1011 or the probe light 1021 is used for the second optical element 152 (phase ring), this provides the advantage of not affecting nonlinear optical observation. Another advantage is that the second optical element 152 (phase ring) is less susceptible to thermal damage even when the light intensity of the excitation light 1011 or the probe light 1021 constituting the first light 1031 is increased.

[0048] Third Embodiment A microscope 400 according to this embodiment will be described with reference to Fig. 4. The microscope 400 according to this embodiment differs from the microscope 100 according to the first embodiment in that it uses a two-photon excitation light source 410 instead of the first light source 110 in order to use two-photon excitation fluorescence for nonlinear optical observation.

[0049] Excitation light 1041 is emitted from the excitation light source 410, and angular displacement is given in two directions perpendicular to the traveling direction by the scanning unit 130. The excitation light source 410 employs a titanium sapphire laser that emits femtosecond pulsed light having an emission wavelength set to a predetermined wavelength between 800 nm and 1100 nm as the excitation light 1041.

[0050] The excitation light 1041 is irradiated onto the object 160 via a path similar to that of the excitation light 1011 in the microscope 100 of the first embodiment. Here, the object 160 is excited by two-photon absorption, and the resulting fluorescence, signal light 1042, is detected by the first detection unit 174 via a path similar to that of the signal light 1012 of the first emission light 1032 in the first embodiment. A spectral filter 473 is inserted on this path to block the excitation light 1041 component that has passed through the object 160. The output signal of the first detection unit 174 is transmitted to the control unit 135. The angular displacement information of the scanning mirror pair 131, 132 and the output signal are analyzed and imaged in the computer 136. By moving the mounting unit 161 in the optical axis direction and combining this with position information, a three-dimensional image of the object 160 can be constructed.

[0051] The microscope 400 includes an optical system for phase-contrast observation similar to that of the first or second embodiment. The first optical member 171 (ring diaphragm) is configured to block a portion of the second light 1081 so as to function as a diaphragm, and to transmit the signal light 1041 so as not to function as a diaphragm. As a result, the signal strength of the nonlinear optical observation is ensured, and simultaneous observation with phase-contrast observation is possible. It is preferable that the wavelength of the second light 1081 is selected to be different from the wavelength of the signal light 1041.

[0052] 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.

[0053] This application claims priority based on Japanese Patent Application No. 2023-202724, filed November 30, 2023, the entire contents of which are incorporated herein by reference.

[0054] 100 microscope 101 excitation light source 102 probe light source 103 optical delay system 104 mirror 105 dichroic mirror 110 light source unit 130 scanning section 131 scanning mirror 132 scanning mirror 135 control box 136 computer 151 dichroic mirror 152 phase ring 153 first condenser lens 160 sample 161 mounting section 170 second condenser lens 171 ring diaphragm 172 dichroic mirror 173 filter 174 photodetector 181 second light source (illumination light source) 185 imaging lens 186 second detection section (camera) 410 excitation light source 473 filter 1011 excitation light 1021 probe light 1022 signal light 1041 excitation light 1042 signal light 1081 second light (illumination light) 1082 second emitted light (transmitted light)

Claims

1. A mounting section on which an object is placed; a first optical system including a first focusing lens that focuses a first light emitted from a first light source and irradiates the object with a first irradiation light; a first detection section located on the opposite side of the mounting section to the first optical system and detecting a first emission light including a first wavelength emitted from the object due to a nonlinear optical effect caused by the first irradiation light via a second focusing lens arranged opposite the first focusing lens and the mounting section; a second optical system including a second focusing lens arranged opposite the opposite side to the first focusing lens and irradiating the object with a second irradiation light emitted from a second light source and including a second wavelength different from the first wavelength; and a second detection section that detects the second emission light emitted from the object by the second irradiation light via the first focusing lens. the first optical system comprises a first separation unit that transmits either the first light or the second emitted light and reflects the other; the second optical system comprises a second separation unit that transmits either the second light or the first emitted light and reflects the other, and a first optical member that is arranged between the second separation unit and the second focusing lens and has a spectral transmittance at the first wavelength higher than the spectral transmittance at the second wavelength and attenuates a portion of the second light; and a second optical member that is arranged between the second detection unit and the first focusing lens and is arranged in a position optically conjugate with the first optical member.

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

3. A microscope as claimed in claim 1 or 2, wherein the first light source is configured to emit a third light including a third wavelength different from both the first wavelength and the second wavelength.

4. The microscope according to claim 3, wherein the first wavelength is shorter than the third wavelength.

5. A microscope according to claim 3, wherein said first optical member has a spectral transmittance at said third wavelength which is lower than the spectral transmittance at said first wavelength.

6. A microscope according to claim 1 or 2, wherein the second optical member includes a phase film that is conjugate to the shape of the first optical member.

7. A microscope according to claim 1 or 2, wherein the second optical member is disposed between the first separation section and the second detection section.

8. A microscope according to claim 1 or 2, wherein the second optical member is located between the first focusing lens and the first separating portion.

9. A microscope according to claim 1 or 2, wherein the second optical member is located between the first separation section and the second detection section.

10. A microscope according to claim 1 or 2, further comprising a scanning unit that scans the position of the first light beam between the first light source and the second separation unit.

11. A microscope as described in claim 1 or 2, wherein the first separation unit is arranged in a section on the optical path of the first light from the first light source to the first focusing lens, and on the optical path of the second emitted light from the first optical system to the second detection unit.

12. A microscope as described in claim 1 or 2, wherein the second separation unit is arranged in a section on the optical path of the second light from the second light source to the second focusing lens and on the optical path of the first emitted light from the second focusing lens to the first detection unit.

13. The microscope of claim 1 or 2, further comprising the first light source.

14. The microscope of claim 1 or 2, further comprising said second light source.

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