Microscope

Abstract

To provide a microscope that allows a user to use a nonlinear optical observation method, while holding a diaphragm used in a phase difference observation method near a sample.SOLUTION: A microscope has an optical system for a phase difference observation and an optical system for nonlinear optical observation arranged opposite to each other with an object therebetween, and comprises: a ring diaphragm that functions as a diaphragm for primary light for phase difference observation, and transmits secondary light including a nonlinear optical effect from the object; and a phase ring that is arranged at a position optically conjugate with the ring diaphragm.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a microscope.

Background Art

[0002] As a method for observing the morphology of a sample such as a cell with poor contrast in bright-field observation method, etc., the phase-contrast observation method is known. In the phase-contrast observation method, a donut-shaped aperture (ring aperture) that partially shields illumination is placed at the pupil position of the condenser, and a ring-shaped phase film (phase ring) in a conjugate relationship with the aperture is placed at the pupil position of the objective lens.

[0003] Also, a method is known in which by utilizing the non-linear optical effect and detecting scattered light including the non-linear response of the sample to the intensity of the irradiation light, the distribution of molecular vibrations etc. contained in the sample is obtained. Examples using the linear optical effect include multi-photon excitation fluorescence, second harmonic generation, coherent anti-Stokes Raman scattering, and induced Raman scattering.

[0004] A microscope having an optical system that combines these methods and shares a part of the illumination and detection optical paths before and after the sample is disclosed in, for example, Patent Document 1.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] When the optical path of the phase difference observation method and the observation method using the non-linear optical effect (hereinafter referred to as the non-linear optical observation method) is shared before and after the sample, the aperture used in the former blocks the illumination light or scattered light of the latter, resulting in a decrease in the signal amount and efficiency. To solve this problem, Patent Document 1 proposes creating a position conjugate to the pupil of the condenser by a relay optical system, placing an aperture there, and branching the optical paths of the phase difference observation method and the non-linear optical observation method between the sample and the aperture. However, there is a problem that the optical system becomes large in the relay optical system described above.

[0007] That is, an object of the present invention is to provide a microscope including an optical system that can simultaneously acquire a phase difference image by a phase difference method and a non-linear optical image by a non-linear optical method and is miniaturized.

Means for Solving the Problems

[0008] The microscope according to an embodiment of the present invention includes a placement unit on which an object is placed, a first optical system including a first condenser lens that condenses the first light emitted from the first light source and irradiates the object with the first irradiation light, a first detection unit that is located on the opposite side of the first optical system with the placement unit interposed therebetween and detects the first emitted light including the first wavelength emitted from the object due to the non-linear optical effect of the first irradiation light through a second condenser lens disposed opposite to the first condenser lens with the placement unit interposed therebetween, a second optical system including a second condenser lens disposed opposite to the opposite side of the first condenser lens and irradiating the object with the second irradiation light including the second wavelength different from the first wavelength emitted from the second light source, a second detection unit that detects the second emitted light emitted from the object by the second irradiation light through the first condenser lens, the first optical system includes a first separation unit that transmits one of the first light and the second emitted light and reflects the other, The second optical system includes a second separation unit that transmits one of the second light and the first emitted light and reflects the other, and a first optical member that is disposed between the second separation unit and the second condenser lens and has a spectral transmittance at the first wavelength higher than that at the second wavelength to attenuate a part of the second light. It includes a second optical member that is disposed between the second detection unit and the first condenser lens and is disposed at a position optically conjugate to the first optical member.

Advantages of the Invention

[0009] According to the present invention, it is possible to provide a microscope including an optical system that can simultaneously acquire a phase difference image by the phase difference method and a non-linear optical image by the non-linear optical method and is miniaturized.

Brief Description of the Drawings

[0010]

Figure 1

Figure 2

Figure 3

Figure 4

Modes for Carrying Out the Invention

[0011] <The First Embodiment> The microscope 100 according to this embodiment is a multimodal microscope capable of acquiring a physical property image including induced Raman scattered light in a common region of interest of a common sample so that an observer can use a non-linear optical observation method together with a morphological image obtained by the phase contrast observation method. The non-linear optical inspection method provided by the microscope of this embodiment can also be applied to non-linear optical scattering other than induced Raman scattering. Non-linear optical scattered light other than induced Raman scattered light includes multi-photon excited fluorescence, second harmonic generation light, coherent anti-Stokes Raman scattered light, and the like. FIG. 1 is a diagram showing a schematic configuration of the microscope 100 according to the first embodiment. The physical property image provided by the microscope 100 of this embodiment includes a molecular vibration spectrum image and a distribution of atomic bonds in the object, and the objects include organic substances and biological substances.

[0012] 1-1 Non-linear optical light detection system The first light source related to the non-linear optical detection system of the microscope 100 according to this embodiment will be described with reference to FIG. 2.

[0013] The first light source 110 includes an excitation light source 101 that emits excitation light 1011, and a dichroic mirror 105 that transmits the excitation light 1011 in a wavelength-selective manner. Here, pulsed light is adopted for the excitation light 1011 in order to efficiently cause a non-linear optical process. The pulse width of the excitation light 1011, which is pulsed light, is on the order of femtoseconds to picoseconds. Also, the pulse repetition frequency may be 1 MHz or more from the viewpoint of mitigating the influence of intensity fluctuations of the excitation light 1011. When a titanium sapphire laser oscillating in the near-infrared band is adopted 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 preferably different by approximately the same amount as the wavelength corresponding to the Raman shift (wave number) of the molecular information of the object. When a titanium sapphire laser is adopted for the excitation light source 101, a probe light source 102 whose wavelength of the probe light 1021 can be swept between 1000 nm and 1100 nm is adopted. Such a probe light source 102 includes optical fiber lasers such as Yb fiber lasers and Er fiber lasers. Also, the pulse repetition frequency of the probe light 1021 is set to be 1 / 2 of the pulse repetition frequency of the excitation light 1011, so that due to the nonlinear optical effect of the object, the probe light 1021 can be modulated at the same frequency as the pulse repetition frequency of the probe light 1021 with respect to the signal light 1012. However, the pulse repetition frequencies of the excitation light 1011 and the probe light 1021 may be made to coincide, and intensity modulation may be applied to the excitation light 1011 or the probe light 1021.

[0015] The probe light 1021 passes through the optical delay system 103 and the mirror 104, is reflected by the dichroic mirror 105, and is combined coaxially with the excitation light 1011. When combining at 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 temporally.

[0016] The scanning unit 130 for scanning the first irradiation light 1031c, which is the first light 1031 including the excitation light 1011 and the probe light 1021 from the first light source 110 and is further condensed by a condenser lens, on the object 160 will be described.

[0017] The scanning unit 130 includes a pair of scanning mirrors 131 and 132 to which angular displacements are given in two directions orthogonal to the traveling directions of the excitation light 1011 and the probe light 1021, respectively. As the pair of scanning mirrors 131 and 132, a galvanometer scanner, a resonant scanner, or a polygon scanner is employed. The pair of scanning mirrors 131 and 132 is controlled by the control unit 135 and is configured to output angular displacement information to the control unit 135.

[0018] The first optical system 190 related to the non-linear optical observation system of the microscope 100 according to the present embodiment will be described with reference to FIG. 1.

[0019] The first light 1031 (excitation light 1011 and probe light 1021) to which the angular displacement is given is reflected by the first separation unit 151 (dichroic mirror) and passes through the second optical member 152 (phase ring) as shown in FIG. 1. The first light 1031 (excitation light 1011 and probe light 1021) incident on the first condenser lens 153 through the second optical member 152 (phase ring) is condensed by the first condenser lens 152 toward the object 160 placed on the placement unit 161.

[0020] By providing a movable stage coupled to the placement 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 placement unit 161. The placement unit 161 is communicably connected to the control unit 135, outputs the position information of the placement unit 160 to the control unit 135, and the control unit 135 can move the placement unit via a movable stage (not shown).

[0021] Next, the second optical system 195 related to the non-linear optical observation system of the microscope 100 according to the present embodiment will be described with reference to FIG. 1.

[0022] When the Raman shift due to the molecular vibration included in the object 160 coincides with the wave number difference between the excitation light 1011 and the probe light 1021, the intensity of the excitation light 1011 slightly decreases and the intensity of the probe light 1021 slightly increases due to the induced Raman scattering effect.

[0023] Hereinafter, an example in which the emission light 1012 (second-order light) having the same wavelength as the excitation light 1011 among the first emission light 1032 modulated by the stimulated Raman scattering effect 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 adopting a modified form (not shown) in which the emission light 1022 (second-order light) having the same wavelength as the probe light 1021 among the first emission light 1032 is used as the signal light 1022.

[0024] The excitation light 1011 and the signal light 1012 among the first emission light 1032 generated by the non-linear optical effect travel as parallel light having an angular displacement by the second condenser lens 170. The signal light 1012 passes through the first optical member 171 (ring aperture) and is wavelength-selectively reflected by the second separation unit 172 (dichroic mirror).

[0025] The emission light 1012 (second-order light) reflected by the second separation unit 172 (dichroic mirror) passes through a spectroscopic 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 detection unit 174. The emission light 1012 is detected as the signal light 1012 by the first detection unit 174.

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

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

[0028] The first detection unit 174 is located on the side opposite to the first optical system 190 with the placement unit 160 interposed therebetween. Further, the first detection unit 174 is configured to detect the first emitted light 1032 including the first wavelength λ1 emitted from the object 160 due to the non-linear optical effect by the first irradiation light 1031c via the second condenser lens 170. The second condenser lens 170 is disposed opposite to the first condenser lens 153 with the placement unit 160 interposed therebetween.

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

[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 the second light 1081 of the second wavelength and the placement unit 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 transmits the second light 1081 in a wavelength-selective manner. Further, the second optical system 195 further includes a first optical member 171 (ring aperture) that partially shields the light beam of the second light 1081 that has passed through the separation unit 172, and a second condenser lens that condenses the light beam of the second light 1081 and irradiates the object 160.

[0032] Here, as the second light source 181, a white LED or a lamp including a plurality of emission wavelengths may be employed. The wavelength band of the second light 1081 may use the visible light band, but it may also be a monochromatic light source provided with a wavelength selection filter. The wavelength of the second light 1081 is preferably different from the excitation light 1011 and the probe light 1021. For example, it can be set to 450 to 650 nm on the shorter wavelength side than the excitation light 1011 and the probe light 1021.

[0033] Between the second light source 181 and the second separation unit 172 (dichroic mirror), elements such as a lens (not shown) are arranged so that the second light 1081 constitutes Köhler illumination by the second condenser lens 170. The first optical member 171 (ring aperture) is disposed at the pupil position of the second condenser lens 170, and a part of the light beam of the second light 1081 is shielded and the remaining part of the light beam is transmitted. The first optical member 171 (ring aperture) is provided with a predetermined transmission region, but it does not necessarily have to be ring-shaped.

[0034] The remaining part of the light beam of the second light 1081 is condensed into the second irradiation light 1081c, which is irradiated onto the object 160, and a part of the second irradiation light 1081c is diffracted by the object 160. The second irradiation light 1081c may be referred to as illumination light 1081c or the second illumination light 1081c. The second emitted light 1082, which includes scattered light and diffracted light and corresponds to the transmitted light that has passed through the object 160, is guided so as to pass through the first condenser lens 153. The second emitted light 1082 guided through the first condenser lens 153 passes through the optical member 152 (phase ring). The second optical member 152 (phase ring) is optically conjugate to the first optical member 171 (ring aperture) via the first condenser lens 153 and the second condenser lens 170. In order to obtain the conjugate relationship between the first optical member 171 (ring aperture) and the second optical member 152 (phase ring), a position adjustment mechanism may be provided in at least one of the first optical member 171 (ring aperture) and the second optical member 152 (phase ring).

[0035] The second optical member 152 (phase ring) includes a phase film having a shape corresponding to the first optical member 171 (ring aperture) and a light attenuation filter. The second emitted light 1082 that has passed through the second optical member 152 passes through the first separation unit 151 (dichroic mirror) and is imaged on the sensor surface of the second detection unit 186 via the imaging lens 185. The second detection unit 186 can obtain a phase difference contrast image corresponding to the refractive index distribution of the object 160. The phase difference contrast image may be referred to as a morphological image.

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

[0037] The first optical member 171 (ring aperture) is configured to have spectral transmittance characteristics such that it shields some light so as to function as an aperture for the second light 1081 and transmits without functioning as an aperture for the signal light 1012 among the first emitted light 1032. By having such optical arrangement and spectral transmittance, the first optical member 171 secures the signal intensity of non-linear optical observation and enables simultaneous observation with phase contrast observation.

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

[0039] Also, the first optical member 171 can be configured by forming a ring-shaped dielectric multilayer film structure on a substrate made of transparent glass so that the reflectance for visible light is higher than that for near-infrared light.

[0040] Also, the first optical member 171 (ring aperture) can be configured such that the transmittance of at least a part of its region has a predetermined spectral transmittance, similar to the filter 173. That is, the spectral transmittance of a part of the first optical member 171 (ring aperture) is made higher for the excitation light 1011 and the second light 1081 than for the probe light 1021. By configuring in this way, the signal light 1012 corresponding to the wavelength of the excitation light 1011 among the first emitted light 1032 can be detected by the first detection unit 174. For example, a ring aperture can be configured by drilling a ring-shaped hole in a band-pass filter corresponding to the wavelength of the excitation light 1011.

[0041] The excitation light 1011 that constitutes the first light 1031 from the first light source 110 exhibits a first wavelength, the second light 1081 from the second light source 181 exhibits a second wavelength, and the probe light 1021 that constitutes the first light 1031 from the first light source 110 can be regarded as exhibiting a third wavelength.

[0042] At this time, as described above, the first wavelength of the excitation light 1011 is longer than the second wavelength of the second light. Also, it can be rephrased that the first light source 110 is configured to emit a 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. Further, the first wavelength can be set to be longer than the third wavelength.

[0043] The first separation unit 151 (dichroic mirror) is an element that constitutes the first optical system 190 and is configured to transmit one of the first light 1031 and the second emitted light 1082 and reflect the other. Also, it can be rephrased that the first separation unit 151 is arranged in a section that is on the optical path of the first light 1031 from the first light source 110 to the first condenser lens 153 and is also on the optical path of the second emitted light 1082 from the first optical system 190 to the second detection unit 186.

[0044] Similarly, the second separation unit 172 (dichroic mirror) is an element that constitutes the second optical system 195 and is configured to transmit one of the second light 1081 and the first emitted light 1032 and reflect the other. Also, it can be rephrased that the second separation unit 172 is arranged in a section that is on the optical path of the second light 1081 from the second light source 181 to the second condenser lens 170 and is also on the optical path of the first emitted light 1032 from the second condenser lens 170 to the first detection unit 174.

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

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

[0047] In this embodiment, the second optical member 152 (phase ring) is placed at a position optically conjugate with the first optical member 171 (ring aperture) formed by the relay optical system 301. As a result, since the excitation light 1011 and the probe light 1021 do not pass through the second optical member 152 (phase ring), the design freedom of the second optical member 152 (phase ring) is improved. For example, it provides the advantage that even if a material that absorbs the excitation light 1011 or the probe light 1021 is used for the second optical member 152 (phase ring), it does not affect non-linear optical observation. Also, even if the light intensity of the excitation light 1011 or the probe light 1021 that constitutes the first light 1031 is increased, it provides the advantage that the second optical member 152 (phase ring) is hardly thermally destroyed.

[0048] <Third Embodiment> The microscope 400 of this embodiment will be described with reference to FIG. 4. The microscope 400 of this embodiment is different from the microscope 100 according to the first embodiment in that a two-photon excitation light source 410 is used instead of the first light source 110 because two-photon excitation fluorescence is used as non-linear optical observation.

[0049] Excitation light 1041 is emitted from the excitation light source 410 and is angularly displaced in two directions orthogonal to the traveling direction by the scanning unit 130. As the excitation light source 410, a titanium sapphire laser that emits femtosecond pulsed light with a predetermined wavelength set between 800 nm and 1100 nm in terms of the emission wavelength of the excitation light 1041 is adopted.

[0050] In the same path as the excitation light 1011 of the microscope 100 of the first embodiment, the excitation light 1041 irradiates the object 160. Here, the object 160 is excited by two-photon absorption, and the signal light 1042, which is the fluorescence thereof, is detected by the first detection unit 174 in the same path as the signal light 1012 among the first emitted lights 1032 in the first embodiment. A spectroscopic 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 together in the computer 136. By moving the placement unit 161 in the optical axis direction and combining the position information thereof, a three-dimensional image of the object 160 can be constructed.

[0051] The microscope 400 includes an optical system for phase difference observation similar to that of the first embodiment or the second embodiment. The first optical member 171 (ring aperture) is configured to block some light so as to function as an aperture for the second light 1081 and transmit light so as not to function as an aperture for the signal light 1041. As a result, the signal intensity of the non-linear optical observation is ensured, and simultaneous observation with the phase difference observation becomes 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.

Explanation of Reference Numerals

[0052] 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 Unit 131 Scanning Mirror 132 Scanning Mirror 135 Control Box 136 Computer 151 Dichroic Mirror 152 Phase Ring 153 First Condensing Lens 160 Sample 161 Mounting Section 170 Second Condensing Lens 171 Ring Aperture 172 Dichroic Mirror 173 Filter 174 Photodetector 181 Second Light Source (Illumination Light Source) 185 Imaging Lens 186 Second Detection Unit (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 placement section on which an object is placed, a first optical system including a first condenser lens that condenses first light emitted from a first light source and irradiates the object with first irradiation light, a first detection unit that is located on the side opposite to the first optical system with the placement section interposed therebetween, and detects first emission light including a first wavelength emitted from the object due to a nonlinear optical effect of the first irradiation light, through a second condenser lens that is disposed to face the first condenser lens with the placement section interposed therebetween, a second optical system including a second condenser lens that is disposed to face the side opposite to the first condenser lens, and irradiates the object with second irradiation light including a second wavelength different from the first wavelength, which is emitted from a second light source, a second detection unit that detects second emission light emitted from the object due to the second irradiation light, through the first condenser lens, the first optical system includes a first separation unit that transmits one of the first light and the second emission light and reflects the other, the second optical system includes a second separation unit that transmits one of the second light and the first emission light and reflects the other, and a first optical member that is disposed between the second separation unit and the second condenser lens, and has a spectral transmittance at the first wavelength higher than a spectral transmittance at the second wavelength and attenuates a part of the second light, a second optical member that is disposed between the second detection unit and the first condenser lens and is disposed at a position optically conjugate with the first optical member, a microscope comprising the same.

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

3. The microscope according to claim 1 or 2, wherein the first light source is configured to emit 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. The microscope according to claim 3, wherein the first optical member has a spectral transmittance at the third wavelength lower than a spectral transmittance at the first wavelength.

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

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

8. The microscope according to claim 1 or 2, wherein the second optical member is located between the first condenser lens and the first separation unit.

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

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

11. The microscope according to claim 1 or 2, wherein the first separation unit is arranged in a section that is on the optical path of the first light from the first light source to the first condenser lens and is also on the optical path of the second emitted light from the first optical system to the second detection unit.

12. The microscope according to claim 1 or 2, wherein the second separation unit is arranged in a section that is on the optical path of the second light from the second light source to the second condenser lens and is also on the optical path of the first emitted light from the second condenser lens to the first detection unit.

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

14. The microscope according to claim 1 or 2, further comprising the second light source.

Images