JP7006010B2 - microscope - Google Patents

Description

The present invention relates to a microscope.

As a method of observing living cells without damage, there is a method of detecting CARS light (coherent anti-Stoke Raman scattered light) (see, for example, Patent Document 1).
Patent Document 1 Japanese Patent Application Laid-Open No. 2005-062155

Observation with a scanning microscope, for example, observation with a microscope utilizing a nonlinear optical effect, takes time to obtain an observed image. Further, in the observation using the nonlinear optical effect, when the optical member is inserted into the path of the irradiation light to the observation target and the emission light from the observation target, the detection efficiency is lowered and the observation takes more time. ..

According to one aspect of the present invention, a pair of objective lenses arranged so as to sandwich an observation object and having a common focus on a region overlapping the observation object, and a coherent first irradiation light of the pair of objective lenses. The first irradiation unit that irradiates the observation object through one of them, and the first emission light emitted by the non-linear optical effect from the observation object irradiated with the first irradiation light are detected through one lens. Of the pair of objective lenses, the detection unit 1 and the second emission light emitted from the observation object irradiated with the first irradiation light by a non-linear optical effect and propagating in an optical path different from the first emission light are emitted from the pair of objective lenses. A microscope comprising a second detector for detecting through the other lens of the light is provided.

The outline of the above invention does not list all the features of the present invention. Sub-combinations of these features can also be inventions.

It is a schematic diagram of a microscope 100. It is a schematic diagram explaining the dark field observation by the microscope 100. It is a schematic diagram explaining CARS light observation with a microscope 100. It is a figure which shows the region of the spatial frequency which can be decomposed by the observation by the reflected CARS light. It is a figure which shows the region of the spatial frequency which can be decomposed by the observation by the transmitted CARS light. It is a schematic diagram explaining the generation principle of CARS light. It is a flow chart which shows the observation procedure using a microscope 100. It is a schematic diagram which shows the distribution of the preliminary observation area 117. It is a schematic diagram which shows the distribution of a detailed observation area 119. It is a schematic diagram which illustrates the distribution of the preliminary observation area 117. It is a schematic diagram which illustrates the distribution of a detailed observation area 119. It is a schematic diagram of a microscope 100. It is a schematic diagram of a microscope 100. It is a schematic diagram of a microscope 100. It is a schematic diagram of a microscope 101. It is a schematic diagram of a microscope 102.

Although the present invention will be described through embodiments of the invention, the following embodiments are not intended to limit the invention in the claims. Not all combinations of features described in the embodiments below are essential to the solution of the invention.

FIG. 1 is a schematic view of a microscope 100 which is an example of an observation device. The microscope 100 includes a stage 110, a control unit 120, an illumination unit 130, an objective optical system 140, an image detection unit 150, a laser device 160, an excitation unit 170, an upper CARS light detection unit 180, and a lower CARS light detection unit 190. ..

In the microscope 100, the illumination unit 130 forms the first irradiation unit, and the laser device 160 forms the second irradiation unit. Further, the image detection unit 150 forms a first detection unit, the upper CARS light detection unit 180 forms a second detection unit, and the lower CARS light detection unit 190 forms a third detection unit.

The stage 110 supports the sample 112 to be observed by the microscope 100. As a result, the sample 112 placed on the stage 110 can be irradiated with the illumination light from the illumination unit 130 from above in the drawing. Further, the sample 112 placed on the stage 110 can be observed from the upper side in the figure by the upper CARS photodetector 180.

The stage 110 has an opening that exposes the lower surface of the sample 112 in the drawing. As a result, the sample 112 placed on the stage 110 can be irradiated with the laser beam from the lower side in the figure by the laser device 160 and the excitation unit 170. Further, the sample 112 placed on the stage 110 can be observed from the lower side in the figure by the image detection unit 150 and the lower CARS light detection unit 190.

Further, the stage 110 is coupled to the stage scanner 111. The stage scanner 111 moves the stage 110 in parallel with the surface on which the sample 112 is placed, as shown by arrows xy in the figure. As a result, in the microscope 100, a wide range of the sample 112 can be observed while the optical axis of the optical system is fixed.

The control unit 120 includes a processing device 122, a keyboard 124, a mouse 126, and a display unit 128. The processing device 122 can be formed by implementing a program for executing a control procedure on a general-purpose personal computer.

The keyboard 124 and the mouse 126 are connected to the processing device 122 and are operated when inputting a user's instruction to the processing device 122. The display unit 128 returns feedback to the user's operation by the keyboard 124 and the mouse 126, and displays the image or character string generated by the processing device 122 toward the user.

The control unit 120 generates an image to be displayed on the display unit 128 based on the detection results of the image detection unit 150, the upper CARS light detection unit 180, and the lower CARS light detection unit 190. Further, the control unit 120 is coupled to each unit of the stage scanner 111, the illumination unit 130, the laser device 160, and the excitation unit 170, and controls these operations according to the instructions received from the user.

The illumination unit 130 includes an illumination light source 131, a collector lens 132, relay lenses 133, 135, 138, a ring diaphragm 134, and a field diaphragm 136. The field diaphragm 136 is arranged at the position P ₁₂ conjugate to the focal position P ₁₁ of the objective optical system 140. The position of the ring diaphragm 134 will be described later. The illumination light source 131 generates white incoherent illumination light by, for example, a halogen lamp or the like. The illumination light collected by the collector lens 132 passes through only the light in the visible light band by the band passing filter 139 via the relay lens 133, 138, the ring diaphragm 134, and the field diaphragm 136, and irradiates the sample 112.

The objective optical system 140 has an upper objective lens 142 and a lower objective lens 144 arranged on opposite sides of the stage 110. The upper objective lens 142 on the upper side in the figure collects the illumination light emitted from the illumination unit 130 on the sample 112 placed on the stage 110. Therefore, the upper objective lens 142 is also called a condenser lens. Even a capacitor lens for lighting that is not aberration-corrected for use as an objective lens may be used as an upper objective lens 142, for example. Further, the lower objective lens 144 on the lower side in the figure condenses the excitation light emitted from the excitation unit 170 inside the sample 112.

In the objective optical system 140, the upper objective lens 142 and the lower objective lens 144 preferably have substantially equal numerical apertures. As a result, when observing the coherent emission light generated by irradiating the sample 112 with the coherent irradiation light, it is possible to prevent the artifact from being superimposed on the image or the spectrum and the detection accuracy from being lowered. More specifically, the ratio R _NA of the numerical aperture of the upper objective lens 142 and the numerical aperture of the lower objective lens 144 preferably satisfies the right equation [0.8 <R _NA <1.2].

The image detection unit 150 includes a dichroic mirror 151, an imaging lens 152, a relay lens 154, a bandpass filter 155, an aperture diaphragm 156, a relay lens 157, and a CCD camera 158. The dichroic mirror 151 reflects the light in the visible light band among the light emitted from the sample 112, and transmits the light in the wavelength band emitted from the laser light sources 161 and 162 to the sample 112. As a result, of the illumination light emitted by the illumination unit 130 to the sample 112, the illumination light transmitted or scattered in the sample 112 is introduced into the image detection unit 150.

The bandpass filter 155 transmits only the light in the wavelength band of the illumination light irradiated to the sample 112 by the illumination unit 130, and blocks the light of other wavelengths. The image forming lens 152 forms an image due to the illumination light scattered by the sample 112 on the primary image plane 153. Further, the relay lenses 154 and 157 reimage the image on the imaging surface of the CCD camera 158. Since the CCD camera 158 has sensitivity in the band of illumination light, the reimaged image is imaged as a dark field observation image.

The laser device 160 has a plurality of laser light sources 161 and 162 and a combiner 163. The laser light source 161 individually generates a pulsed laser having an emission wavelength λ _S , and the laser light source 162 individually generates a pulse laser having an emission wavelength λ _P. The emission wavelengths λ _S and λ _P of the laser light sources 161 and 162 are different wavelengths from each other. Further, the emission wavelengths λ _S and λ _P of the laser light sources 161 and 162 are also different from the wavelength band transmitted by the band pass filter 139.

As the laser light sources 161 and 162, for example, a mode-lock picosecond Nd: _YVO4 laser, a mode-lock picosecond itribium laser, or the like can be used. One of the laser light sources 161 and 162 may be replaced with an optical parametric oscillator that converts the wavelength of the pulsed laser generated by the other of the laser light sources 161 and 162.

Of the picosecond pulses generated by the laser light sources 161 and 162, the pulse laser having the shorter wavelength λ _P is used as the pump light in CARS light observation. Further, among the picosecond pulses generated by the laser light sources 161 and 162, the pulse laser having the longer wavelength λ _S is used as Stokes light in CARS light observation.

The laser light emitted from the laser light sources 161 and 162 is incident on the combiner 163 to form a single beam. As a result, the sample 112 can be irradiated with excitation light that is a combination of pump light and Stokes light to generate CARS light.

The laser device 160 may be provided with a delayed optical path for the purpose of synchronizing the pump light generated by the laser light sources 161 and 162 with the Stokes light. The delayed optical path can be formed by a plurality of reflectors whose distances from each other can be changed. Further, in the laser apparatus 160, the Stokes light may be widened by using a photonic crystal fiber.

The excitation unit 170 has a galvano scanner 171 and a scan lens 172. The galvano scanner 171 includes a reflecting mirror that swings around two axes having different directions from each other, and two-dimensionally displaces the optical path of the incident irradiation light in a direction intersecting the optical axis.

The scan lens 172 focuses the irradiation light emitted from the galvano scanner 171 on a predetermined primary image plane 173. As a result, the observation area of the sample 112 can be scanned by the irradiation light emitted from the laser device 160, and the irradiation light can be irradiated to the observation area having a predetermined area.

Each of the upper CARS photodetector 180 and the lower CARS photodetector 190 has a dichroic mirrors 181, 191 and relay lenses 182, 192, 183, 193, passband filters 184, 194 and a photomultiplier tube 185, 195, respectively. .. The dichroic mirrors 181 and 191 transmit the light in the visible light band generated by the illumination unit 130 as the illumination light, transmit the excitation light from the excitation unit 170, and reflect the CARS light generated in the sample 112. As a result, the dichroic mirrors 181 and 191 introduce the CARS light generated in the sample 112 into the upper CARS photodetector 180 or the lower CARS photodetector 190.

Further, the band-passing filters 184 and 194 transmit CARS light and block light having a wavelength other than CARS light, including illumination light from the illumination unit 130 and excitation light from the excitation unit 170. As a result, the photomultiplier tubes 185 and 195 can selectively detect CARS light. Therefore, in the microscope 100, CARS light observation and dark field observation can be performed at the same time.

In the illumination unit 130, a reflector 137 is arranged between the field diaphragm 136 and the relay lens 138. Further, also in the excitation unit 170, a reflecting mirror 174 is arranged between the scan lens 172 and the imaging lens 175. The reflectors 137 and 174 prevent the structure of the microscope 100 from becoming excessively high by bending the optical path of the illumination light or the excitation light.

The microscope 100 as described above can be used for dark field observation using the illumination unit 130 and the image detection unit 150 for the sample 112. The microscope 100 can also be used for CARS light observation using the laser device 160, the excitation unit 170, the upper CARS photodetector 180, and the lower CARS photodetector 190.

Further, in the microscope 100, the reflecting mirrors 137 and 174 of the illumination unit 130 and the excitation unit 170, and the dichroic mirrors 151, 181 and 191 of the image detection unit 150, the upper CARS light detection unit 180 and the lower CARS light detection unit 190. Are all arranged on the optical axis Q of the objective optical system 140. Therefore, the objective optical system 140 is commonly used for both dark field observation and CARS light observation.

Here, in the microscope 100, the ring diaphragm 134 forming the illumination light for dark field observation is not between the upper CARS light detection unit 180 and the upper objective lens 142, but a relay optical system including relay lenses 138 and 135. It is arranged in the lighting unit 130 through the interposition. That is, the ring diaphragm 134 is arranged at the position P ₂ optically conjugate with the position P ₁ of the exit pupil of the upper objective lens 142.

Then, the aperture diaphragm 156 forming the dark field observation image is not between the lower objective lens 144 and the lower CARS light detection unit 190, but is via a relay optical system including an imaging lens 152 and a relay lens 154. It is arranged in the image detection unit 150. That is, the aperture stop 156 is arranged at the position P ₄ optically conjugate with the position P ₃ of the exit pupil of the lower objective lens 144.

Further, the dichroic mirror 181 of the upper CARS light detection unit 180 is arranged between the upper objective lens 142 and the illumination unit 130, and the dichroic mirror 191 of the lower CARS light detection unit 190 is the lower objective lens 144 and image detection. It is arranged between the unit 150 and the unit 150.

As shown by the arrow M in the figure, the ring diaphragm 134 can be moved in a direction parallel to the optical axis Q. Further, the optical member G ₁ shown by the dotted line in the drawing can be inserted and removed between the ring diaphragm 134 and the relay lens 135. As the optical member G ₁ , a lens, parallel flat glass, or the like is used. As a result, in the microscope 100, the optical path length between the upper objective lens 142 and the ring diaphragm 134 can be changed. In particular, when the optical member G1 is inserted or removed, the image formation relationship between the ring diaphragm ₁₃₄ and the illumination light source 131 required for Koehler illumination can be maintained even if the optical path length between the upper objective lens 142 and the ring diaphragm 134 is significantly changed. can.

Therefore, for example, when the upper objective lens 142 is moved up and down to change the distance from the relay lens 138 for focus adjustment, the position of the ring diaphragm 134 is moved in a direction parallel to the optical axis Q to move the upper objective. Adjust the optical path length between the lens 142 and the ring diaphragm 134.

Further, when different optical characteristics such as observation magnification are required, or when the upper objective lens 142 comes into contact with the sample 112 or the like and is contaminated, the upper objective lens 142 can be used as another objective lens having a different magnification or number of openings. When replaced, the position of the exit pupil position P1 of the _upper objective lens 142 may change significantly, and the appropriate ring size of the ring aperture 134 may also change. In that case, replace the ring diaphragm ₁₃₄ with an appropriate size, and add or replace the optical member G1 to roughly match the optical path length between the upper objective lens 142 and the ring diaphragm 134. After that, the position of the ring diaphragm 134 is moved in the direction parallel to the optical axis Q, and the optical path length between the upper objective lens 142 and the ring diaphragm 134 is finely adjusted.

At this time, the ring diaphragm 134 and the optical member G ₁ may be integrally configured as a replaceable ring diaphragm block. In that case, the upper objective lens 142 can be replaced at once with an appropriate ring aperture block, which simplifies the operation and is automatically linked to the replacement of the upper objective lens 142. It is convenient because it is easy to simplify the exchange mechanism even when the mechanism is to be exchanged.

To adjust the position of the ring diaphragm 134, the user may move the ring diaphragm 134 to a position where the observed image becomes clearest without leaking the direct light of the illumination light while observing the sample 112 in the dark field. Further, a mechanism may be provided for evaluating the contrast of the dark field image and moving the ring diaphragm 134 to a position where the contrast is highest. By making the position of the ring diaphragm 134 adjustable in this way, the position of the ring diaphragm 134 is accurately aligned with the position P ₁ of the entrance pupil of the upper objective lens 142 and the position P ₂ optically coupled to the dark field. It is possible to prevent a decrease in resolution as a field microscope.

Further, the aperture stop 156 can also be moved in a direction parallel to the optical axis Q. Further, the optical member G ₂ shown by the dotted line in the drawing can be inserted and removed between the aperture stop 156 and the relay lens 154. As the optical member G ₂ , a lens, parallel flat glass, or the like is used. As a result, in the microscope 100, the optical path length between the lower objective lens 144 and the aperture diaphragm 156 can be changed. In particular, when the optical member G ₂ is inserted or removed, the optical path length between the aperture aperture 156 and the relay lens 157 can be maintained even if the optical path length between the lower objective lens 144 and the aperture stop 156 is significantly changed. At 158, the emission light from the sample 112 can be appropriately received.

Therefore, for example, when the lower objective lens 144 is moved up and down to change the distance from the imaging lens 152, for example, the position of the aperture diaphragm 156 is moved in a direction parallel to the optical axis Q. The optical path length between the lower objective lens 144 and the aperture stop 156 is adjusted.

Further, when different optical characteristics such as observation magnification are required, or when the lower objective lens 144 comes into contact with the sample 112 or the like and is contaminated, the lower objective lens 144 is used as another objective having a different magnification or number of openings. When replaced with a lens _, the position of the exit pupil position P3 of the lower objective lens 144 may change significantly, and the appropriate opening size of the aperture aperture 156 may also change. In that case, the opening of the aperture diaphragm 156 may be changed to an appropriate size, the aperture diaphragm of an appropriate size may be replaced, or the optical member G2 may be added or replaced, and the _lower objective lens 144 may be replaced. Approximately match the optical path length between the aperture stop 156 and the aperture stop 156, and then move the position of the aperture stop 156 in the direction parallel to the optical axis Q to adjust the optical path length between the lower objective lens 144 and the aperture stop 156. Make fine adjustments.

At this time, the aperture stop 156 and the optical member G ₂ may be integrally configured as a replaceable aperture stop block. In that case, the lower objective lens 144 can be replaced at once with an appropriate aperture stop block, which simplifies the operation and is linked to the replacement of the lower objective lens 144. It is convenient because it is easy to simplify the exchange mechanism even when the mechanism is automatically exchanged.

The replacement of the ring diaphragm block and the aperture diaphragm block is automatically performed by the electric mechanism together with the replacement of the upper objective lens 142 and the lower objective lens 144, which minimizes the load on the operator who operates the microscope 100. It is convenient.

With such an arrangement of the ring diaphragm 134 and the aperture diaphragm 156, in the microscope 100, the ring diaphragm 134 is excluded from the optical path between the upper CARS photodetector 180 on the upper side in the drawing and the upper objective lens 142. Further, the aperture stop 156 is excluded from the optical path from the lower CARS photodetector 190 on the lower side in the figure to the lower objective lens 144.

In the microscope 100, for example, when the combination of wavelength characteristics shown in Table 1 below is used, dark field observation and CARS light observation can be performed at the same time. In this case, the wavelength of the CARS light generated from the sample 112 is 800 to 1010 [nm]. However, it goes without saying that the combination of the characteristics of the excitation light and the filters is not limited to the following. Further, even if an eccentric diaphragm having a biased opening that shields the center from light is used instead of the ring diaphragm 134, the sample 112 can be observed in the dark field using the microscope 100.

FIG. 2 is a schematic diagram illustrating dark field observation with the microscope 100 set as described above. In the dark field observation with the microscope 100, the illumination unit 130 and the image detection unit 150 are used in addition to the objective optical system 140 and the control unit 120.

As shown by the dotted line in the figure, the illumination light emitted by the illumination light source 131 in the illumination unit 130 is illuminated through the relay lens 133, 135, 138, the ring diaphragm 134, the field diaphragm 136, and the band passage filter 139, respectively. It is ejected from the part 130. As a result, the illumination light whose irradiation angle is limited to the annular shape of only the peripheral portion of the numerical aperture of the upper objective lens is irradiated to the sample 112 on the stage 110 from above in the figure.

Most of the illumination light applied to the sample 112 goes straight when passing through the sample 112. Then, it is captured by the lower objective lens 144, reflected by the dichroic mirror 151 of the image detection unit 150, and heads toward the CCD camera 158, but is not detected because it is shielded by the aperture stop 156 and does not reach the CCD camera 158. However, a part of the illumination light is scattered as it passes through the sample 112, changes its direction, passes through the aperture stop 156, reaches the CCD camera 158, and is detected. Therefore, the dark field image can be observed by the illumination light scattered by the sample 112.

The image detection unit 150 detects such scattered light and detects a bright and dark image according to the shape of the sample 112. In such dark-field observation, even when the sample 112 is transparent and the contrast is low, microstructures, micropathogens, and microscratches can be easily confirmed.

FIG. 3 is a schematic diagram illustrating CARS light observation with a microscope 100. In CARS light observation with the microscope 100, in addition to the objective optical system 140 and the control unit 120, a laser device 160, an excitation unit 170, an upper CARS light detection unit 180, and a lower CARS light detection unit 190 are used.

As shown by the dotted line in the figure, the excitation light emitted from the excitation unit 170 passes through the dichroic mirrors 151 and 191 and irradiates the sample 112 on the stage 110 from the lower part of the figure. By condensing the excitation light inside the sample 112 by the lower objective lens 144, CARS light having a wavelength corresponding to the composition of the molecules contained in the sample 112 is generated in the sample 112.

Therefore, in the microscope 100, the sample 112 can be observed by using the upper CARS photodetector 180 and the lower CARS photodetector 190. Here, in the microscope 100, the optical elements such as the ring diaphragm 134 and the aperture diaphragm 156 that reduce the amount of light are also on the optical path of the excitation light that generates the CARS light in the sample 112, and the light of the CARS light generated in the sample 112. It is not placed on the street either. Therefore, the utilization efficiency of the excitation light can be improved. Moreover, since the generated CARS light can be efficiently detected, the work time required for observation can be shortened.

The CARS light observed by the lower CARS photodetector 190 arranged on the same side as the excitation unit 170 with respect to the sample 112 is, so to speak, the reflected CARS light reflected by the sample 112. On the other hand, the CARS light observed by the upper CARS photodetector 180 arranged on the opposite side of the excitation unit 170 with respect to the sample 112 is, so to speak, a transmitted CARS light that has passed through the sample 112.

FIG. 4 is a diagram showing a region of spatial frequency that can be decomposed by observation with reflected CARS light. FIG. 5 is a diagram showing a region of spatial frequency that can be decomposed by observation with transmitted CARS light. Here, the region of the spatial frequency that can be decomposed is a region including the spatial frequency that contributes to the formation of the image among the plurality of spatial frequencies when the observation target is decomposed into a plurality of spatial frequencies, that is, Fourier transformed. ..

In addition, in FIGS. 4 and 5, in the scale shown on the right side in the figure, the upper end side in the figure shows that the light intensity is high, and the lower end side in the figure shows that the light intensity is low. Further, in each of the observation images displayed near the center of each figure, it is shown that the light intensity on the center side is high and the light intensity on the outer peripheral side is low in the figure. It corresponds to the shade shown on the scale.

As shown in FIG. 4, the decomposable spatial frequency region of the observation by the reflected CARS light and the decomposable spatial frequency region of the observation by the transmitted CARS light shown in FIG. 5 are different from each other and are exclusive to each other. .. Therefore, it is preferable to appropriately use the upper CARS photodetector 180 and the lower CARS photodetector 190 according to the purpose of observing the sample 112. Further, by combining the detection result of the upper CARS light detection unit 180 and the detection result of the lower CARS light detection unit 190, a wider frequency region can be observed.

Further, since the laser device 160 is used as a common light source in the upper CARS light detection unit 180 and the lower CARS light detection unit 190, the same position in the observation target can be detected at the same time. As a result, it is possible to obtain a more accurate image as compared with the case of using different devices without causing a positional shift between the image due to the reflected CARS light and the image due to the transmitted CARS light.

FIG. 6 is a schematic diagram illustrating the CARS process in which the observation target generates CARS light in the laser microscope 100. In the CARS process, the observed object (sample 112) is irradiated with excitation light containing two laser beams, pump light and Stokes light, which have different light frequencies ω ₁ and ω ₂ , and the light frequency of the pump light is ω ₁ . It occurs when the difference [ω ₁ -ω ₂ ] from the optical frequency ω ₂ of Stokes light coincides with the angular frequency ω ₀ of the natural vibration of the molecule contained in the observed object.

When the vibrational mode of a specific molecular structure contained in the observed object is excited by the CARS process, the molecular vibration interacts with the probe light, which is a third laser beam having an optical frequency ω ₃ , to be tertiary. CARS light derived from non-linear polarization is generated.

Further, since the pump light can also be used as the probe light, CARS light is generated under the condition of [ω ₁ = ω ₃ ]. The CARS light generated in the observed object has an optical frequency satisfying [ω _CARS = ω ₁ − ω ₂ + ω ₃ ].

Therefore, by detecting the CARS light emitted from the observed object, the presence of a specific molecular structure, for example, a functional group contained in the observed object can be detected. Further, by repeatedly detecting the CARS light while changing the position where the excitation light is applied to the observed object, the distribution of a specific molecular structure in the observed object can be imaged.

Since the CARS light has a higher light intensity than the spontaneous Raman scattered light or the like, it can be detected in a short time when a photoelectric conversion element is used. Therefore, not only the time required for detection is shortened, but also observation at a video rate is possible.

This makes it possible to detect not only the distribution of a specific molecular structure but also changes in the distribution. Further, by setting the band of the excitation light that irradiates the observed object to the infrared band that causes less damage to the living cells, the living cells of the observed object can be observed while being alive.

Further, the CARS light generated by the nonlinear optical effect is generated in an extremely narrow region where the excitation light is narrowed down by the lower objective lens 144. Therefore, the region targeted for CARS light detection is a narrow region in both the direction intersecting the optical axis of the irradiation light and the direction parallel to the optical axis. Therefore, the observation of the observed object by CARS light has a three-dimensionally high resolution.

Therefore, the observation plane may be formed inside the object to be observed by using the irradiation light in the infrared band or the near infrared band. Further, by sequentially moving the observation plane in the depth direction of the object to be observed, an image reflecting the three-dimensional distribution of a specific molecular structure can be generated.

As described above, as a phenomenon that causes a non-linear optical effect when the sample 112 is irradiated with coherent irradiation light, a phenomenon that produces Raman scattered light such as induced Raman scattering in addition to CARS light, and fluorescence due to multiphoton excitation. And the phenomenon of generating harmonics such as the second harmonic and the third harmonic are known. By detecting, for example, Raman scattered light among the lights generated by these non-linear phenomena, an image reflecting the three-dimensional distribution of a specific molecular structure in the sample 112 can be generated. Further, even with light other than Raman scattered light, an image reflecting the three-dimensional structure of the sample 112 can be generated. Therefore, in the microscope 100, by appropriately adjusting the wavelength of the laser light emitted by the laser device 160, the wavelength characteristics of various filters, and the like, the microscope 100 is used for multiphoton excitation fluorescence, the second harmonic, and the third. Observation of the sample 112 by harmonics, induced Raman scattering, etc. can be performed.

FIG. 7 is a flow chart showing an observation procedure of the sample 112 using the microscope 100. First, the sample 112 is held in the stage 110 (step S101). The sample 112 once held in the stage 110 is not moved until the series of observation procedures described below is completed.

Next, the sample 112 is observed over a wide area. In this embodiment, as shown in FIG. 2, the illumination unit 130 is turned on and the sample 112 is observed in the dark field (step S102). Since the time required for dark field observation is short and the sample 112 can be observed in real time while driving the stage scanner 111 and changing the position of the stage 110, the spatial region where the sample 112 exists (the sample 112 with respect to the focal point of the objective optical system). Position) can be grasped quickly.

That is, in the observation method using the nonlinear optical effect such as CARS light observation, since the excited region is narrow and the depth of focus is shallow, the molecule to be detected is not included in the sample 112, so that CARS from the sample is not included. It is not possible to distinguish between the case where the light cannot be detected and the case where the CARS light from the sample cannot be detected because the excitation light is out of focus from the sample 112. However, if the spatial region where the sample 112 exists is grasped by observing a wide range including the surface of the sample 112 in the dark field, the invalid observation of CARS light observation of the region where the sample 112 does not exist can be prevented. can.

Further, since the microscope 100 can simultaneously perform wide area observation such as dark field observation and observation by nonlinear optical effect such as CARS light observation, it is confirmed by wide area observation that the focus of the objective optical system is inside the sample 112. At the same time, it is possible to observe by a non-linear optical effect. Therefore, in the illustrated procedure, preliminary CARS light observation is performed using CARS light observation as an observation method (step S103).

Since the CARS light is generated by the optical linear optical effect generated when the excitation light is converged very finely, the observable range is narrow. Therefore, in the microscope 100, the excitation light is scanned by a galvano scanner 171 or the like, and an observation image is generated by the CARS light detected from the scanned region. As described above, the observation with CARS light generates an observation image by scanning, so that the time required for observation per unit area is long.

Therefore, in the preliminary CARS light observation using the microscope 100 (step S103), the entire sample 112 or a wide area is overviewed by observing the CARS light for a plurality of preliminary observation regions set at intervals. Here, since the individual preliminary observation areas 117 are narrow, the time required for preliminary CARS light observation can be shortened. Further, in the microscope 100, by using the stage scanner 111 together, the preliminary observation area can be arranged at a wide interval. In this way, the state of the entire sample 112 can be grasped in a short time.

FIG. 8 is a schematic diagram illustrating the distribution of the preliminary observation region 117 in the sample 112 in the preliminary CARS light observation when the sample 112 is observed with the microscope 100. In the preliminary CARS optical observation, a plurality of preliminary observation regions 117, which are set at intervals and each region is narrow, are observed over the entire sample 112. As a result, in the preliminary CARS optical observation, the entire sample 112 can be overviewed in a short time.

In the preliminary CARS optical observation, in addition to widening the distance between the preliminary observation regions 117, the scanning speed in each preliminary observation region 117 may be increased. Further, each preliminary observation region 117 may be a single observation point or a region having a two-dimensional spread formed by scanning.

As described above, in the microscope 100, by controlling the stage scanner 111 and the galvano scanner 171 by the control unit 120, it is possible to form a first observation system for acquiring information associated with the position of the observation region in the sample 112. In the example shown in FIG. 1, the control unit 120, the laser device 160, the excitation unit 170, the upper CARS photodetector unit 180, and the lower CARS photodetector unit 190 can form the first observation system.

Further, in the preliminary CARS light observation with the microscope 100, since CARS light observation and dark field image observation can be performed at the same time, the irradiation position of the excitation light in the sample 112, particularly, the excitation light is focused and irradiated in the sample 112. You can know in real time whether or not you are doing it. Therefore, in the preliminary CARS light observation, the focal position of the excitation light in the sample 112 can be continuously monitored to prevent the irradiation position of the excitation light from deviating from the sample 112. As a result, observation by the CARS process, which takes time to acquire the observation image, can be efficiently performed.

Further, for example, when observing a cell sheet in which live cells and dead cells coexist as sample 112, the region in which it is clear that the cells are dead cells at the stage of preliminary CARS light observation is the specific observation region 118 described later. It can be omitted from the extraction candidates of. Similarly, in the sample 112, the region in which it is obvious that the cells are living cells at the stage of preliminary CARS light observation can be omitted from the extraction candidates of the specific observation region 118. Therefore, by observing a small area with high accuracy, the same observation accuracy as when observing the entire sample 112 with high accuracy can be obtained, and the effective observation throughput can be improved.

Referring to FIG. 7 again, as described above, by using the dark field image observation and the preliminary CARS light observation in combination, the region matching the predetermined conditions in the sample 112, for example, the discoloration and deformation in the sample 112, can be seen. The specific observation region 118 to be observed in detail, in which mutations such as alteration are observed, is extracted from the preliminary observation region 117 and identified (step S104). In this case, the mutation to be detected may be one found by dark field image observation or one found by CARS light observation.

Further, when a mutation is detected by dark field image observation and preliminary CARS light observation, the control unit 120 acquires information related to the position of the mutation in the sample 112. When a plurality of specific observation regions 118 are specified for one sample 112, the depth direction in which the regions exist in the sample 112, for example, with reference to the surface thereof, depending on the number of the specific observation regions 118. The position information linked to the position of can be acquired. As a result, the specific observation region 118 can be reliably and quickly observed in the detailed CARS optical observation described later.

FIG. 9 is a schematic diagram illustrating the distribution of the detailed observation region 119 in the sample 112 in the detailed CARS optical observation. Each of the detailed observation regions 119 in the sample 112 includes a specific observation region 118 whose position was located in the preliminary CARS light observation. The position of the specific observation region 118 is, for example, the scanning position by the galvano scanner 171 and the stage scanner 111, and the position information in the depth direction of the sample 112 linked to the depth of the sample 112 acquired by preliminary CARS optical observation. It can be specified based on Z ₁ , Z ₂ , and Z ₃ .

In the illustrated example, each of the detailed observation areas 119 is wider than the specific observation area 118 to which each corresponds. Further, in the CARS light observation in the detailed observation region 119, the scanning speed of the excitation light by the galvano scanner 171 may be lowered. As a result, in the detailed observation area 119, the specific observation area 118 and its surroundings can be observed with a high resolution of CARS light observation. In this way, in the microscope 100, the stage scanner 111 and the galvano scanner 171 are controlled by the control unit 120 to form a second observation system for acquiring an image of the observation region in the sample 112.

The CARS optical observation can also form an image that reflects the three-dimensional distribution of the molecule to be detected. Therefore, at least one of the specific observation area 118 and the detailed observation area 119 may be set as a three-dimensional area. For example, the width (height) in the depth direction of the three-dimensional detailed observation region 119 may be determined according to the interval in the depth direction of the sample 112 in the preliminary CARS light observation.

FIG. 10 is a schematic diagram illustrating another shape of the preliminary observation region 117 in the sample 112 when the preliminary CARS light observation is performed with the microscope 100. In the illustrated preliminary CARS light observation, each of the preliminary observation regions 117 three-dimensionally arranged in the sample 112 at intervals has a width in the depth direction of the sample 112. Thereby, in the preliminary CARS optical observation, the tendency of the spatial distribution such as the mutation in each specific observation region 118 extracted from the preliminary observation region 117 can be grasped.

FIG. 11 is a schematic diagram illustrating the shape of the detailed observation region 119 specified by the preliminary CARS light observation by the preliminary observation region 117 as described above. Each of the illustrated detailed observation regions 119 includes the corresponding preliminary observation region 117 inside, and is formed in a sterically wider range than the preliminary observation region 117. As a result, the surroundings of the preliminary observation region 117 where the mutation has occurred can be observed in detail, and an image of the mutation generated in the sample 112 can be quickly and surely obtained.

The width (height) of the detailed observation region 119 shown in the figure in the depth direction may be determined by the distance between the three-dimensionally arranged preliminary observation regions 117 in the depth direction of the sample 112. The figure is a case where the distance in the depth direction of the preliminary observation area 117 is 1 times. Alternatively, the specific observation area 118 may be arranged in the center and its width may be double the interval. As a result, the width of the detailed observation region 119 in the sample 112 in the depth direction is sufficient to minimize the region that may be missed.

FIG. 12 is a diagram showing a setting when observing the sample 112 with a phase contrast using the microscope 100. The settings shown in FIG. 12 are the same as those of the microscope 100 shown in FIG. 1, except for the portion described below. Therefore, common elements are given the same reference number to omit duplicate explanations.

In the microscope 100 used when dark field observation and CARS light observation are used in combination, the aperture diaphragm 156 incorporated in the image detection unit 150 as a second optical member is removed, and a phase ring 146 is arranged instead. It can also be used for phase difference observation. The phase ring 146 is optically conjugated to the position _P3 of the exit pupil of the lower objective lens 144 and is incorporated in the position _P4 where the real image of the exit pupil is formed. In general, the diameter of the ring diaphragm 134 used for phase contrast observation is smaller than that of the ring diaphragm 134 used for dark field observation.

By setting as described above, the sample 112 placed on the stage 110 can be observed with a phase contrast using the microscope 100. Further, as in the case of the setting shown in FIG. 1, CARS optical observation can be executed at the same time while maintaining the phase difference observation setting.

The phase ring 146 is placed between the lower objective lens 144 and the dichroic mirror 191 at a position where the exit pupil position _P3 of the lower objective lens 144 is optically coupled and a real image of the exit pupil can be formed. However, CARS optical observation can be performed. In this case, since the excitation light applied to the sample 112 is attenuated by passing through the phase ring 146, the sample 112 can be excited to generate CARS light, but the light intensity of the detected CARS light is lowered. do.

FIG. 13 is a diagram showing a setting when the sample 112 is observed by differential interference contrast using the microscope 100. The settings shown in FIG. 13 are the same as those of the microscope 100 shown in FIG. 1, except for the portion described below. Therefore, common elements are given the same reference number to omit duplicate explanations.

In the illustrated microscope 100, as the first optical member, a polarizer 232 and a Wollaston prism 234 are incorporated in the illumination unit 130 instead of the ring diaphragm 134. Further, in the microscope 100 of this setting, the Wollaston prism 236 and the analyzer 238 are incorporated in the image detection unit 150 as the second optical member instead of the aperture stop 156. This makes it possible to observe the differential interference contrast of the sample 112 using the microscope 100. The Wollaston prisms 234 and 236 may be of the Nomalski type.

The Wollaston prism 234 is located in the vicinity of P ₂ in which the intersection of the normal ray and the abnormal ray separated by the Wollaston prism 234 is determined to be the position P ₂ optically coupled to the position P ₁ of the exit pupil of the upper objective lens 142. The Polarizer 232 is arranged on the side of the illumination light source 131 immediately adjacent to the Polarizer 232. The polarizer 232 converts the illumination light generated by the illumination light source 131 into linear polarization, and the Wollaston prism 234 separates the illumination light into two linear polarizations whose vibration directions are orthogonal to each other.

The Wollaston prism 236 is determined so that the intersection of the normal light beam and the abnormal light ray synthesized by the Wollaston prism 236 is at the position P ₄ optically coupled to the position P ₃ of the exit _pupil of the lower objective lens 144. It is installed in a nearby position, and the analyzer 238 is located on the side of the CCD camera 158 immediately next to it. The Wollaston prism 236 combines the two separated linear polarizations into one, and the analyzer 238 transmits only the polarization component in a specific direction.

By mounting the optical member as described above, in the image detection unit 150 of the microscope 100, the two linear polarizations separated by the Wollaston prism 234 on the illumination unit 130 side pass through the sample 112 as the sample 112. A phase difference is generated according to the phase distribution of the above, and interference occurs when passing through the Wollaston prism 236 and the analyzer 238 on the image detection unit 150 side. Therefore, the image detected by the image detection unit 150 has a contrast corresponding to the differential amount of the refractive index distribution of the sample 112.

FIG. 14 is a diagram showing a setting in the case of observing the modulation contrast of the sample 112 using the microscope 100. The settings shown in FIG. 14 are the same as those of the microscope 100 shown in FIG. 1, except for the portion described below. Therefore, common elements are given the same reference number to omit duplicate explanations.

In the illustrated microscope 100, as a first optical member, a polarizing plate 231 and a slit diaphragm 235 are incorporated in the illumination unit 130 instead of the ring diaphragm 134. Further, in the microscope 100, as a second optical member, a modulator 233 is incorporated in the image detection unit 150 instead of the aperture stop 156. As a result, the modulated contrast of the sample 112 can be observed using the microscope 100.

The slit diaphragm 235 is incorporated at a position P ₂ optically coupled to the position P ₁ of the exit pupil of the upper objective lens 142, and has a rectangular slit that opens at a position off the optical axis. The modulator 233 is incorporated at a position P ₄ that is optically conjugate to the position P ₃ of the exit pupil of the lower objective lens 144. The slit diaphragm 235 and the modulator 233 are arranged at optically conjugate positions. The modulator 233 has a transparent region that is transparent to transmitted light, a gray region that attenuates transmitted light and transmits it, and a dark region that blocks transmitted light. The gray area of the modulator 233 is arranged at a position where the slit image of the slit diaphragm 235 is formed. As a result, the transmitted light is refracted and deflected according to the gradient of the refractive index of the sample 112, and the ratio of the transmitted light transmitted through the modulator 233 changes, so that the detected image detected by the image detection unit 150 becomes bright and dark.

In addition, a polarizing plate may be attached to a part of the opening of the slit diaphragm 235, and the polarizing plate 231 may be arranged on the illumination light source 131 side immediately adjacent to the slit diaphragm 235. In this case, the contrast of the detected image can be adjusted by rotating the polarizing plate 231.

FIG. 15 is a diagram showing other settings when observing the sample 112 in the dark field using the microscope 101. The settings in FIG. 15 are the same as those of the microscope 100 shown in FIG. 1, except for the portion described below. Therefore, common elements are given the same reference number to omit duplicate explanations.

In the illustrated setting, the image detection unit 150 is arranged between the upper objective lens 142 and the upper CARS photodetection unit 180. In this case, the aperture stop 156 of the image detection unit 150 is arranged at the position P ₅ optically conjugate with the position P ₁ of the exit pupil of the upper objective lens 142. Further, the dichroic mirror 151 used in the image detection unit 150 of the microscope 100 is replaced with a dichroic mirror 159 having a different wavelength characteristic.

The dichroic mirror 159 transmits the CARS light generated from the sample 112, transmits a part of the illumination light from the illumination unit 130, and reflects a part of the light in the visible light band among the light emitted from the sample 112. do. As a result, the image detection unit 150 detects the illumination light scattered by the sample 112 and returned to the upper objective lens 142 again to generate a dark field image. The dichroic mirror 159 has, for example, the wavelength characteristics shown in Table 2 below.

In the above example, the image detection unit 150 is arranged between the upper objective lens 142 and the upper CARS light detection unit 180. However, even if the image detection unit 150 is arranged between the upper CARS light detection unit 180 and the illumination unit 130, dark field observation and CARS light observation can be performed at the same time.

FIG. 16 is a schematic diagram showing the structure of another microscope 102. The microscope 102 has the same structure as the microscope 100 except for the portion described below. Therefore, common elements are given the same reference number to omit duplicate explanations.

The microscope 102 includes a stage 110, a control unit 120, an objective optical system 140, an observation system including an illumination unit 130 and an image detection unit 150, a laser device 160, an excitation unit 170, and an upper CARS light detection. It is common with the microscope 100 shown in FIG. 1 and the like in that it includes a unit 180 and another observation system including a lower CARS light detection unit 190.

Compared to the microscope 100, in the microscope 102, the illumination unit 130 forming the first irradiation unit has the excitation unit 170 as the second irradiation unit, the upper objective lens 142, and the lower objective with respect to the stage 110. It is arranged on the same side in the direction parallel to the optical axis of the lens 144. Further, the image detection unit 150 is arranged adjacent to the upper CARS light detection unit 180 on the opposite side of the illumination unit 130 from the stage 110 in the above direction.

The microscope 102 laid out in this way can observe the sample 112 in the same manner by the same operation as the microscope 100 shown in FIG. Further, since the laser device 160 and the lighting unit 130, each of which is a heat source and consumes a large amount of power, can be arranged together, thermal and power management becomes easy.

Although the present invention has been described above using the embodiments, the technical scope of the present invention is not limited to the scope described in the above embodiments. It will be apparent to those skilled in the art that various changes or improvements can be made to the above embodiments. It is clear from the description of the claims that the form with such changes or improvements may be included in the technical scope of the present invention.

The order of execution of each process such as operation, procedure, step, and step in the apparatus, system, program, and method shown in the claims, specification, and drawings is particularly "before" and "prior to". It should be noted that it can be realized in any order except when it is clearly stated, and when the output of the previous process is used in the subsequent process. Even when the scope of claims, the specification, and the operation flow in the drawings are described using "first", "next", etc. for convenience, it means that it is essential to carry out in this order. It's not a thing.

100, 101, 102 microscope, 110 stage, 111 stage scanner, 112 sample, 117 preliminary observation area, 118 specific observation area, 119 detailed observation area, 120 control unit, 122 processing device, 124 keyboard, 126 mouse, 128 display unit, 130 Illumination unit, 131 Illumination light source, 132 Collector lens 133, 135, 138, 154, 157, 182, 183, 192, 193 Relay lens, 134 Ring aperture, 136 Field aperture, 137, 174 Reflector, 139, 155 band Passage filter, 140 objective optical system, 142 upper objective lens, 144 lower objective lens, 146 phase ring, 150 image detector, 153, 173 primary image plane, 152, 175 imaging lens, 156 aperture aperture, 158 CCD camera, 151, 159, 181, 191 dichroic mirror, 160 laser device, 161, 162 laser light source, 163 combiner, 170 exciter, 171 galvano scanner, 172 scan lens, 180 upper CARS light detector, 184, 194 band pass filter, 190 Lower CARS optical detector, 185, 195 photoelectron multiplying tube, 231 polarizing plate, 232 polarizer, 233 modulator, 234, 236 Wollaston prism, 235 slit aperture, 238 analyzer

Claims (4)

A pair of objective lenses arranged with the observation object in between and having a common focal point in the area overlapping the observation object.
A first irradiation unit that irradiates the observation object with the coherent first irradiation light through one of the pair of objective lenses.
A first detection unit that detects the first emission light emitted by the non-linear optical effect from the observation object irradiated with the first irradiation light through the one lens.
The second emission light emitted from the observation object irradiated with the first irradiation light by a nonlinear optical effect and propagating in an optical path different from the first emission light is emitted from the other of the pair of objective lenses. The second detector that detects through the lens of
A second irradiation unit that irradiates the observation object with an incoherent second irradiation light, and a second irradiation unit.
A third detection unit that detects the third emission light emitted by the linear optical effect from the observation object irradiated with the second irradiation light through any of the pair of objective lenses.
At a position on at least one of the second irradiation light and the third emission light and not on the optical path of the first irradiation light, the first emission light and the second emission light. A first dichroic mirror that is arranged and guides the second irradiation light to the pair of objective lenses, and a second dichroic mirror that separates the third emission light from the emission light of the pair of objective lenses.
A first optical member for optically controlling the second irradiation light to visualize a transparent observation object, and
A second optical member for optically controlling the third emission light to visualize a transparent observation object is provided.
The first optical member is an objective that transmits the second irradiation light of the pair of objective lenses in a section before being incident on the first dichroic mirror on the optical path of the second irradiation light. Arranged in a position conjugate with the pupil of the lens,
The second optical member is on the optical path of the third emission light, and in a section after being separated by the second dichroic mirror, the third emission light of the pair of objective lenses is emitted. A microscope placed in a position conjugate with the pupil of a transmitting objective lens . The first optical member is movable along an optical path in order to adjust the optical path length with the pupil of the lens that transmits the second irradiation light among the pair of objective lenses, and the second optical member is movable. The microscope according to claim 1 , wherein the member is movable along an optical path in order to adjust the optical path length with the pupil of the other objective lens that transmits the third emission light of the pair of objective lenses. .. The microscope according to claim 1 or 2, wherein at least one of the first optical member and the second optical member integrally includes an optical component that changes the optical path length to the pair of objective lenses. The position of the pair of objective lenses arranged between the first optical member and the pair of objective lenses and facing the pupil of the lens facing the first optical member is kept away from the pair of objective lenses. Further equipped with a first relay optical system,
The position of the pair of objective lenses arranged between the second optical member and the pair of objective lenses and facing the pupil of the lens facing the second optical member is kept away from the pair of objective lenses. The microscope according to any one of claims 1 to 3, further comprising a second relay optical system.

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