JP2019035669A - Observation apparatus and observation method - Google Patents

Abstract

To observe an object efficiently in terms of time.SOLUTION: An observation apparatus comprises: a first observation system that observes an observation object to acquire information associated with an observation area within the observation object; and a second observation system that observes the observation area identified on the basis of the information acquired by the first observation system. In the observation apparatus, an observation range for each unit time of the first observation system may be wider than that of the second observation system. Also, in the observation apparatus, the observation range of the first observation system includes a surface of the observation object, and the information may include the information to identify a spatial area in which the observation object exists.SELECTED DRAWING: Figure 2

Description

  The present invention relates to an observation apparatus and an observation method.

As a method for observing a living cell or the like without damaging it, there is a method for detecting CARS light (coherent anti-Stokes Raman scattering light) (for example, see Patent Document 1).
Japanese Patent Application Laid-Open No. 2005-062155

  In observation with a scanning microscope, for example, observation with a microscope using a nonlinear optical effect, it takes time until an observation image is obtained. Further, in observation using the nonlinear optical effect, if an optical member is inserted in the path of the irradiation light to the observation target and the emission light from the observation target, the detection efficiency is reduced and the observation takes more time. .

  According to the first aspect of the present invention, the first observation system for observing the observation target and acquiring information associated with the observation region inside the observation target, and the information acquired by the first observation system. An observation device is provided that includes a second observation system that observes the observation region specified based on the observation region.

  According to the second aspect of the present invention, the first observation stage of observing the observation target and acquiring information associated with the observation area inside the observation target, and the information acquired in the first observation stage An observation method comprising: a second observation step of observing an observation region identified based on the observation region.

  The summary of the invention does not enumerate all the features of the present invention. A sub-combination of these feature groups can also be an invention.

1 is a schematic diagram of a microscope 100. FIG. 2 is a schematic diagram for explaining dark field observation with a microscope 100. FIG. 2 is a schematic diagram illustrating CARS light observation with a microscope 100. FIG. It is a figure which shows the area | region of the spatial frequency which can be decomposed | disassembled by observation by reflected CARS light. It is a figure which shows the area | region of the spatial frequency which can be decomposed | disassembled by observation by transmitted CARS light. It is a schematic diagram explaining the generation principle of CARS light. 3 is a flowchart showing an observation procedure using a microscope 100. 5 is a schematic diagram showing a distribution of a preliminary observation region 117. FIG. It is a schematic diagram which shows distribution of the detailed observation area | region 119. FIG. 6 is a schematic view illustrating the distribution of a preliminary observation region 117. It is a schematic diagram which illustrates distribution of the detailed observation area | region 119. 1 is a schematic diagram of a microscope 100. FIG. 1 is a schematic diagram of a microscope 100. FIG. 1 is a schematic diagram of a microscope 100. FIG. 1 is a schematic diagram of a microscope 101. FIG. 2 is a schematic diagram of a microscope 102. FIG.

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

  FIG. 1 is a schematic diagram of a microscope 100 that is an example of an observation apparatus. 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 a first irradiation unit, and the laser device 160 forms a second irradiation unit. 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 a sample 112 that is an observation target of the microscope 100. Thereby, the illumination light from the illumination unit 130 can be applied to the sample 112 placed on the stage 110 from above in the figure. Further, the sample 112 placed on the stage 110 can be observed from the upper side in the drawing by the upper CARS light detection unit 180.

  The stage 110 has an opening that exposes the lower surface of the sample 112 in the drawing. Thereby, the laser beam can be irradiated to the sample 112 placed on the stage 110 from the lower side in the figure by the laser device 160 and the excitation unit 170. In addition, 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.

  The stage 110 is coupled to the stage scanner 111. The stage scanner 111 moves the stage 110 parallel to the surface on which the sample 112 is placed, as indicated by arrows xy in the drawing. Thereby, in the microscope 100, the wide range of the sample 112 can be observed with the optical axis of the optical system 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 mounting 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 a user instruction is input to the processing device 122. The display unit 128 returns feedback to the user's operation using 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. 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 in accordance with instructions received from the user.

The illumination unit 130 includes an illumination light source 131, a collector lens 132, relay lenses 133, 135, and 138, a ring stop 134, and a field stop 136. Field stop 136 is disposed in a position conjugate _{P 12} with respect to the focal position _{P 11} of the objective optical system 140. The position of the ring aperture 134 will be described later. The illumination light source 131 generates white incoherent illumination light using, for example, a halogen lamp. The illumination light collected by the collector lens 132 passes through the relay lenses 133 and 138, the ring stop 134, and the field stop 136, and passes through only the light in the visible light band by the band pass filter 139 and is irradiated on the sample 112.

  The objective optical system 140 includes an upper objective lens 142 and a lower objective lens 144 that are disposed on opposite sides of the stage 110. The upper upper objective lens 142 in the drawing condenses 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. Note that even an illumination condenser lens that is not subjected to aberration correction intended for use as an objective lens may be used as the upper objective lens 142, for example. In addition, the lower lower objective lens 144 in the drawing 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 the same numerical aperture. Thereby, when observing the coherent emission light generated by irradiating the sample 112 with the coherent irradiation light, it is possible to prevent the detection accuracy from being lowered due to the artifact superimposed on the image or the spectrum. More specifically, the ratio R _NA between 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 band pass filter 155, an aperture stop 156, a relay lens 157, and a CCD camera 158. The dichroic mirror 151 reflects light in the visible light band out of the light emitted from the sample 112 and transmits light in the wavelength band irradiated on the sample 112 from the laser light sources 161 and 162. Thereby, the illumination light transmitted or scattered in the sample 112 out of the illumination light irradiated on the sample 112 by the illumination unit 130 is introduced into the image detection unit 150.

  The band pass filter 155 transmits only the light in the wavelength band of the illumination light irradiated on the sample 112 by the illumination unit 130 and blocks light of other wavelengths. The imaging lens 152 forms an image of the illumination light scattered by the sample 112 on the primary image plane 153. Further, the relay lenses 154 and 157 re-image 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 re-imaged image is captured as a dark field observation image.

The laser device 160 includes a plurality of laser light sources 161 and 162 and a combiner 163. A laser light source 161 is a pulsed laser emission wavelength lambda _S, the laser light source 162, the pulsed laser emission wavelength lambda _P, respectively generated separately. The emission wavelengths λ _S and λ _P of the laser light sources 161 and 162 are different from each other. Further, the emission wavelengths λ _S and λ _P of the laser light sources 161 and 162 are different from the wavelength band transmitted by the band pass filter 139.

As the laser light sources 161 and 162, for example, a mode-locked picosecond Nd: YVO ₄ laser, a mode-locked picosecond yttrium laser, or the like can be used. Note that one of the laser light sources 161 and 162 may be replaced with an optical parametric oscillator that converts the wavelength of the pulse laser generated by the other of the laser light sources 161 and 162.

Of picosecond pulses generated by the laser light source 161, a pulse laser having a wavelength lambda _P the shorter is used as the pump light in the CARS light observation. Of 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 beams emitted from the laser light sources 161 and 162 are incident on the combiner 163 and become a single beam. Thereby, the excitation light combining pump light and Stokes light can be irradiated to the sample 112 to generate CARS light.

  The laser device 160 may include a delay optical path for the purpose of synchronizing the pump light generated by the laser light sources 161 and 162 and the Stokes light. The delay optical path can be formed by a plurality of reflecting mirrors whose intervals can be changed. In the laser device 160, the Stokes light may be broadened using a photonic crystal fiber.

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

  The scan lens 172 focuses the irradiation light emitted from the galvano scanner 171 onto a predetermined primary image plane 173. Thereby, the observation region of the sample 112 can be scanned with the irradiation light emitted from the laser device 160, and the irradiation light can be irradiated onto the observation region having a predetermined area.

  Each of the upper CARS light detection unit 180 and the lower CARS light detection unit 190 includes dichroic mirrors 181 and 191, relay lenses 182, 192, 183, and 193, bandpass filters 184 and 194, and photomultiplier tubes 185 and 195. . The dichroic mirrors 181 and 191 transmit light in the visible light band generated by the illumination unit 130 as illumination light, transmit excitation light from the excitation unit 170, and reflect CARS light generated in the sample 112. Accordingly, the dichroic mirrors 181 and 191 introduce the CARS light generated in the sample 112 into the upper CARS light detection unit 180 or the lower CARS light detection unit 190.

  Further, the bandpass filters 184 and 194 transmit the CARS light and block light having a wavelength other than the CARS light, including the illumination light from the illumination unit 130 and the excitation light from the excitation unit 170. Thereby, 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 simultaneously.

  In the illumination unit 130, a reflecting mirror 137 is disposed between the field stop 136 and the relay lens 138. Also in the excitation unit 170, a reflecting mirror 174 is disposed between the scan lens 172 and the imaging lens 175. The reflecting mirrors 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 of the sample 112 using the illumination unit 130 and the image detection unit 150. The microscope 100 can also be used for CARS light observation using the laser device 160, the excitation unit 170, the upper CARS light detection unit 180, and the lower CARS light detection unit 190.

  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 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 that forms the illumination light for dark field observation is not provided 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 illumination part 130 by passing. That is, the ring diaphragm 134 is disposed at a position P ₂ that is optically conjugate with the position P ₁ of the exit pupil of the upper objective lens 142.

An aperture stop 156 that forms a dark field observation image is not provided between the lower objective lens 144 and the lower CARS light detection unit 190 but via a relay optical system including the imaging lens 152 and the relay lens 154. Arranged in the image detection unit 150. That is, the aperture stop 156 is disposed at a position P ₄ optically conjugate with the exit pupil position P ₃ of the lower objective lens 144.

  The dichroic mirror 181 of the upper CARS light detection unit 180 is disposed 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 connected to the lower objective lens 144 and image detection. It is arranged between the unit 150.

As indicated by an arrow M in the figure, the ring stop 134 can be moved in a direction parallel to the optical axis Q. Furthermore, an optical member G ₁ indicated by a dotted line in the figure, it is also possible to swap between the ring diaphragm 134 and the relay lens 135. As the optical element G _1, lens, using a parallel flat glass. Thus, in the microscope 100, the optical path length between the upper objective lens 142 and the ring stop 134 can be changed. Especially in insertion of the optical member G _1, to keep the significant deviations of the optical path length also, imaging relationship of the illumination light source 131 and the aperture ring 134 required for Koehler illumination between the upper objective lens 142 and the ring stop 134 it can.

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

Further, when different optical characteristics such as observation magnification are required, or when the upper objective lens 142 is contaminated by contacting the sample 112 or the like, the upper objective lens 142 is replaced with another objective lens having a different magnification or numerical aperture. When exchange, to sometimes position of the position P ₁ of the exit pupil of the upper objective lens 142 is changed greatly, it may vary also the size of the suitable ring ring diaphragm 134. In that case, the ring aperture 134 is replaced with an appropriate size, and the optical member G ₁ is added or replaced, so that the optical path length between the upper objective lens 142 and the ring aperture 134 is approximately adjusted. Thereafter, the position of the ring diaphragm 134 is moved in a 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.

In this case, the ring aperture 134 and the optical member G ₁ is may be configured as a replaceable ring diaphragm blocks together. In that case, it is possible to replace the upper objective lens 142 together with an appropriate ring diaphragm block, so that the operation can be simplified, and further, automatically in conjunction with the replacement of the upper objective lens 142. It is convenient to use a mechanism that is exchanged automatically because the exchange mechanism can be easily simplified.

In adjusting the position of the ring stop 134, the user may move the ring stop 134 to a position where the observation image becomes clear without direct leakage of the illumination light while the sample 112 is observed in the dark field. Further, a mechanism may be provided in which the contrast of the dark field image is evaluated and the ring diaphragm 134 is moved to a position where the contrast becomes highest. In this way, by making the position of the ring diaphragm 134 adjustable, the position of the ring diaphragm 134 is accurately adjusted to the position P ₂ optically conjugate with the position P ₁ of the entrance pupil of the upper objective lens 142, so A reduction in resolution as a field microscope can be prevented.

The aperture stop 156 can also be moved in a direction parallel to the optical axis Q. Furthermore, an optical member G ₂ indicated by a dotted line in the figure, it is also possible to swap between the aperture stop 156 and the relay lens 154. As the optical element G _2, lens, using a parallel flat glass. Thus, in the microscope 100, the optical path length between the lower objective lens 144 and the aperture stop 156 can be changed. Especially in insertion of the optical member G _2, be varied greatly optical path length between the lower objective lens 144 and aperture stop 156, it is possible to keep the optical path length of the aperture stop 156 and the relay lens 157, CCD camera At 158, the light emitted from the sample 112 can be appropriately received.

  Thus, for example, when the lower objective lens 144 is moved up and down to change the distance from the imaging lens 152 for focus adjustment, the position of the aperture stop 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 is contaminated due to contact with the sample 112 or the like, the lower objective lens 144 is changed to another objective having a different magnification or numerical aperture. replacing the lens, to sometimes the position of the exit pupil position P ₃ of the lower objective lens 144 is changed greatly, may vary also the size of the appropriate aperture of the aperture stop 156. In this case, changing the aperture of the aperture stop 156 to an appropriate size, or replace the aperture of appropriate size, or by exchanging or adding optical member G _2, lower objective lens 144 The optical path length between the lower objective lens 144 and the aperture stop 156 is adjusted by roughly adjusting the optical path length between the lower objective lens 144 and the aperture stop 156 after the position of the aperture stop 156 is moved in the direction parallel to the optical axis Q. Fine tune.

In this case, aperture stop 156 and an optical member G ₂ may be configured as an exchangeable aperture stop blocks together. In that case, it is possible to replace the lower objective lens 144 together with an appropriate aperture stop block, so that the operation can be simplified and further linked to the replacement of the lower objective lens 144. Therefore, it is convenient for the mechanism to be automatically replaced because it is easy to simplify the replacement mechanism.

  The exchange of the ring diaphragm block and the aperture diaphragm block is automatically performed by the electric mechanism together with the exchange of the upper objective lens 142 and the lower objective lens 144, so that the load on the operator who operates the microscope 100 is the least. Convenient.

  With such an arrangement of the ring stop 134 and the aperture stop 156, in the microscope 100, the ring stop 134 is excluded from the optical path between the upper CARS light detection unit 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 light detector 190 on the lower side to the lower objective lens 144 in the drawing.

In the microscope 100, for example, when the combination of wavelength characteristics described in Table 1 below is used, dark field observation and CARS light observation can be performed simultaneously. 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. In addition, the sample 112 can be observed in the dark field using the microscope 100 even when an eccentric stop having a biased opening with a light shielded center is used instead of the ring stop 134.

  FIG. 2 is a schematic diagram for explaining dark field observation with the microscope 100 set as described above. In the dark field observation in 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 indicated by a dotted line in the drawing, the illumination light emitted from the illumination light source 131 in the illumination unit 130 is illuminated through the relay lenses 133, 135, and 138 through the ring diaphragm 134, the field diaphragm 136, and the band-pass filter 139, respectively. Injected from the unit 130. As a result, the sample 112 on the stage 110 is irradiated with illumination light whose irradiation angle is limited to an annular shape only in the peripheral portion of the numerical aperture of the upper objective lens from above in the drawing.

  Most of the illumination light irradiated on the sample 112 travels 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 directed toward the CCD camera 158, but is shielded by the aperture stop 156 and does not reach the CCD camera 158, and thus is not detected. However, a part of the illumination light is scattered when passing through the sample 112, the direction of travel is changed, passes through the aperture stop 156, reaches the CCD camera 158, and is detected. Therefore, a 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 corresponding to the shape of the sample 112. In such dark field observation, even if the sample 112 is transparent and the contrast is low, it is possible to easily confirm a microstructure, a micropathogen, and a micro scratch.

  FIG. 3 is a schematic diagram for explaining CARS light observation with the microscope 100. In CARS light observation in 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 indicated by a dotted line in the figure, the excitation light emitted from the excitation unit 170 passes through the dichroic mirrors 151 and 191 and is irradiated on the sample 112 on the stage 110 from below in the figure. By converging the excitation light inside the sample 112 by the lower objective lens 144, CARS light having a wavelength corresponding to the composition of molecules contained in the sample 112 is generated in the sample 112.

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

  Note that the CARS light observed by the lower CARS light detection unit 190 disposed on the same side as the excitation unit 170 with respect to the sample 112 is, in other words, reflected CARS light reflected by the sample 112. On the other hand, the CARS light observed by the upper CARS light detection unit 180 disposed on the opposite side of the excitation unit 170 with respect to the sample 112 is transmitted CARS light, that is, the transmitted CARS light.

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

  4 and 5, in the scale shown on the right side of the figure, the upper end side in the figure indicates that the light intensity is high, and the lower end side in the figure indicates that the light intensity is low. In addition, in each of the observation images displayed near the center of each figure, the light intensity on the center side is high and the light intensity on the outer peripheral side is low in the figure, and the shades of the display image are Corresponds to the shading shown on the scale.

  As shown in FIG. 4, the resolvable spatial frequency region for observation with reflected CARS light and the resolvable spatial frequency region for observation with transmitted CARS light shown in FIG. 5 are different from each other and are mutually exclusive. . Therefore, it is preferable to appropriately use the upper CARS light detection unit 180 and the lower CARS light detection unit 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.

  In addition, since the upper CARS light detection unit 180 and the lower CARS light detection unit 190 use the laser device 160 as a common light source, the same position in the observation target can be detected simultaneously. Thereby, a more accurate image can be acquired compared with the case where it uses with a separate apparatus, without generating a position shift between the image by reflected CARS light, and the image by transmitted CARS light.

FIG. 6 is a schematic diagram for explaining the CARS process in which the observation target generates CARS light in the laser microscope 100. CARS process, different optical frequencies omega _{1 together,} by irradiating the excitation light including the two laser beams of the pump light and stokes light having a omega ₂ in the object to be observed (sample 112), and the optical frequency omega ₁ of the pump light This occurs when the difference [ω ₁ −ω ₂ ] with the optical frequency ω ₂ of the Stokes light coincides with the angular frequency ω ₀ of the natural vibration of the molecule contained in the observed object.

When the vibration mode of a specific molecular structure included in the object to be observed is excited by the CARS process, the molecular vibration interacts with the probe light, which is the third laser light having the optical frequency ω ₃ , so that the third order CARS light derived from nonlinear polarization is generated.

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

  Therefore, by detecting the CARS light emitted from the object to be observed, it is possible to detect the presence of a specific molecular structure, such as a functional group, included in the object to be observed. In addition, by repeatedly detecting CARS light while changing the position where the observation object is irradiated with the excitation light, the distribution of a specific molecular structure in the observation object can be imaged.

  Since CARS light has higher light intensity than 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.

  Thereby, not only the distribution of the specific molecular structure but also the change of the distribution can be detected. In addition, by making the excitation light band irradiated on the object to be observed an infrared band that causes little damage to the living cells, the living cells of the object to be observed can be observed.

  Furthermore, the CARS light generated by the nonlinear optical effect is generated in a very narrow region where the excitation light is narrowed down by the lower objective lens 144. For this reason, the area | region used as the object of CARS light detection becomes a narrow area | region regarding both the direction crossing the optical axis of irradiation light, and a direction parallel to an optical axis. Therefore, the observation of the object to be observed with the CARS light has a three-dimensionally high resolution.

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

  Note that, as described above, when the sample 112 is irradiated with coherent irradiation light, as a phenomenon that produces a nonlinear optical effect, in addition to CARS light, a phenomenon in which Raman scattered light such as stimulated Raman scattering is generated, or fluorescence due to multiphoton excitation. There are known phenomena such as the phenomenon of generating the second harmonic, the third harmonic, and the like. By detecting, for example, Raman scattered light among the light generated by these nonlinear phenomena, an image reflecting the three-dimensional distribution of a specific molecular structure in the sample 112 can be generated. Further, an image reflecting the three-dimensional structure of the sample 112 can be generated even with light other than Raman scattered light. For this reason, 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, using the microscope 100, multiphoton excitation fluorescence, second harmonic, third The sample 112 can be observed by harmonics, stimulated Raman scattering, or the like.

  FIG. 7 is a flowchart showing the observation procedure of the sample 112 using the microscope 100. First, the sample 112 is held on the stage 110 (step S101). The sample 112 once held on the stage 110 is not moved until a 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). The time required for dark field observation is short, and the sample 112 can be observed in real time while the stage scanner 111 is driven to change the position of the stage 110. Can be quickly grasped.

  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. A case where light cannot be detected cannot be distinguished from a case where the CARS light from the sample cannot be detected because the excitation light is out of focus from the sample 112. However, by observing a wide area including the surface of the sample 112 in the dark field, if the spatial area where the sample 112 exists is grasped, the invalid observation of observing the area where the sample 112 does not exist as CARS light is prevented. it can.

  Furthermore, since the microscope 100 can simultaneously perform wide-area observation such as dark field observation and observation by nonlinear optical effects such as CARS light observation, it is confirmed that the focal point of the objective optical system is inside the sample 112 by wide-area observation. At the same time, it is possible to observe by the nonlinear 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 the galvano scanner 171 or the like, and an observation image is generated by the CARS light detected from the scanned region. In this way, observation with CARS light generates an observation image by scanning, and thus the time required for observation per unit area is long.

  Therefore, in preliminary CARS light observation using the microscope 100 (step S103), the entire sample 112 or a wide area is overviewed by performing CARS light observation on a plurality of preliminary observation areas set at intervals. Here, since each preliminary observation area 117 is narrow, the time required for preliminary CARS light observation can be shortened. Further, in the microscope 100, by using the stage scanner 111 in combination, the preliminary observation areas can be arranged at wide intervals. Thus, the state of the entire sample 112 can be grasped in a short time.

  FIG. 8 is a schematic view 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 light observation, a plurality of preliminary observation areas 117 which are set at intervals and are narrow in each area are observed over the entire sample 112. Thereby, in preliminary CARS light observation, the whole sample 112 can be overviewed in a short time.

  In the preliminary CARS light observation, in addition to widening the interval between the preliminary observation regions 117, the scanning speed in each preliminary observation region 117 may be increased. Furthermore, each preliminary observation region 117 may be one observation point or a region having a two-dimensional expansion formed by scanning.

  As described above, in the microscope 100, the first observation system that acquires information associated with the position of the observation region in the sample 112 can be formed by controlling the stage scanner 111 and the galvano scanner 171 by the control unit 120. In the example illustrated in FIG. 1, the control unit 120, the laser device 160, the excitation unit 170, the upper CARS light detection unit 180, and the lower CARS light detection unit 190 can form a first observation system.

  Further, in the preliminary CARS light observation in the microscope 100, since the CARS light observation and the dark field image observation can be simultaneously performed, the irradiation position of the excitation light in the sample 112, in particular, the excitation light is focused and irradiated in the sample 112. Whether or not you are in real time. Therefore, in the preliminary CARS light observation, the focal position of the excitation light in the sample 112 can be continuously monitored, and the irradiation position of the excitation light can be prevented from deviating from the sample 112. Thereby, the observation by CARS process which requires time for acquisition of an observation image can be performed efficiently.

  Further, for example, when a cell sheet in which live cells and dead cells are mixed is observed as the sample 112, a region where a dead cell is obvious at the stage of preliminary CARS light observation is a specific observation region 118 described later. Can be omitted from the extraction candidates. Similarly, in the sample 112, an area where it is obvious that it is a living cell at the stage of preliminary CARS light observation can be omitted from the extraction candidates for the specific observation area 118. Therefore, by observing a small area with high accuracy, the same observation accuracy as that 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 dark field image observation and preliminary CARS light observation in combination, an area that satisfies a predetermined condition in sample 112, for example, discoloration, deformation, A specific observation area 118 to be observed in detail, in which a mutation such as alteration is recognized, is extracted from the preliminary observation area 117 and specified (step S104). In this case, the mutation to be detected may be found by dark field image observation or may be found by CARS light observation.

  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, based on the surface thereof, depending on the number of the specific observation regions 118. The position information linked to the position can be acquired. Thereby, in the detailed CARS light observation mentioned later, the specific observation area | region 118 can be observed reliably and rapidly.

FIG. 9 is a schematic view illustrating the distribution of the detailed observation region 119 in the sample 112 in the detailed CARS light observation. Each of the detailed observation areas 119 in the sample 112 includes a specific observation area 118 whose position is specified in the preliminary CARS light observation. The position of the specific observation region 118 is, for example, the position information in the depth direction of the sample 112 linked to the depth of the sample 112 acquired by the scanning position by the galvano scanner 171 and the stage scanner 111 and the preliminary CARS light observation. It can be specified based on Z ₁ , Z ₂ , 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 CARS light observation in the detailed observation region 119, the scanning speed of the excitation light by the galvano scanner 171 may be lowered. Thereby, in the detailed observation area | region 119, the specific observation area | region 118 and its periphery can be observed with the high resolution of CARS light observation. As described above, in the microscope 100, the control unit 120 controls the stage scanner 111 and the galvano scanner 171 to form a second observation system that acquires an image of the observation region in the sample 112.

  CARS light observation can also form an image reflecting the three-dimensional distribution of molecules 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 distance in the depth direction of the sample 112 in the preliminary CARS light observation.

  FIG. 10 is a schematic view 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 preliminary CARS light observation shown in the figure, each of the preliminary observation regions 117 arranged in a three-dimensional manner at intervals in the sample 112 has a width in the depth direction of the sample 112. Thereby, in preliminary CARS light observation, it is possible to grasp the tendency of spatial distribution such as mutation in each specific observation area 118 extracted and specified from the preliminary observation area 117.

  FIG. 11 is a schematic view illustrating the shape of the detailed observation region 119 specified by the preliminary CARS light observation using the preliminary observation region 117 as described above. Each of the illustrated detailed observation regions 119 includes a corresponding preliminary observation region 117 inside, and is formed in a three-dimensionally wider range than the preliminary observation region 117. As a result, the periphery of the preliminary observation region 117 in which a mutation has occurred can be observed in detail, and an image of the mutation that has occurred in the sample 112 can be acquired quickly and reliably.

  Note that the width (height) in the depth direction of the detailed observation region 119 shown in the figure may be determined by the interval in the depth direction of the sample 112 between the three-dimensionally arranged preliminary observation regions 117. The figure shows a case where the interval in the depth direction of the preliminary observation region 117 is set to one time. Alternatively, the specific observation region 118 may be arranged in the center and its width may be twice the interval. As a result, regarding the width in the depth direction of the detailed observation region 119 in the sample 112, the region that can be missed is sufficiently minimized.

  FIG. 12 is a diagram illustrating a setting when the phase difference observation is performed on the sample 112 using the microscope 100. Note that the settings shown in FIG. 12 are the same as those of the microscope 100 shown in FIG. Therefore, common elements are denoted by the same reference numerals, and redundant description is omitted.

The microscope 100 used when dark field observation and CARS light observation are used together removes the aperture stop 156 incorporated in the image detection unit 150 as the second optical member, and arranges the phase ring 146 instead. It can also be used for phase difference observation. Phase ring 146 is a position P ₃ optically conjugate of the exit pupil of the lower objective lens 144 are incorporated in a position P _4, which can real image of the exit pupil. In general, the diameter of the ring stop 134 used for phase difference observation is smaller than the ring stop 134 used for dark field observation.

  By setting as described above, the phase difference observation of the sample 112 placed on the stage 110 can be performed using the microscope 100. Similarly to the setting shown in FIG. 1, CARS light observation can be performed simultaneously while maintaining the phase difference observation setting.

Incidentally, between the lower objective lens 144 and the dichroic mirror 191, a position P ₃ optically conjugate of the exit pupil of the lower objective lens 144, a phase ring 146 is disposed at a position that allows real image of the exit pupil Even CARS light observation can be executed. 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 reduced. To do.

  FIG. 13 is a diagram illustrating a setting when the differential interference observation is performed on the sample 112 using the microscope 100. The settings shown in FIG. 13 are the same as those of the microscope 100 shown in FIG. Therefore, common elements are denoted by the same reference numerals, and redundant description is omitted.

  In the illustrated microscope 100, a polarizer 232 and a Wollaston prism 234 are incorporated in the illumination unit 130 instead of the ring diaphragm 134 as a first optical member. In the microscope 100 with this setting, a Wollaston prism 236 and an analyzer 238 are incorporated in the image detection unit 150 as the second optical member, instead of the aperture stop 156. As a result, the differential interference observation of the sample 112 can be performed using the microscope 100. The Wollaston prisms 234 and 236 may be Nomarski type.

The Wollaston prism 234 is in the vicinity of P ₂ determined so that the intersection of the ordinary ray and the extraordinary ray separated thereby is an optically conjugate position P ₂ with the exit pupil position P _{1 of the} upper objective lens 142. The polarizer 232 is disposed on the side of the illumination light source 131 immediately adjacent thereto. The polarizer 232 converts the illumination light generated by the illumination light source 131 into linearly polarized light, and the Wollaston prism 234 separates the illumination light into two linearly polarized lights whose vibration directions are orthogonal to each other.

Wollaston prism 236, thereby to be of the ordinary ray and the extraordinary ray intersection of lower objective position of the exit pupil of the lens 144 P ₃ and optically P ₄ that is determined to be a conjugate position P ₄ synthesis The analyzer 238 is incorporated in a nearby position, and is disposed on the side of the CCD camera 158 immediately adjacent thereto. The Wollaston prism 236 combines the two separated linearly polarized lights 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 linearly polarized lights separated by the Wollaston prism 234 on the illumination unit 130 side pass through the sample 112. A phase difference corresponding to the phase distribution of the image detection unit 150 is generated, and interference occurs when passing through the Wollaston prism 236 and the analyzer 238 on the image detection unit 150 side. Therefore, a contrast corresponding to the differential amount of the refractive index distribution of the sample 112 is generated in the image detected by the image detection unit 150.

  FIG. 14 is a diagram illustrating settings when the sample 112 is observed with modulation contrast using the microscope 100. The settings shown in FIG. 14 are the same as those of the microscope 100 shown in FIG. Therefore, common elements are denoted by the same reference numerals, and redundant description is omitted.

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

Slit diaphragm 235 has a rectangular slit which is incorporated in the position P ₁ and the optically conjugate position P ₂ of the exit pupil of the upper objective lens 142, is open to the outside from the optical axis position. The modulator 233 is incorporated at a position P ₄ optically conjugate with the exit pupil position P ₃ of the lower objective lens 144. The slit diaphragm 235 and the modulator 233 are disposed at optically conjugate positions. The modulator 233 has a transparent region that is transparent to the transmitted light, a gray region that attenuates and transmits the transmitted light, and a dark region that blocks the transmitted light. The gray region of the modulator 233 is arranged at a position where a 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 is changed, so that the detected image detected by the image detection unit 150 is light 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 disposed 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 illustrating another setting in the case where the sample 112 is observed 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. Therefore, common elements are denoted by the same reference numerals, and redundant description is omitted.

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

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 out of the light emitted from the sample 112. To do. Thereby, 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 wavelength characteristics described in Table 2 below, for example.

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

  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 parts described below. Therefore, common elements are denoted by the same reference numerals, and redundant description is omitted.

  The microscope 102 has a common stage 110, control unit 120, objective optical system 140, one observation system including an illumination unit 130 and an image detection unit 150, a laser device 160, an excitation unit 170, and upper CARS light detection. 1 and the other observation system including the lower CARS light detection unit 190, and is common to the microscope 100 shown in FIG.

  Compared with the microscope 100, in the microscope 102, the illumination unit 130 that forms the first irradiation unit has an excitation unit 170 that serves as a second irradiation unit, an upper objective lens 142, and a lower objective. They are arranged on the same side in the direction parallel to the optical axis of the lens 144. The image detection unit 150 is disposed adjacent to the upper CARS light detection unit 180 on the opposite side of the stage 110 with respect to the illumination unit 130 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. Furthermore, since the laser device 160 and the illumination unit 130 that are each a heat source and consume large power can be arranged together, thermal and power management becomes easy.

  As mentioned above, although this invention was demonstrated using embodiment, the technical scope of this invention is not limited to the range as described in the said embodiment. It will be apparent to those skilled in the art that various modifications or improvements can be added to the above-described embodiment. It is apparent from the scope of the claims that the embodiments added with such changes or improvements can be included in the technical scope of the present invention.

  The order of execution of each process such as operations, procedures, steps, and stages in the apparatus, system, program, and method shown in the claims, the description, and the drawings is particularly “before” or “prior to”. It should be noted that it can be realized in any order except when it is clearly indicated, and when the output of the previous process is used in the subsequent process. Regarding the operation flow in the claims, the description, and the drawings, even if it is described using “first”, “next”, etc. for convenience, it means that it is essential to carry out in this order. It is not a thing.

100, 101, 102 Microscope, 110 stage, 111 stage scanner, 112 samples, 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 Illuminator, 131 Illumination light source, 132 Collector lens, 133, 135, 138, 154, 157, 182, 183, 192, 193 Relay lens, 134 Ring diaphragm, 136 Field diaphragm, 137, 174 Reflector, 139, 155 bands Pass 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 stop, 158 CCD camera 151, 159, 181, 191 Dichroic mirror, 160 laser device, 161, 162 laser light source, 163 combiner, 170 excitation unit, 171 galvano scanner, 172 scan lens, 180 upper CARS light detection unit, 184, 194 band pass filter, 190 Lower CARS photodetector, 185, 195 Photomultiplier tube, 231 Polarizer, 232 Polarizer, 233 Modulator, 234, 236 Wollaston prism, 235 Slit stop, 238 Analyzer

Claims (14)

A first observation system for observing the observation object and acquiring information associated with an observation region inside the observation object;
An observation apparatus comprising: a second observation system that observes an observation region specified based on the information acquired by the first observation system.   The observation apparatus according to claim 1, wherein the first observation system has a wider observation range per unit time than the second observation system. The observation range of the first observation system includes the surface of the observation target,
The observation apparatus according to claim 2, wherein the information includes information for specifying a spatial region where the observation target exists. The first observation system observes a plurality of preliminary observation areas arranged at intervals in the observation target;
The observation apparatus according to claim 2, wherein the second observation system observes a main observation region including at least one of the plurality of preliminary observation regions.   The observation apparatus according to claim 4, wherein the plurality of preliminary observation areas are three-dimensionally arranged in the observation target.   The observation apparatus according to claim 4, wherein a range of the main observation region is limited to the inside of the observation target based on the information.   The observation according to any one of claims 4 to 6, wherein the main observation region includes at least one of the plurality of preliminary observation regions and has a wider observation range than one of the plurality of preliminary observation regions. apparatus. The first observation system includes:
A first irradiation unit for generating irradiation light for irradiating the observation target;
The observation apparatus according to claim 1, further comprising: a first detection unit configured to detect emission light emitted from the observation target irradiated with the irradiation light by a linear optical phenomenon.   The observation apparatus according to claim 8, wherein the first observation system observes the observation object by any one of dark field observation, phase difference observation, differential interference observation, and modulation contrast observation.   The observation device according to any one of claims 1 to 9, wherein the second observation system has a higher resolution than the first observation system. The second observation system is
A second irradiation unit for generating irradiation light for irradiating the observation target;
The observation apparatus according to claim 10, further comprising: a second detection unit configured to detect emission light emitted from the observation target irradiated with the irradiation light by a nonlinear optical phenomenon.   The observation device according to claim 11, wherein the second detection unit detects any of third harmonics, stimulated Raman scattering light, and CARS light generated in the observation target.   The observation apparatus according to any one of claims 1 to 12, wherein the observation object is a cell sheet including a plurality of cells. A first observation stage of observing the observation object and obtaining information associated with an observation area inside the observation object;
An observation method comprising: a second observation step of observing an observation region specified based on the information acquired in the first observation step.