JP2016206652A - Imaging method of three-dimensional structure of sample and microscope device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technology that makes it possible to simply photograph a three-dimensional structure of a sample without complicating computation processing in a localization microscopy.SOLUTION: A method photographing a three-dimensional structure of a sample is configured to: label a substance capable of switching a first state having fluorescence and a second state not having the fluorescence when excited by light of a prescribed wavelength; illuminate the substance with the light of the prescribed wavelength using an illumination optical system; excite at least a part of the substance put in the first state; use an image formation optical system to image-form the excited substance; acquire a plurality of images having parallax occurring in a process of forming the image; specify a position of the substance in the plurality of images in an in-plane direction perpendicular to an optical axis in a sample; use the position of the substance in the plurality of images in the in-plane direction perpendicular to the optical axis in the sample to specify a position of the substance in the optical axis direction in the sample; repeat processes from a process of exciting the substance to a process of specifying the position of the substance in the optical axis direction; and obtain a three-dimensional structure of an entire sample.SELECTED DRAWING: Figure 2

Description

  The present invention relates to a method for imaging a three-dimensional structure of a sample by super-resolution microscopy.

  It is known that high resolution exceeding the diffraction limit of visible light can be achieved by super-resolution microscopy. In localized microscopy, which is one of super-resolution microscopy, when a fluorescent substance in the field of view is excited, fluorescence is emitted randomly from the substance. This is imaged in a plurality of times on the image sensor using an imaging optical system, and the position information of the bright spot of each image is obtained. Then, by superimposing the bright spot information detected in each image on one image, an image having a fine structure exceeding the resolution by the wavelength of visible light is obtained.

Conventionally, there also exists a technique for obtaining a three-dimensional image of a sample by using a local microscope.
For example, Non-Patent Document 1 is a method using a cylindrical lens. In the method using the cylindrical lens, astigmatism generated by arranging the cylindrical lens on the optical path is used. That is, the light from the sample has an elliptical shape due to astigmatism, and the ellipticity changes depending on the coordinates in the z direction. From this, the information on the position in the z direction, that is, the height is obtained from the ellipticity, and the height information in the z direction is added to the image in the xy direction obtained by the local microscope, thereby generating a three-dimensional image.

  Non-Patent Document 2 is a method using a double helix. In the method using a double helix, PSF (Point Spread Function) is formed into a double helix by performing phase modulation. For this reason, two points appear in the image on the xy plane for a certain z, and the rotation angle of the two points depends on the coordinates in the z direction. Thus, height information is obtained from the relationship between the two rotation angles and the z-direction position, that is, height information, and the z-direction height information is added to the xy-direction image obtained by the local microscopy. A three-dimensional image is generated.

“Three-Dimensional Super-Resolution Imaging by Stochastic Optical Reconstruction Microscopy”, Science, 2008 February 8, Vol.319 no.5864 pp.810-813 “Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function”, PNAS, 2009 January 9, Vol.106 pp.2995-2999

  In the method of generating a three-dimensional image by local microscopy using the above-mentioned cylindrical lens or double helix, the xy position of the bright spot can be obtained by Gaussian fitting or centroid detection as in the case of normal local microscopy. Can not. That is, since the PSF shape changes depending on the z position, calculation processing such as fitting for obtaining the xy position of the bright spot becomes complicated.

  In the local microscopy, if the bright spot density is low, a large number of images are required, which increases the imaging time. Therefore, in order to shorten the imaging time, it is necessary to adjust the experimental conditions such as labeling (that is, fluorescent labeling) of the fluorescent substance so that the bright spot density is increased, and similarly, the xy position of the bright spot is obtained. Therefore, calculation processing such as fitting becomes complicated.

  It is an object of the present invention to provide a technique that enables easy imaging of a three-dimensional structure of a sample without complicating arithmetic processing in localized microscopy. It is a second object of the present invention to provide a technique that enables easy imaging of a three-dimensional structure of a sample without complicating arithmetic processing even when the bright spot density is high.

  According to the method for imaging a three-dimensional structure of a sample according to one aspect of the present invention, a first state having fluorescence and a second state having no fluorescence are excited when excited by light of a predetermined wavelength. Labeling the structure of the sample with a switchable substance, and illuminating the substance using an illumination optical system with light of the predetermined wavelength to excite at least a part of the substance in the first state Forming an image of the excited substance using an imaging optical system, obtaining a plurality of images having parallax generated in the imaging process, and the substance of the substance in the plurality of images Identifying the position of the substance in the sample using the step of identifying the position of the substance in the in-plane direction perpendicular to the optical axis in the sample and the position of the substance in the in-plane direction of the substance in the plurality of images A step of specifying a position in the direction, and a step of exciting the substance to a step of specifying the position of the substance in the optical axis direction in the sample to obtain a three-dimensional structure of the whole sample. It is characterized by having.

  According to the present invention, it is possible to easily image a three-dimensional structure of a sample without complicating arithmetic processing in a localized microscope. Further, even when the bright spot density is high, it is possible to easily image the three-dimensional structure of the sample without complicating the arithmetic processing.

1 shows a schematic configuration of a binocular stereomicroscope equipped with a Galileo parallel optical system. It is a figure explaining the method of obtaining a three-dimensional super-resolution image. It is a schematic block diagram of a TIRF microscope apparatus. It is a schematic block diagram of the microscope apparatus in the case of using a polarizing plate. It is a schematic block diagram of the other microscope apparatus in the case of using a polarizing plate. It is the flowchart which showed the imaging process of the three-dimensional structure of the sample by the microscope apparatus which concerns on 1st Embodiment. It is a schematic block diagram of the microscope apparatus which concerns on 2nd Embodiment. It is another schematic block diagram of the microscope apparatus which concerns on 2nd Embodiment. It is a schematic block diagram of the microscope apparatus which concerns on 2nd Embodiment to which sheet illumination is applied. It is a schematic block diagram of the microscope apparatus which can switch an eccentric position. It is a schematic block diagram of the microscope apparatus which employ | adopted the disk scanning system. It is a schematic block diagram of the microscope apparatus to which sheet illumination is applied. It is the flowchart which showed the imaging process of the three-dimensional structure of the sample by the microscope apparatus which concerns on 3rd Embodiment.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.
<First Embodiment>
(Galileo microscope)
FIG. 1 is a schematic configuration diagram of a microscope apparatus according to the present embodiment. FIG. 1 shows a schematic configuration of a binocular stereomicroscope equipped with a Galileo parallel optical system.

  First, the structure of the sample is labeled with a substance capable of switching between a first state having fluorescence at a predetermined wavelength and a second state having no fluorescence.

  The light source 2 emits light having a predetermined wavelength. The light emitted from the light source 2 is applied to the sample surface 5 through the illumination optical system 3. The excitation light is reflected by a dichroic mirror (DM) 6 and irradiated onto the sample surface 5 through the objective lens 4. When the excitation light is irradiated, a part of the substance labeling the sample, that is, the substance in the first state is excited. This also applies to the microscope apparatus described below.

  Fluorescence is emitted from the excited material and enters the camera 8 through the two imaging lenses 7A and 7B. The camera 8 is disposed at the imaging position of the imaging lenses 7A and 7B. In this way, the image signal of the sample is acquired by the imaging element of the camera 8, and the obtained image signal is transferred to an information processing apparatus such as a personal computer (PC) 9. The PC 9 performs necessary processing on the image signal transferred from the camera 8 to obtain a super-resolution image of the sample.

  That is, since the fluorescent material is excited on the sample surface 5 and shines at random, when the camera 8 captures an image a plurality of times, an image of a bright spot that shines at each timing is obtained. The PC 9 generates a single image by superimposing the positional information on the XY plane of each bright spot of the plurality of images obtained in this way. Thereby, the structure of the sample can be imaged with a high resolution exceeding the resolution of the light wavelength of a normal microscope apparatus.

  Here, the microscope apparatus shown in FIG. 1 is a binocular stereomicroscope, and parallax is generated between two optical paths (optical paths 10A and 10B) obtained by dividing light from the structure in the sample. For this reason, in the microscope apparatus according to the present embodiment, the position information of the sample in the Z direction, that is, the parallax in the two images (hereinafter referred to as image pairs) obtained through the imaging lenses 7A and 7B, respectively, Calculate the height information of the sample. This will be described with reference to FIG.

FIG. 2 is a diagram for explaining a method for obtaining a three-dimensional super-resolution image by the microscope apparatus according to the present embodiment.
By utilizing the fact that fluorescence is randomly emitted from a substance by excitation light, the XY direction structure of the sample can be obtained with high resolution. It should be noted that when calculating the X-direction and Y-direction structures, that is, the position information of the bright spot, a calculation may be made using a known multi-emitter compatible algorithm. Examples of multi-emitter compatible algorithms include Bayesian estimation and compressed sensing. As described above, the microscope apparatus according to the present embodiment calculates the position information in the Z direction of the sample using the parallax of the acquired image pair, and converts the position information into the XY direction structure obtained by applying a known algorithm or the like. In addition, super-resolution image data of the three-dimensional structure of the sample is obtained.

  Specifically, the parallax Δx of the image pair (images 1 and 2) obtained by imaging at the timing of n = k is converted into position information in the Z direction, and the bright spot imaged at n = k Height information. In the example shown in FIG. 2, the height information in the Z direction is represented according to the scale.

  In FIG. 2, for simplicity of explanation, the parallax Δx for one bright spot is shown in the image pair obtained at the timing of n = k. To obtain the height information of each bright spot in the image.

  Similarly, for the image pair obtained by imaging at the next timing n = k + 1, the parallax is converted into position information in the Z direction, and the height information of the bright spot in the image pair obtained at n = k + 1 is obtained. Get.

  In this way, imaging is performed a plurality of times as in the conventional super-resolution microscopy, and one image is generated by superimposing position information in the X, Y, and Z directions obtained at each imaging timing n. Thus, a super-resolution three-dimensional shape image of the sample is obtained.

  According to the conventional localized microscope, when obtaining a super-resolution three-dimensional image, the calculation processing for obtaining positional information in the X, Y, and Z directions is very complicated. It was. However, according to the present embodiment, the position information in the Z direction is obtained from each of the pair of images with parallax, and the processing for matching with the position information in the X and Y directions obtained by performing the imaging a plurality of times as in the past is performed. It is possible to image a three-dimensional structure of the entire sample with high resolution. That is, a three-dimensional super-resolution image can be easily generated without complicating the arithmetic processing.

Here, a description will be given of a case where the bright spot density is increased by adjusting the experimental conditions in order to shorten the imaging time by reducing the number of images taken by the local microscopy.
An algorithm for accurately obtaining the position of each bright spot is known in “Simultaneous multiple-emitter fitting for single molecule super-resolution imaging”, Biomedical Optics Express, 2011 May 1, Vol. 2 pp.1377-1393 and the like. An algorithm that can be used even when a plurality of bright spots are included in the analysis region is called a multi-emitter correspondence algorithm. When this algorithm is applied, the position of each bright spot can be detected even when each bright spot is not sufficiently separated from other bright spots. Therefore, a large number of bright spot positions can be obtained by one imaging, and the total number of imaging necessary for obtaining a super-resolution image can be reduced. Examples of such multi-emitter compatible algorithms include Bayesian estimation and compressed sensing.

  When the position information in the X direction and the Y direction is calculated using the above-described multi-emitter algorithm, bright spots can be detected with high density. Thereby, the imaging time of a super-resolution image can be shortened. By calculating the position information in the Z direction from the parallax of the image pair and using it together with the position information in the X and Y directions obtained by the multi-emitter compatible algorithm, the high-density imaging time can be shortened and simplified. A three-dimensional structure of the sample can be obtained by calculation.

  In the above, as a mechanism for emitting fluorescence at random, any existing method such as PALM, STORM, dSTORM, and spontaneous blinking can be used. In addition, the state where the bright spot density is high is a distance within the spatial resolution of image formation from the nearest substance in which at least a part of the substance capable of emitting fluorescence (first state) is in the first state. It is a state excited by.

  In the above description, the Galileo microscope of FIG. 1 has been described as an example. However, a method of generating a three-dimensional super-resolution image in the localized microscope method according to the present embodiment is performed by using another type of microscope apparatus. Can also be realized. This will be specifically described below.

(TIRF (Total Internal Reflection Fluorescence) microscope)
FIG. 3 is a schematic configuration diagram of the TIRF microscope apparatus.
In the TIRF microscope, the light emitted from the light source 2 is excited through the illumination optical system 3. The excitation light is condensed at a position deviated from the optical axis of the rear focal plane (pupil plane) of the objective lens 4 through the DM 6 and irradiated onto the sample surface 5. At this time, the sample surface 5 is arranged so as to have total reflection illumination. In the TIRF microscope, parallax exists in two images obtained by imaging the light transmitted through the imaging lenses 7A and 7B with the imaging element of the camera 8. Thus, similar to the Galileo microscope of FIG. 1, the PC 9 can obtain an image pair with parallax.

  The parallax Δx of each bright spot is converted into position information in the Z direction to obtain height information of each bright spot, and the height information is similarly obtained for a plurality of image pairs. A resolution image can be obtained. About the method, it is the same as that of said Galileo type | mold microscope, and is as having demonstrated with reference to FIG.

  In the TIRF microscope, since only a finite range is illuminated with respect to the optical axis direction of imaging, only the substance near the sample surface 5 is excited. For this reason, when the sample has a thick shape of a predetermined thickness or more in the Z direction, a TIRF microscope that can suppress background light from outside the focal plane is suitable.

(Method using polarizing plate)
In addition to the Galileo microscope of FIG. 1 and the TIRF microscope of FIG. 3, it is also possible to obtain an image with parallax by the configuration of the optical system of the microscope, and similarly obtain a three-dimensional shape image.
FIG. 4 is a schematic configuration diagram of a microscope apparatus when a polarizing plate is used. As shown in the optical path diagram of FIG. 4, when the sample surface 5 is irradiated with excitation light emitted from the light source 2 and excited by the illumination optical system 3, the fluorescence emitted from the sample surface 5 passes through the DM 6. Thus, only light polarized in a predetermined direction passes through the polarizing plates 11A and 11B. In the embodiment, the polarizing plate 11A of FIG. 4 which is one polarizing plate transmits only s-polarized light, and the polarizing plate 11B which is the other polarizing plate transmits only p-polarized light. In this way, the light separated for each polarization component by the polarization beam splitter (PBS) 12 enters each of the two cameras 8A and 8B. Each of the cameras 8A and 8B images the light of the s-polarized component or the p-polarized component, and the PC 9 generates an image of the s-polarized component or the p-polarized component from the image signal transferred from the cameras 8A and 8B. In the embodiment, the camera 8A images the light of the s-polarized component, the camera 8B images the light of the p-polarized component, and the PC 9 images the s-polarized component and the p-polarized component from the image signals transferred from the cameras 8A and 8B, respectively. Is generated.

  Here, the images of the s-polarized component and the p-polarized component are imaged through different pupil positions, so there is parallax. Therefore, even with the microscope apparatus having the configuration shown in FIG. 4, a plurality of image pairs are obtained using the parallax generated in the imaging step, and the three-dimensional super-resolution image of the sample is obtained by the method described in FIG. Can be obtained.

  FIG. 5 is a schematic configuration diagram of another microscope apparatus using a polarizing plate. The microscope apparatus shown in FIG. 5 differs from that shown in FIG. 4 in that one camera 8 images two polarization components, p-polarized light and s-polarized light.

  Similar to the microscope having the configuration shown in FIG. 4, the s-polarized light component transmitted through the polarizing plate 11A and the p-polarized light component transmitted through the polarizing plate 11B are separated by the PBS 12 and reflected by the mirror 18A and the mirror 18B, respectively. The light enters one camera 8. The camera 8 in FIG. 5 can simultaneously capture an image pair in which parallaxes of the s-polarized component and the p-polarized component exist. Therefore, similarly to the microscope having the configuration shown in FIG. 4, a plurality of image pairs can be acquired and a three-dimensional super-resolution image of the sample can be generated by the method described in FIG.

  FIG. 6 is a flowchart showing an imaging process of the three-dimensional structure of the sample by the microscope apparatus according to the present embodiment. FIG. 6 shows an operation flow until the above-described microscope apparatus configured to acquire an image pair having parallax simultaneously obtains three-dimensional super-resolution image data of the entire sample.

  First, in step S1, a substance to be used for observation is selected, and the structure of the sample is labeled with the selected substance. In step S2, a part of the substance, that is, the substance in the first state is excited, and in step S3, the fluorescence emitted from the excited substance is applied to the image sensor of the camera 8 using the imaging lens 7. Make an image. Thereby, an image pair with parallax is acquired. In step S4, the excitation process in step S2 and the imaging process in step S3 are repeated a plurality of times until it is determined that imaging has been completed. If it is determined that the imaging has been completed, the process proceeds to step S5.

  In step S5, position information in the X and Y directions of the bright spot in each image is calculated. In step S6, for each image pair, the parallax is obtained from the position information in the X direction and the Y direction obtained in step S5, and the position information in the Z direction is calculated therefrom. Thus, when the position information in the X, Y, and Z directions is acquired, the process ends.

  In FIG. 6, first, a plurality of image pairs are acquired by the processing from step S2 to step S4, and thereafter, in step S5 and step S6, the position information of the bright spot in the X, Y and Z directions is obtained from each image pair. Although it is seeking, it is not limited to this. For example, each time an image pair is acquired, the position information in the X, Y, and Z directions of the bright spot is obtained from the acquired image pair, and this operation is repeated to obtain image data of a three-dimensional structure of the entire sample. You can also

  As described above with reference to FIG. 2, the series of processing shown in FIG. 6 eliminates the need for complicated calculations, and allows simple calculations to be performed in the X, Y, and Z directions of the bright points of a plurality of image pairs. Position information can be obtained, and thereby a three-dimensional structure of the entire sample can be obtained with high resolution. That is, a three-dimensional super-resolution image of the sample can be obtained.

<Second Embodiment>
(Long focal depth)
As described above, in the first embodiment described above, the measurement by the stereo method is applied to the local microscope, and the image pair with parallax is used, thereby imaging the three-dimensional structure with high resolution. It is carried out. This embodiment is different from the microscope apparatus according to the first embodiment in that a means for further increasing the depth of focus is provided. This will be specifically described with reference to the drawings.

FIG. 7 is a schematic configuration diagram of the microscope apparatus according to the present embodiment.
The difference between the configuration of the microscope apparatus according to the present embodiment and that of the first embodiment is that an axicon lens 21 is disposed in the imaging optical system as shown in FIG. Since the axicon lens 21 is arranged in the imaging optical system, the depth of focus increases.

  In localized microscopy, which is one of the super-resolution microscopy methods, the farther away from the focal position in the Z direction, the stronger the influence of blur light (the greater the blur), and the lower the localization accuracy. On the other hand, as shown in FIG. 7, when the axicon lens 21 is arranged in the imaging optical system, the microscope apparatus has a long focal depth. That is, the arrangement of the axicon lens 21 changes the shape of the PSF of the microscope apparatus into a shape extending in the Z direction, thereby avoiding deterioration in localization accuracy due to a blurred image, and a thick sample. Also, super-resolution in the Z direction can be obtained.

  Here, the performance degradation due to the blur light and the resolution in the Z direction are in a trade-off relationship, and the resolution in the Z direction is lowered when the depth of focus is increased. However, in this embodiment, the measurement by the stereo method is performed in combination with the local microscope. For this reason, in addition to being compatible with known multi-emitter analysis that does not require complicated calculations as in the first embodiment, it is possible to suppress blur light by increasing the depth of focus and The effect is obtained that resolution can be obtained independently.

  In the configuration diagram shown in FIG. 7, the division of light for obtaining an image pair with parallax is performed after the long focal depth is increased by the axicon lens 21. The light induced by the axicon lens 21 to increase the focal depth of the image is divided by the beam splitter 22, guided by the mirrors 18 </ b> A, 18 </ b> B, etc., and enters the camera 8. However, the timing of dividing the light is not limited to this, and may be performed before the long focal depth is increased.

  FIG. 8 is another schematic configuration diagram of the microscope apparatus according to the present embodiment. In the configuration shown in FIG. 8, axicon lenses 21 </ b> A and 21 </ b> B are arranged on individual optical paths after being divided into two by the beam splitter 22.

  As shown in FIG. 8, even when the individual focal depth is increased by the axicon lenses 21A and 21B after the light from the sample is divided by the beam splitter 22, the long focal depth is increased before the division of FIG. The effect similar to the structure to be made can be acquired.

  In the first embodiment, a microscope apparatus provided with various illumination optical systems has been described. However, the microscope apparatus according to the present embodiment is characterized in that an axicon lens 21 is disposed in the imaging optical system. It is what. For this reason, as in this embodiment, the method of increasing the depth of focus by disposing the axicon lens 21 in the imaging optical system is applied to the various microscope apparatuses described in the first embodiment. Is possible.

  Furthermore, by adopting a configuration that further increases the depth of focus for a microscope apparatus that applies measurement by the stereo method to local microscopy, for example, the level required for sectioning resolution of a sectioning microscope is compared with the conventional level. It can also be lowered. This will be described with reference to FIG.

FIG. 9 is a schematic configuration diagram of a microscope apparatus according to this embodiment to which sheet illumination is applied.
The specific configuration and operation of the microscope apparatus to which the sheet illumination is applied will be described in a third embodiment described later. In the sheet illumination, the sample is illuminated with the light excited by the excitation objective lens 4A. The detection objective lens 4B detects fluorescence. The excitation objective lens 4A spreads the laser light in a sheet shape and illuminates it from the side of the sample (upward direction in FIG. 9), photographs the plane from above (right direction in FIG. 9), and moves the sample in the Z direction ( By moving in the vertical direction in FIG. 9, even a thick sample can be observed.

  In the microscope apparatus to which such sheet illumination is applied, when the axicon lens 21 is arranged in the imaging optical system as shown in FIG. 9, the accuracy of the image is hardly deteriorated due to the influence of the blur light due to the long focal depth. Become. That is, since the background light of the image is suppressed and even a thick sample can be an observation target, the hurdle of sectioning ability required for a sectioning microscope such as a microscope apparatus to which sheet illumination is applied is used as an axicon lens 21. It can be lowered compared to the case without it.

  In addition, the axicon lens 21 is also arranged in the imaging optical system for a microscope using a TIRF (see FIG. 3) described above, a temporal focus optical system or HiLo (thin oblique illumination light), and a combination thereof. The effect of increasing the depth of focus can be enjoyed.

<Third Embodiment>
In the first embodiment described above, the microscope apparatus simultaneously captures image pairs with parallax. On the other hand, the present embodiment is different in that an image pair is obtained by performing imaging in a plurality of times. Similarly to the first embodiment, the microscope apparatus according to the present embodiment can have various configurations. Hereinafter, specific description will be given with reference to the drawings.

(Method by switching the eccentric opening)
FIG. 10 is a schematic configuration diagram of a microscope apparatus capable of switching the eccentric position.
The light emitted from the light source 2 is irradiated onto the sample surface 5 by the illumination optical system 3. The excitation light is irradiated onto the sample surface 5, and the fluorescence emitted from the substance excited by the excitation light passes through the DM 6 and passes through the eccentric opening 14A of the member 13A or the eccentric opening 14B of the member 13B and passes to the camera 8. To be imaged.

  The members 13A and 13B are respectively provided with eccentric openings 14A and 14B on the upper side and the lower side in FIG. 10 with respect to the optical axis. The PC 9 switches between the two members 13A and 13B arranged on the optical path. Each member is provided with eccentric apertures 14A and 14B, which transmit light rays at different pupil positions. The transmitted light is imaged by the imaging lens 7 and enters the camera. The imaging element of the camera 8 images light transmitted through the eccentric openings 14A and 14B of the members 13A and 13B, respectively.

  By providing such a configuration, an image pair with parallax can be obtained in the same manner as the microscope apparatus having the configuration including the two polarizing plates 11A and 11B in FIG. Here, it is preferable that the two images with parallax are simultaneously taken as in the first embodiment from the viewpoint of the detection accuracy of the bright spot. However, if the imaging interval between two images is about milliseconds, it is possible to acquire an image pair with relatively high accuracy. Therefore, even in the microscope apparatus having the configuration shown in FIG. 10, a plurality of image pairs are acquired by imaging, and the position information in the Z direction of each bright spot is obtained from the parallax as in the first embodiment. Accordingly, it is possible to easily obtain a three-dimensional super-resolution image of the sample without complicating the arithmetic processing.

(Disk scan method)
FIG. 11 is a schematic configuration diagram of a microscope apparatus that further employs a disk scanning method in the configuration of FIG.
The light emitted from the light source 2 enters the objective lens 4 through the microlens array 16 and the rotating disk 15 that rotate about the axis 19. A large number of pinholes are spirally arranged on the rotating disk 15. The microlens array 16 also has a microlens arranged in a spiral so as to be paired with the pinholes of the rotating disk 15, thereby condensing the light emitted from the light source 2 into the pinholes.

  When the rotating disk 15 (and the microlens array 16) is rotated around the axis 19, the light passing through a large number of pinholes is sequentially irradiated onto the sample surface 5 through the objective lens 4. Thereby, the same effect as the case where the sample surface 5 is scanned can be obtained, and the camera 8 can perform imaging at high speed.

  As shown in the figure, also in the configuration of FIG. 11, members 13A and 13B having eccentric openings 14A and 14B, respectively, are provided on the optical path, as in FIG. By switching and imaging the members 13A and 13B arranged on the optical path according to the control of the PC 9, each transmits light rays at different pupil positions as in the microscope apparatus of FIG. Can be obtained. Therefore, even a microscope apparatus that employs a disk scanning method can achieve the same effects as the microscope apparatus of FIG.

  However, in the disk scanning method, only a finite range is imaged in the optical axis direction. For this reason, when the sample has a shape of a predetermined thickness or more in the Z direction, a microscope employing a disk scanning method is suitable.

  FIG. 11 illustrates a microscope apparatus when the disk scanning method is adopted in the configuration of FIG. 10, but is not limited to this. For example, it is possible to use a disk scanning method for the microscope apparatus according to the first embodiment.

(Sheet lighting)
FIG. 12 is a schematic configuration diagram of a microscope apparatus in which sheet illumination is further applied to the configuration of FIG.
In the microscope apparatus described so far, the objective lens 4 performs excitation light irradiation and fluorescence detection. On the other hand, in the sheet illumination 20, the excitation objective lens 4A performs excitation light irradiation, and the detection objective lens 4B disposed perpendicular to the excitation objective lens performs fluorescence detection. The cylindrical lens 17 in FIG. 12 is provided to squeeze the excitation light beam and illuminate the sheet surface on the sample surface (not shown in FIG. 12).

  Even in a microscope apparatus that employs the sheet illumination 20, similar to the microscope apparatus having the configuration shown in FIG. 10 or FIG. 11, the aperture plates 13A and 13B are switched and images are transmitted from the components transmitted through the eccentric openings 14A and 14B, respectively. To obtain an image pair with parallax. By using the sheet illumination 20, since the sample is irradiated with the sheet-like excitation light, a three-dimensional super-resolution image of the sample can be generated while suppressing damage to the sample.

  Since the sheet illumination 20 illuminates only a finite range with respect to the optical axis direction of imaging, only the substance near the sample surface 5 is excited. For this reason, when the sample has a thin shape having a predetermined thickness or more in the Z direction, a microscope using the sheet illumination 20 is suitable.

  In addition, in FIG. 12, although the microscope apparatus at the time of applying the sheet illumination 20 to the structure of FIG. 10 is illustrated, it is not limited to this. For example, the sheet illumination 20 can be applied to the microscope apparatus according to the first embodiment.

  FIG. 13 is a flowchart showing an imaging process of the three-dimensional structure of the sample by the microscope apparatus according to the present embodiment. FIG. 13 shows an operation flow until the microscope apparatus configured to obtain an image pair with parallax by performing imaging in a plurality of times as described above to obtain three-dimensional super-resolution image data of the entire sample.

  Compared with FIG. 6 which shows the operation | movement flow of the microscope apparatus which concerns on said 1st Embodiment, the process to step S11-step S13 is the same as the process of step S1-step S3 of FIG. 6, respectively.

  In step S14, it is determined whether or not an image pair having a parallax necessary for calculation of position information in the Z direction has been acquired. The determination can be made based on whether or not the PC 9 performs the switching control of the members 13A and 13B. If the image has not been acquired (No in step S14), the process proceeds to step S15, and control for giving parallax to the image, that is, switching between the member 13A and the member 13B arranged on the optical path. And control returns to step S12. If the acquisition of the image pair has been completed (Yes in step S14), the process proceeds to step S16.

The processing after step S16 is the same as the processing after step S4 in FIG.
The series of processing shown in FIG. 13 can also provide the same effect as the case of acquiring image pairs in FIG. 6 at the same time.

  Similarly to FIG. 6, for example, every time an image pair is acquired, position information in the X, Y, and Z directions of the bright spot is obtained from the acquired image pair, and this operation is repeated for FIG. It is also possible to obtain an image data having a three-dimensional structure.

  The present invention is not limited to the above-described embodiments as they are, and can be embodied by modifying the constituent elements without departing from the scope of the invention in the implementation stage. Moreover, various inventions can be formed by appropriately combining a plurality of constituent elements disclosed in the embodiment. For example, all the constituent elements shown in the embodiments may be appropriately combined. Furthermore, constituent elements over different embodiments may be appropriately combined. It goes without saying that various modifications and applications are possible without departing from the spirit of the invention.

2 Light source 3 Illumination optical system 4, 4A, 4B Objective lens 5 Sample surface 6 Dichroic mirror 7, 7A, 7B Imaging lens 8 Camera 9 PC
10A, 10B Optical path 11A, 11B Polarizing plate 12 Polarizing beam splitter 14A, 14B Eccentric aperture 15 Rotating disk 16 Micro lens array 17 Cylindrical lens 18A, 18B Mirror 19 Axis 20 Sheet illumination 21 Axicon lens

Claims (11)

A method for imaging a three-dimensional structure of a sample,
Labeling the structure of the sample with a substance capable of switching between a first state having fluorescence and a second state having no fluorescence when excited by light of a predetermined wavelength;
Illuminating the substance using illumination optics with the light of the predetermined wavelength to excite at least a portion of the substance in the first state;
Imaging the excited material using an imaging optical system;
Obtaining a plurality of images having parallax generated in the imaging step;
Identifying the position of the substance in the plurality of images in the in-plane direction perpendicular to the optical axis in the sample;
Identifying the position of the substance in the optical axis direction in the sample using the position of the substance in the sample in the plurality of images in the in-plane direction;
Repeating steps from exciting the substance to identifying the position of the substance in the sample in the optical axis direction to obtain a three-dimensional structure of the entire sample;
A method for imaging a three-dimensional structure. In the imaging step, inducing an increase in the depth of focus of the imaging;
The method for imaging a three-dimensional structure according to claim 1, further comprising:   3. The method for imaging a three-dimensional structure according to claim 2, wherein the step of inducing an increase in the depth of focus of the imaging is performed by generating a Bessel beam using an axicon lens.   The step of acquiring the plurality of images includes dividing the light from the sample and using each of the divided light to simultaneously acquire a plurality of images with parallax. 4. The method for imaging a three-dimensional structure according to any one of items 3 to 4.   5. The method for imaging a three-dimensional structure according to claim 4, wherein the step of inducing an increase in the focal depth of the imaging is performed before the step of dividing the light from the sample.   5. The method for imaging a three-dimensional structure according to claim 4, wherein the step of inducing an increase in the depth of focus of the imaging is performed after the step of dividing the light from the sample.   7. The illumination optical system has a finite thickness in the optical axis direction of image formation, and illuminates a finite range with respect to the optical axis direction of the illumination optical system. An imaging method of the three-dimensional structure.   In the step of exciting, at least a part of the substance in the first state is excited at a distance within a spatial resolution of the imaging with the nearest substance in the first state. The three-dimensional structure imaging method according to any one of claims 1 to 7.   9. The method for imaging a three-dimensional structure according to claim 1, wherein the position in the in-plane direction is specified by performing analysis by applying an algorithm corresponding to a multi-emitter.   2. The imaging method according to claim 1, wherein the imaging optical system forms an image only in a finite range in the optical axis direction. A microscope apparatus for imaging a three-dimensional structure of a sample,
A labeling unit for labeling the structure of the sample with a substance capable of switching between a first state having fluorescence and a second state having no fluorescence when excited by light of a predetermined wavelength;
An illumination optical system that illuminates the substance with light of the predetermined wavelength and excites at least a part of the substance in the first state;
An imaging optical system for imaging the excited substance;
Obtaining a plurality of images having parallax generated in the step of imaging by the imaging optical system;
Identifying the position of the substance in the plurality of images in the in-plane direction perpendicular to the optical axis in the sample;
Using the position of the substance in the sample in the plurality of images in the in-plane direction, the position of the substance in the sample in the optical axis direction is specified,
An image processing unit that repeats from the step of exciting the substance to the step of specifying the position of the substance in the optical axis direction in the sample, and obtaining a three-dimensional structure of the entire sample;
A microscope apparatus.