CN113358611A - Embedded three-dimensional photoelectric correlation imaging device and method - Google Patents
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Abstract
一种嵌入式三维光电关联成像装置及方法，装置包括：电镜真空腔室、物镜模块、真空光学窗、扫描模块、光源模块、光电探测模块以及电镜成像模块；其中，物镜模块与电镜成像模块均设置在电镜真空腔室的内部，真空光学窗开设在电镜真空腔室的侧壁，并与物镜模块实现光路连通；扫描模块用于对光源模块发射的光束进行二维扫描，并将二维扫描光束通过真空光学窗传输至物镜模块，以使物镜模块对样品不同层面进行扫描；扫描模块还用于将物镜模块对样品不同层面扫描后得到的荧光和明场光线传输至光电探测模块进行光电探测；电镜成像模块用于对样品进行电镜成像。
An embedded three-dimensional photoelectric correlation imaging device and method, the device comprises: an electron microscope vacuum chamber, an objective lens module, a vacuum optical window, a scanning module, a light source module, a photoelectric detection module and an electron microscope imaging module; wherein, the objective lens module and the electron microscope imaging module are both It is arranged inside the vacuum chamber of the electron microscope, and the vacuum optical window is opened on the side wall of the vacuum chamber of the electron microscope, and is connected with the objective lens module in the optical path; the scanning module is used for two-dimensional scanning of the light beam emitted by the light source module, and the two-dimensional scanning The light beam is transmitted to the objective lens module through the vacuum optical window, so that the objective lens module scans different layers of the sample; the scanning module is also used to transmit the fluorescence and brightfield light obtained after the objective lens module scans different layers of the sample to the photodetection module for photoelectric detection. ; The electron microscope imaging module is used for electron microscope imaging of the sample.
Description
Technical Field
The present disclosure relates to the field of photoelectric imaging, and in particular, to an embedded three-dimensional photoelectric correlation imaging apparatus and method.
Background
The fluorescent microscopic imaging realizes the positioning and observation of biological macromolecules and molecular compounds by marking specific protein or subcellular structures in cells through dyes or fluorescent molecules, and the resolution of most of fluorescent imaging is limited by the optical diffraction limit, so that the structural analysis of the biological macromolecules cannot be carried out. And cryo-electron tomography (cryo-ET) is a core technology for analyzing a biomacromolecule structure, and is used for reconstructing a three-dimensional image of a sample and analyzing the biomacromolecule structure by tilting the sample at different angles in a transmission electron microscope and acquiring a series of two-dimensional images. However, since the penetration depth of the electron beam is limited, the thickness of the sample cannot exceed 300nm, and most biological samples need to be thinned. At present, the thinning of a Focused Ion Beam (FIB) is the most promising thinning mode of a frozen sample, the FIB is generally integrated in a cavity of a Scanning Electron Microscope (SEM), a certain angle is formed between an ion emission gun and the sample, the upper surface and the lower surface of an interested area in the sample are cut and thinned, a flat sheet sample required by a transmission electron microscope can be obtained, and the prepared sheet sample is sent into the frozen transmission electron microscope through a freezing transmission system to be subjected to tomography imaging. In order to improve the efficiency and success rate of sample preparation, the fluorescence microscopy imaging and the electron microscopy imaging are correlated, and the navigation is provided for the accurate thinning of the FIB by combining the positioning advantage of the fluorescence microscope on target molecules and the resolution advantage of an electron microscope.
In carrying out the disclosed concept, applicants have discovered that the existing association of fluorescence microscopy and electron microscopy imaging has at least the following drawbacks: the axial resolution of the imaging by using the wide-field fluorescence microscope is very low, only two-dimensional information of a sample can be obtained, and the problem of three-dimensional information loss exists. In the imaging process, the sample is moved into the electron microscope from the light mirror, so that the risk of sample pollution is increased, and the requirement of immediately checking the fluorescence of the sample after thinning is difficult to realize.
BRIEF SUMMARY OF THE PRESENT DISCLOSURE
In view of the prior art, the present disclosure provides an embedded three-dimensional photoelectric correlation imaging apparatus and method, which are used to at least partially solve the above technical problems.
One aspect of the present disclosure provides an embedded three-dimensional photoelectric correlation imaging apparatus, including: the device comprises an electron microscope vacuum chamber 1, an objective lens module 2, a vacuum optical window 3, a scanning module 4, a light source module 5, a photoelectric detection module 6 and an electron microscope imaging module 7; the objective lens module 2 and the electron microscope imaging module 7 are both arranged inside the electron microscope vacuum chamber 1, and the vacuum optical window 3 is arranged on the side wall of the electron microscope vacuum chamber 1 and is communicated with the objective lens module 2 in a light path; the scanning module 4 is used for performing two-dimensional scanning on the light beam emitted by the light source module 5 and transmitting the two-dimensional scanning light beam to the objective lens module 2 through the vacuum optical window 3, so that the objective lens module 2 scans different layers of the sample; the scanning module 4 is also used for transmitting fluorescence and bright field light obtained after the objective lens module 2 scans different layers of the sample to the photoelectric detection module 6 for photoelectric detection; the electron microscope imaging module 7 is used for carrying out electron microscope imaging on the sample.
Optionally, the objective lens module 2 includes: 2-1 of a microscope objective, 2-2 of a vacuum piezoelectric displacement platform and 2-5 of a vacuum flange; wherein, the microscope objective 2-1 is arranged on the vacuum piezoelectric displacement platform 2-2, and the vacuum piezoelectric displacement platform 2-2 is arranged on the vacuum flange 2-5; the vacuum flange 2-5 is provided with a light path channel, the vacuum optical window 3 is connected with one end of the light path channel, and the vacuum piezoelectric displacement table 2-2 is used for driving the microscope objective 2-1 to axially scan different layers of the sample through two-dimensional scanning light beams.
Optionally, the objective lens module 2 further includes: a first mirror 2-3 and a second mirror 2-4 for deflecting the two-dimensional scanning beam passing through the optical path.
Optionally, the scanning module 4 comprises: the device comprises a light source interface 4-1, a dichroic mirror 4-2, an X-direction scanning galvanometer 4-3, a first relay mirror 4-4, a second relay mirror 4-5, a Y-direction scanning mirror 4-6, a scanning lens 4-7 and a microscope 4-8; the light source interface 4-1 is used for receiving the light beam emitted by the light source module 5; the dichroic mirror 4-2 is used for reflecting the light beam to the X-direction scanning galvanometer 4-3, the X-direction scanning galvanometer 4-3 is used for carrying out X-direction scanning on the light beam and reflecting the light beam to the second relay mirror 4-5 and the first relay mirror 4-4, the Y-direction scanning mirror 4-6 is used for carrying out Y-direction scanning on the light beam emitted by the first relay mirror 4-4 to obtain a two-dimensional scanning light beam, and the two-dimensional scanning light beam is reflected to the vacuum optical window 3 after passing through the scanning lens 4-7 and the microscope 4-8; the first relay mirror 4-4 and the second relay mirror 4-5 make the X-direction scanning galvanometer 4-3 conjugate with the Y-direction scanning galvanometer 4-6.
Optionally, the scanning module 4 further comprises: and the pinhole 4-9 is used for filtering the fluorescence and bright field light rays returned along the optical path of the scanning module 4 and transmitting the filtered fluorescence and bright field light rays to the photoelectric detection module 6 for photoelectric detection.
Alternatively, the pinholes 4-9 comprise fixed pinholes or variable pinholes.
Optionally, the light source module 5 is a multi-path coupled laser light source.
Optionally, the photo detection module 6 comprises at least one point detector, wherein when the photo detection module 6 comprises a plurality of point detectors, a dichroic mirror is arranged between different point detectors to distinguish different detection wavelengths.
Optionally, the point detector comprises a photomultiplier tube or an avalanche diode.
The present disclosure provides a photoelectric correlation imaging method based on the above embedded three-dimensional photoelectric correlation imaging apparatus, including: placing the sample in an electron microscope imaging position 8, and performing electron microscope imaging on the sample by using an electron microscope imaging module 7 to obtain an electron microscope image; moving the sample to an optical lens imaging position 9, and scanning different layers of the sample by using an objective lens module 2 based on a two-dimensional scanning light beam transmitted by a scanning module 4 to obtain a three-dimensional fluorescence image and a three-dimensional bright field image; and calculating coordinate transformation from the three-dimensional fluorescence image to the electron microscope image based on the three-dimensional bright field image, and associating the three-dimensional bright field image with the electron microscope image.
Drawings
Fig. 1 schematically shows a structure diagram of an embedded three-dimensional photoelectric correlation imaging device provided by an embodiment of the disclosure;
fig. 2 schematically illustrates a structural view of an objective lens module provided by an embodiment of the present disclosure;
FIG. 3 schematically illustrates a block diagram of a scanning module provided by an embodiment of the present disclosure;
FIG. 4 schematically illustrates a flow chart of an embedded three-dimensional photoelectric correlation imaging method of an embodiment of the present disclosure;
fig. 5 schematically shows a result graph of embedded three-dimensional photoelectric correlation imaging of an embodiment of the present disclosure.
[ description of reference ]
1-electron microscope vacuum chamber;
2-an objective lens module;
2-1-microobjective; 2-2-vacuum piezoelectric displacement table; 2-3-a first mirror; 2-4-second mirror; 2-5-vacuum flange;
3-a vacuum optical window;
4-a scanning module;
4-1-light source interface; a 4-2-dichroic mirror; 4-3-X direction scanning galvanometer; 4-4-a first relay mirror; 4-5-a second relay mirror; a 4-6-Y direction scanning mirror; 4-7-a scanning lens; 4-8-microscopic cylindrical lens; 4-9-pinhole;
5-a light source module;
6-a photoelectric detection module;
7-electron microscope imaging module
8-electron microscope imaging position;
9-optical imaging station.
Detailed Description
For the purpose of promoting a better understanding of the objects, aspects and advantages of the present disclosure, reference is made to the following detailed description taken in conjunction with the accompanying drawings.
Fig. 1 schematically shows a structure diagram of an embedded three-dimensional photoelectric correlation imaging device provided by an embodiment of the disclosure.
As shown in fig. 1, the embedded three-dimensional photoelectric correlation imaging apparatus may include:
the device comprises an electron microscope vacuum chamber 1, an objective lens module 2, a vacuum optical window 3, a scanning module 4, a light source module 5, a photoelectric detection module 6 and an electron microscope imaging module 7.
The vacuum optical window 3 is used for connecting light paths inside and outside the vacuum chamber 1 of the electron microscope without influencing the vacuum degree in the vacuum chamber 1 of the electron microscope, the light source module 5 is used for providing a light source required for exciting fluorescence and bright field illumination of a sample, the scanning module 4 is used for carrying out two-dimensional scanning on light beams emitted by the light source module 5, scanning points focused on the sample into a plane and transmitting the two-dimensional scanning light beams to the objective lens module 2 through the vacuum optical window 3, so that the objective lens module 2 scans different layers of the sample to realize three-dimensional imaging, the scanning module 4 is also used for collecting fluorescence and bright field light rays obtained after the objective lens module 2 scans different layers of the sample, and the fluorescence and bright field light are transmitted to the photoelectric detection module 6, and the photoelectric detection module 6 is used for performing photoelectric detection on the fluorescence and bright field light, converting the optical signal into an electric signal and acquiring and imaging. The electron microscope imaging module 7 is used for carrying out electron microscope imaging on the sample.
The embedded three-dimensional photoelectric correlation imaging device provided by the embodiment of the disclosure is further described with reference to the drawings.
Fig. 2 schematically illustrates a structural view of an objective lens module provided by an embodiment of the present disclosure.
As shown in fig. 2, the objective lens module 2 may include, for example:
2-1 of microscope objective, 2-2 of vacuum piezoelectric displacement platform and 2-5 of vacuum flange. The microscope objective 2-1 is arranged on the vacuum piezoelectric displacement platform 2-2, and the vacuum piezoelectric displacement platform 2-2 is arranged on the vacuum flange 2-5. The vacuum flange 2-5 is provided with a light path channel, the vacuum optical window 3 is connected with one end of the light path channel, and the vacuum piezoelectric displacement table 2-2 is used for driving the microscope objective 2-1 to axially scan different layers of a sample through a two-dimensional scanning light beam so as to ensure three-dimensional optical imaging. Wherein, the microscope objective 2-1 can be any commercial microscope objective
With reference to fig. 2, the objective lens module 2 may further include:
the first reflector 2-3 and the second reflector 2-4 are used for turning the two-dimensional scanning beam passing through the optical path channel to avoid collision.
According to the embodiment of the disclosure, the vacuum flange 2-5 can be further provided with a groove and threads for mounting the vacuum optical window 3 and a clamp, the vacuum piezoelectric displacement table 2-2, the reflectors 2-3 and 2-4 are mounted on the clamp, the clamp is mounted on the vacuum flange 2-5 through screws, the vacuum optical window 3 is fixed on the vacuum flange 2-5 in an adhesive manner, the vacuum flange 2-5 has the same shape and size as the original vacuum flange on the vacuum chamber 1 of the electron microscope, but a vacuum optical window mounting opening and an optical path channel are additionally processed, and a two-dimensional scanning beam can pass through the vacuum optical window without being shielded. Through the vacuum flange of this kind of structure, can guarantee better that vacuum optical window 3 connects the objective module in electron microscope vacuum chamber 1, do not influence the vacuum in electron microscope vacuum chamber 1 simultaneously.
Fig. 3 schematically shows a structural diagram of a scanning module provided in an embodiment of the present disclosure.
As shown in fig. 3, the scanning module 4 may include, for example:
the device comprises a light source interface 4-1, a dichroic mirror 4-2, an X-direction scanning galvanometer 4-3, a first relay mirror 4-4, a second relay 4-5, a Y-direction scanning mirror 4-6, a scanning lens 4-7 and a microscope 4-8.
Referring to fig. 1 to 3, the light source interface 4-1 is connected to the light source module 5 through an optical fiber, and is configured to receive the light beam emitted by the light source module 5. The dichroic mirror 4-2 is used for reflecting the collimated light beam to the X-direction scanning galvanometer 4-3. The X-direction scanning galvanometer 4-3 is used for scanning the light beam in the X direction and reflecting the light beam to the second relay mirror 4-5 and the first relay mirror 4-4. The Y-direction scanning mirror 4-6 is used for carrying out Y-direction scanning on the light beam emitted by the first relay mirror 4-4 to obtain a two-dimensional scanning light beam. The two-dimensional scanning light beam is reflected to the vacuum optical window 3 after passing through the scanning lens 4-7 and the microscope 4-8, enters the light path of the vacuum flange 2-3 through the vacuum optical window 3, is then reflected by the second reflecting mirror 2-4 and the first reflecting mirror 2-3, enters the microscope objective 2-1 and is focused on a sample, the microscope objective 2-1 is installed on the vacuum piezoelectric displacement table 2-2, and different layers of the sample are imaged through axial scanning of the vacuum piezoelectric displacement table 2-2, so that three-dimensional imaging is realized.
The first relay lens 4-4 and the second relay lens 4-5 ensure the conjugate relation between the X-direction scanning galvanometer 4-3 and the Y-direction scanning galvanometer 4-6, so that light spots are kept relatively static on the X-direction scanning galvanometer 4-3 and the Y-direction scanning galvanometer 4-6, and pupil drift is avoided. The relay mirror may be a lens, a spherical mirror, or an off-axis parabolic mirror, and in a specific example of the present disclosure, the relay mirror is selected to be an off-axis parabolic mirror.
According to the embodiment of the present disclosure, the scanning module 4 may further include a pinhole 4-9, for example, for filtering the fluorescence and bright field light returning along the optical path of the scanning module 4, and transmitting the filtered fluorescence and bright field light to the photodetection module 6 for photodetection. Specifically, with reference to fig. 1 to 3, the excited fluorescence and bright field light in the sample return to the dichroic mirror 4-2 along the original path, and reach the pinhole 4-9 through the dichroic mirror 4-2, and the pinhole 4-9 filters light from outside the focal plane of the sample, and enters the optical detection module 6 for photoelectric detection.
In the disclosed embodiment, the pinholes 4-9 may comprise fixed pinholes or variable pinholes, for example, an electrically variable pinhole is selected as the pinhole 4-9.
In the embodiment of the present disclosure, the light source module 5 may be a multi-path coupled laser light source. The photo detection module 6 comprises at least one point detector, wherein, when the photo detection module 6 comprises a plurality of point detectors, a dichroic mirror is arranged between the different point detectors to distinguish different detection wavelengths, and the point detectors may comprise photomultiplier tubes or avalanche diodes, for example two photomultiplier tubes are selected as the detectors.
In the embodiment of the present disclosure, the electron microscope imaging module 7 may include, for example, an electron emission gun and an ion emission gun. The ion emission gun and the sample are arranged at a certain angle, so that the upper surface and the lower surface of an interested area in the sample can be cut and thinned, and a flat sheet sample required by a transmission electron microscope can be obtained.
In the embodiment of the present disclosure, an electron microscope imaging position 8 and an optical lens imaging position 9 are further disposed in the electron microscope vacuum chamber 1, before three-dimensional photoelectric correlation imaging is performed, a three-dimensional space coordinate distance between the electron microscope imaging position 8 and the optical lens imaging position 9 needs to be manually calibrated, the distance is only related to imaging device hardware, is irrelevant to a sample, and is only used for rapidly positioning a target area, the precision requirement is low, the calibration is applicable for a long time after one time, and re-calibration is not needed until the imaging device hardware changes.
The embedded three-dimensional photoelectric correlation imaging device provided by the embodiment of the disclosure is provided with the objective lens module 2 and the scanning module 4 matched with the objective lens module, and can realize axial scanning on different layers of a sample by using a two-dimensional scanning light beam obtained by the scanning module 4 and combining the objective lens module 2 so as to obtain three-dimensional information of the sample, thereby solving the problem of three-dimensional information loss in the existing photoelectric correlation imaging. Simultaneously, objective module 2 and electron microscope imaging module 7 all set up in electron microscope vacuum chamber 1's inside for electron microscope imaging and optical imaging all can be accomplished at electron microscope vacuum chamber 1, have reduced the sample and have shifted the number of times, reduce the risk of sample pollution. In addition, due to the combination of the objective lens module and the scanning module, the imaging device can be applied to most commercial double-beam scanning electron microscopes on the market, and the cost is reduced.
Based on the same inventive concept, the embodiment of the disclosure also provides an embedded three-dimensional photoelectric correlation imaging method.
Fig. 4 schematically shows a flowchart of an embedded three-dimensional photoelectric correlation imaging method according to an embodiment of the present disclosure.
As shown in fig. 4, the method may include, for example, operations S401 to S403.
In operation S401, the sample is placed in the electron microscope imaging position 8, and the electron microscope imaging module 7 is used to perform electron microscope imaging on the sample, so as to obtain an electron microscope image.
In the embodiment of the disclosure, the sample loaded in the electron microscope chamber is moved to the electron microscope imaging position 8, the state of the sample is quickly browsed in a low power mode, the damage conditions of the sample such as the presence or absence of the film breakage of the carrier net and the like are observed, a region with a complete sample is found, the mode is switched to a medium power mode, the region is moved to the center of a field of view, and an electron microscope image excited by an ion beam and the current position coordinate are stored. The electron microscope image is shown in fig. 5 c.
In operation S402, the sample is moved to the optical lens imaging position 9, and different layers of the sample are scanned by using the objective module 2 based on the two-dimensional scanning beam transmitted by the scanning module 4, so as to obtain a three-dimensional fluorescence image and a three-dimensional bright field image.
In the embodiment of the present disclosure, the sample is moved to the imaging position 9 of the optical mirror according to the coordinate distance between the imaging position 9 of the optical mirror and the imaging position 8 of the electron microscope, and the three-dimensional bright field image and the three-dimensional fluorescence image of the sample are simultaneously acquired by using the embedded optical imaging device, where the maximum projections of the three-dimensional bright field image and the three-dimensional fluorescence image are shown as a and b in fig. 5. The bright field image contains coordinate grid information, and the selection of the target area can be quickly confirmed again.
In operation S403, a coordinate transformation from the three-dimensional fluorescence image to the electron microscope image is calculated based on the three-dimensional bright field image, and the three-dimensional bright field image is associated with the electron microscope image.
In the embodiment of the disclosure, first, in an electron microscope image excited by an ion beam and a fluorescence image generated by imaging with a light mirror, 3 to 8 corresponding locating points are found, the locating points are particles with a regular shape of hundreds of nanometers to several micrometers, and can emit fluorescence after being irradiated by laser, for example, b and c in fig. 5, 6 locating marks are selected for locating, coordinates of the locating points in the two images are recorded, and software can be used for recording. Then, based on the recorded coordinates, coordinate transformation from the three-dimensional fluorescence image to the electron microscope image excited by the ion beam is calculated through coordinate transformation parameter fitting, so that three-dimensional photoelectric correlation imaging is realized, and the image after the photoelectric correlation imaging is shown as d in fig. 5.
The photoelectric correlation imaging method provided by the embodiment of the disclosure can acquire three-dimensional information of a sample, reduces pollution of the sample in the imaging process, and has high image quality after photoelectric correlation imaging.
The above-mentioned embodiments are intended to illustrate the objects, aspects and advantages of the present disclosure in further detail, and it should be understood that the above-mentioned embodiments are only illustrative of the present disclosure and are not intended to limit the present disclosure, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present disclosure should be included in the scope of the present disclosure.
Claims (10)
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| CN115575364A (en) * | 2022-09-30 | 2023-01-06 | 中国科学院生物物理研究所 | Ion Beam Processing Method Based on Optical Microscopic Imaging |
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