CN111521608A - Super-resolution microscopic imaging method and microscope - Google Patents
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- G02B21/365—Control or image processing arrangements for digital or video microscopes
- G02B21/367—Control or image processing arrangements for digital or video microscopes providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison
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Abstract
The embodiment of the invention discloses a super-resolution microscopic imaging method and a microscope. Wherein the super-resolution microscopic imaging method comprises the steps of carrying out expansion treatment on a biological sample; processing the expanded biological sample slice to form a biological sample slice; forming a structured light illuminating polished section by using the non-diffraction light beam, and acquiring a super-resolution single image of the structured light illuminating polished section; and fusing all the super-resolution single images to obtain the super-resolution image of the biological sample. The technical scheme of the embodiment of the invention can solve the problems that the existing microscopic imaging speed is too low and the existing microscopic imaging is not suitable for carrying out fluorescence in-situ sequencing on a large-volume and high-flux biological sample, and realize the rapid and high-precision microscopic imaging of the biological sample.
Description
Technical Field
The embodiment of the invention relates to a microscopic imaging technology, in particular to a super-resolution microscopic imaging method and a microscope.
Background
The resolution of optical microscopy is limited to about half the detection wavelength, about 200nm, by the diffraction effects of light. In order to search for more life activities occurring between subcellular structures, molecular complexes, etc., super-resolution microscopic imaging techniques with higher resolution have been rapidly developed in recent years.
In the prior art, a super-resolution Microscopy based on single-molecule positioning is a commonly used Microscopy, and comprises a random Optical Reconstruction Microscopy (STORM) and a photo-activated Localization Microscopy (PALM), and the imaging resolution of the super-resolution Microscopy can reach 20nm × 20nm × 50 nm. But the imaging speed is slow, and hundreds or even thousands of original images can be needed to be synthesized by a single-frame super-resolution image. Moreover, due to the repeated activation and re-quenching processes, phototoxicity is high, and the method is not suitable for performing fluorescence in situ sequencing (merfish) on large-volume and high-flux biological samples.
Disclosure of Invention
The embodiment of the invention provides a super-resolution microscopic imaging method and a microscope, which are used for realizing rapid high-precision biological sample microscopic imaging.
In a first aspect, an embodiment of the present invention provides a super-resolution microscopic imaging method, including: performing expansion treatment on a biological sample;
slicing the expanded biological sample to form a biological sample slice;
forming a structured light illuminating polished section by using the non-diffraction light beam, and acquiring a super-resolution single image of the structured light illuminating polished section;
and fusing all the super-resolution single images to obtain the super-resolution image of the biological sample.
Optionally, the swelling treatment of the biological sample comprises:
embedding the biological sample in a gel solution of a dense cross-linked electrolyte;
the control gel swells by folding to expand linearly, thereby swelling the biological sample.
Optionally, the gel comprises a polyacrylic gel, which swells upon absorption of water.
Optionally, the non-diffracted light beam is a Bessel light beam, an Airy light beam, a Mathieu light beam, or a Weber light beam.
Optionally, the forming a structured light illumination slide by using the non-diffracted beam, and acquiring a super-resolution single image of the structured light illumination slide includes:
modulating the non-diffracted beam into a structured light sheet;
irradiating the biological sample slice by using the structured light slide to form a structured light slide; acquiring a super-resolution single image of the structured light illuminating light sheet;
and moving the biological sample slice to acquire super-resolution single images corresponding to all the structured light illuminating polished sections.
Optionally, the moving the biological sample section comprises translating the biological sample section and/or rotating the biological sample section about an axis perpendicular to a planar direction in which the biological sample section lies. In a second aspect, an embodiment of the present invention further provides a super-resolution microscope, including a sample preprocessing module, a microscopic imaging module, and a data processing module;
the sample pretreatment module is used for performing expansion treatment on a biological sample and slicing the expanded biological sample to form a biological sample slice;
the microscopic imaging module comprises an object stage and at least one pair of imaging units, each pair of imaging units comprises a first imaging unit and a second imaging unit, the first imaging unit comprises a first light source, a first light modulation unit, a first objective lens and a first camera which are arranged along a first optical axis, the second imaging unit comprises a second light source, a second light modulation unit, a second objective lens and a second camera which are arranged along a second optical axis, and the first optical axis is vertical to the second optical axis;
the first light modulation unit is used for modulating the light emitted by the first light source into a first diffraction-free light beam and modulating the first diffraction-free light beam into a first structure light sheet, the first structure light sheet is used for providing illumination for the second objective, and the second camera is used for acquiring a super-resolution single image formed by the second objective;
the second light modulation unit is used for modulating the light emitted by the second light source into a second diffraction-free light beam and modulating the second diffraction-free light beam into a second structured light sheet, the second structured light sheet is used for providing illumination for the first objective lens, and the first camera is used for acquiring a super-resolution single image formed by the first objective lens;
the data processing module is used for fusing all the super-resolution single images to obtain a super-resolution image of the biological sample.
Optionally, the microscopic imaging module includes two pairs of imaging units, and two planes in which the first optical axis and the second optical axis of the two pairs of imaging units are located have an included angle that is not zero.
Optionally, the first light modulation unit includes a cone lens, a rotating galvanometer, a phase template, and a rotating galvanometer or a spatial light modulator;
the second light modulation unit comprises a cone lens, a rotary galvanometer, a phase template and a rotary galvanometer or a spatial light modulator.
Optionally, the system further comprises a third light source, a third objective and a third camera;
the third light source is used for providing illumination for the third objective lens;
the third camera is used for obtaining the imaging of the third objective lens and observing the biological sample.
According to the super-resolution microscopic imaging method provided by the embodiment of the invention, the biological sample is subjected to expansion treatment, so that the volume of the biological sample is expanded without influencing a structure to be imaged; slicing the expanded biological sample to form a biological sample slice; forming a structured light illuminating polished section by using the non-diffraction light beam to obtain a super-resolution single image of the structured light illuminating polished section; and fusing all the super-resolution single images to obtain the super-resolution image of the biological sample. The method solves the problem that the existing microscopic imaging speed is too low and is not suitable for carrying out fluorescence in-situ sequencing on a large-volume and high-flux biological sample, and realizes the rapid and high-precision microscopic imaging of the biological sample.
Drawings
FIG. 1 is a schematic flow chart of a super-resolution microscopy imaging method provided by an embodiment of the invention;
FIG. 2 is a schematic structural diagram of a super-resolution microscope provided by an embodiment of the present invention;
fig. 3 and fig. 4 are schematic partial structural diagrams of a super-resolution microscope according to an embodiment of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention. It should be further noted that, for the convenience of description, only some of the structures related to the present invention are shown in the drawings, not all of the structures.
The terminology used in the embodiments of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. It should be noted that the terms "upper", "lower", "left", "right", and the like used in the description of the embodiments of the present invention are used in the angle shown in the drawings, and should not be construed as limiting the embodiments of the present invention. In addition, in this context, it is also to be understood that when an element is referred to as being "on" or "under" another element, it can be directly formed on "or" under "the other element or be indirectly formed on" or "under" the other element through an intermediate element. The terms "first," "second," and the like, are used for descriptive purposes only and not for purposes of limitation, and do not denote any order, quantity, or importance, but rather are used to distinguish one element from another. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
Fig. 1 is a schematic flow chart of a super-resolution microscopic imaging method according to an embodiment of the present invention, which is applicable to fluorescence in situ sequencing of a large-volume high-throughput biological sample, and the super-resolution microscopic imaging method includes:
step S110 is to perform a swelling process on the biological sample.
It can be understood that, in order to break the limit of resolution of the ordinary wide-field microscope limited by diffraction limit, super-resolution imaging can be realized by enlarging the sample, and by destroying the interaction between proteins, the polymer is filled with liquid, and the volume of the polymer can expand hundreds of times, for example, when the volume expands 1000 times, the length, the width and the height of the polymer respectively expand 10 times, so that 10 times super-resolution imaging can be realized.
Optionally, the swelling treatment of the biological sample comprises:
s111, embedding the biological sample in a gel solution of a dense cross-linked electrolyte;
and S112, controlling the gel to expand through folding linear expansion, so that the biological sample is expanded.
Optionally, the gel comprises a polyacrylic gel, which swells upon absorption of water.
The polyacrylic acid gel can expand many times after absorbing water, and is used for realizing the expansion of biological samples. In other embodiments, other types of polymers may be used, and the embodiments of the present invention are not limited.
And step S120, slicing the expanded biological sample to form a biological sample slice.
It will be appreciated that the stage of the microscope may not be able to carry the expanded biological sample, and that three-dimensional imaging of the biological sample may be achieved by forming slices of the biological sample, performing super-resolution imaging of the slices, and then fusing the images of all the slices.
And S130, forming the structured light illuminating light sheet by using the non-diffraction beam, and acquiring a super-resolution single image of the structured light illuminating light sheet.
It can be understood that the non-diffraction light beam has the advantages of small cross-sectional diameter, and unchanged cross-sectional profile and light intensity during propagation, and can effectively reduce the thickness of the light sheet and reduce phototoxicity. In this embodiment, optionally, the non-diffracted light beam may be a Bessel light beam, an Airy light beam, a Mathieu light beam, or a Weber light beam, and may be selected according to actual conditions in specific implementation. For example, the light beam may be modulated in the form of a cone lens, a phase template, or the like to form a Bessel light beam, and then the switching of the Bessel light beam is controlled by rotating a galvanometer to form a structured light sheet, or the light emitted by the light source may be directly modulated into a structured light sheet formed by a non-diffracted light beam by using a Spatial Light Modulator (SLM), which is not limited in the embodiment of the present invention.
Optionally, the forming of the structured light illumination polished section by using the non-diffracted beam, and the obtaining of the super-resolution single image of the structured light illumination polished section includes:
s131, modulating the non-diffraction light beam into a structured light polished section;
s132, irradiating the biological sample slice by using a structured light slide to form a structured light illuminating slide;
s133, acquiring a super-resolution single image of the light sheet;
and S134, moving the biological sample slice to acquire super-resolution single images corresponding to all the structured light illuminating polished sections.
The light beam may be modulated in the form of a cone lens, a phase template, or the like to form a non-diffracted light beam (e.g., a Bessel light beam), and then the switching of the Bessel light beam is controlled by a rotating galvanometer to form a structured light sheet, or the light emitted from a light source may be directly modulated into a structured light sheet formed by a non-diffracted light beam by using a Spatial Light Modulator (SLM), which is not limited in the embodiments of the present invention
Optionally, moving the biological sample section comprises translating the biological sample section and/or rotating the biological sample section about an axis perpendicular to the planar direction in which the biological sample section lies.
By translating the biological sample slice, super-resolution microscopic imaging of each position can be obtained, and by rotating the biological sample slice, the same resolution ratio in each direction can be ensured, and the microscopic imaging effect is improved. When one section imaging is finished, other sections can be continuously imaged, and finally, a three-dimensional image of the whole biological sample is obtained.
And S140, fusing all the super-resolution single images to obtain a super-resolution image of the biological sample.
Before all the super-resolution single images are fused, filtering processing can be carried out on each super-resolution single image, and the super-resolution single images are processed by a computer and other equipment to obtain the super-resolution images of the whole biological sample.
According to the technical scheme of the embodiment, the biological sample is subjected to expansion treatment, so that the volume of the biological sample is expanded without influencing a structure to be imaged; slicing the expanded biological sample to form a biological sample slice; forming a structured light illuminating polished section by using the non-diffraction light beam to obtain a super-resolution single image of the structured light illuminating polished section; and fusing all the super-resolution single images to obtain the super-resolution image of the biological sample. The method solves the problem that the existing microscopic imaging speed is too low and is not suitable for carrying out fluorescence in-situ sequencing on a large-volume and high-flux biological sample, and realizes the rapid and high-precision microscopic imaging of the biological sample.
Fig. 2 is a schematic structural diagram of a super-resolution microscope applicable to fluorescence in situ sequencing of a large-volume high-throughput biological sample according to an embodiment of the present invention, which includes a sample pre-processing module 10, a microscopic imaging module 20, and a data processing module 30; the sample pretreatment module 10 is used for performing expansion treatment on a biological sample and slicing the expanded biological sample to form a biological sample slice; the microscopic imaging module 20 includes a stage 21 and at least one pair of imaging units 22 (in fig. 2, the microscopic imaging module 20 includes the pair of imaging units 22 as an example, and the invention is not limited thereto), each pair of imaging units 22 includes a first imaging unit 23 and a second imaging unit 24, the first imaging unit 23 includes a first light source 231, a first light modulation unit 232, and a first objective 234 and a first camera 235 arranged along a first optical axis a, the second imaging unit 24 includes a second light source 241, a second light modulation unit 242, and a second objective 244 and a second camera 245 arranged along a second optical axis b, and the first optical axis a and the second optical axis b are perpendicular; the first light modulation unit 232 is configured to modulate the light emitted from the first light source 231 into a first undiffracted light beam, and modulate the first undiffracted light beam into a first structured light-to-light sheet, for example, the first modulation unit 232 shown in fig. 2 includes a cone lens 2321 and a deflecting galvanometer 2322, and in other embodiments, the first modulation unit may also be a spatial light modulator. The first structured light sheet is used for providing illumination for the second objective 244, and the second camera 245 is used for acquiring a super-resolution single image formed by the second objective 244; the second light modulation unit 242 is configured to modulate light emitted from the second light source 241 into a second undiffracted light beam, and modulate the second undiffracted light beam into a second structured light sheet, for example, the second modulation unit 242 shown in fig. 2 includes a axicon 2421 and a deflecting galvanometer 2422, and in other embodiments, the second modulation unit may also be a spatial light modulator. The second structured light sheet is used for providing illumination for the first objective 234, and the first camera 235 is used for acquiring a super-resolution single image formed by the first objective 234; the data processing module 30 is used for fusing all the super-resolution single images to obtain a super-resolution image of the biological sample.
The first light source 231 and the second light source 241 may be lasers, such as fiber lasers, and output ends of the first light source and the second light source may be respectively provided with collimating mirrors 236 and 246, in order to adjust beam quality, relay mirrors 237 and 247 may be further provided, a dichroic mirror 238 and a filter 239 are further provided between the first objective 234 and the first camera 235, the dichroic mirror 238 is used for reflecting the illumination beam and transmitting the fluorescence beam, and the filter 239 is used for filtering out noise; a dichroic mirror 248 and a filter 249 are further disposed between the second objective 244 and the second camera 245, the dichroic mirror 248 is used for reflecting the illumination beam and transmitting the fluorescence beam, and the filter 249 is used for filtering out noise, it being understood that the relay mirror may be a single lens or a lens group. The stage 21 is used for carrying the biological sample slice and also for driving the biological sample slice to translate or rotate. In this embodiment, the structured light illuminating light sheet generates a stripe structure in one direction at a time, and in order to realize super-resolution imaging in two directions, after scanning imaging is completed once, the biological sample slice may be rotated by 90 ° around a certain axis perpendicular to the plane direction of the biological sample slice, and then secondary imaging is performed, and then the two imaging results are fused, so that super-resolution images in two directions can be obtained.
According to the technical scheme of the embodiment, the biological sample is subjected to expansion treatment through the sample pretreatment module, so that the volume of the biological sample is expanded without influencing a structure to be imaged; slicing the expanded biological sample to form a biological sample slice; illuminating the light sheet by using the structured light through the microscopic imaging module to obtain a super-resolution single image of the structured light illuminated light sheet; and fusing all the super-resolution single images through the data processing module to obtain the super-resolution image of the biological sample. The method solves the problem that the existing microscopic imaging speed is too low and is not suitable for carrying out fluorescence in-situ sequencing on a large-volume and high-flux biological sample, and realizes the rapid and high-precision microscopic imaging of the biological sample.
On the basis of the above technical solution, optionally, the microscopic imaging module includes two pairs of imaging units, and two planes in which the first optical axis and the second optical axis are located in the two pairs of imaging units have an included angle that is not zero.
Illustratively, taking two planes of the first optical axis and the second optical axis of the two pairs of imaging units are perpendicular as an example, fig. 3 and fig. 4 are respectively partial structural schematic diagrams of a super-resolution microscope provided by an embodiment of the present invention, where fig. 3 shows a structural schematic diagram of an imaging unit in xoz plane, fig. 4 shows a structural schematic diagram of an imaging unit in yoz plane, and planes of the optical axes a and b are perpendicular to planes of the optical axes c and d. By arranging the two groups of imaging units, the sample slice can be prevented from rotating, super-resolution single images in two directions can be acquired at one time, and the imaging speed is improved.
Optionally, the first light modulation unit 232 includes a cone lens, a rotating galvanometer, a phase template, and a rotating galvanometer or a spatial light modulator; the second light modulation unit 242 may be selected according to actual situations when it includes an axicon and a rotating galvanometer, a phase template and a rotating galvanometer or a spatial light modulator, which is not limited in this embodiment of the present invention.
Optionally, with continuing reference to fig. 2, the super-resolution microscope provided in this embodiment further includes a third light source 40, a third objective lens 41, and a third camera 42; a third light source 40 for providing illumination to a third objective lens 41; the third camera 42 is used to acquire an image of the third objective lens 41 for observing the biological sample.
Illustratively, the super-resolution microscope further comprises a reflecting mirror 43 and a relay mirror 44, wherein the reflecting mirror 43 can change the direction of an optical path, the size of the super-resolution microscope is reduced, and the relay mirror 44 can improve the quality of a light beam. The third objective lens 41 has a smaller magnification and the third camera 42 is used to acquire an image of the biological sample over a larger range for viewing the biological sample.
It is to be noted that the foregoing is only illustrative of the preferred embodiments of the present invention and the technical principles employed. It will be understood by those skilled in the art that the present invention is not limited to the particular embodiments described herein, but is capable of various obvious changes, rearrangements and substitutions as will now become apparent to those skilled in the art without departing from the scope of the invention. Therefore, although the present invention has been described in greater detail by the above embodiments, the present invention is not limited to the above embodiments, and may include other equivalent embodiments without departing from the spirit of the present invention, and the scope of the present invention is determined by the scope of the appended claims.
Claims (10)
1. A super-resolution microscopic imaging method is characterized by comprising the following steps:
performing expansion treatment on a biological sample;
slicing the expanded biological sample to form a biological sample slice;
forming a structured light illuminating polished section by using the non-diffraction light beam, and acquiring a super-resolution single image of the structured light illuminating polished section;
and fusing all the super-resolution single images to obtain the super-resolution image of the biological sample.
2. The super resolution microscopy imaging method according to claim 1, wherein the subjecting of the biological sample to dilation comprises:
embedding the biological sample in a gel solution of a dense cross-linked electrolyte;
the control gel swells by folding to expand linearly, thereby swelling the biological sample.
3. The super-resolution microscopy imaging method according to claim 2, wherein the gel comprises a polyacrylic gel that swells upon absorption of water.
4. The super resolution microscopy imaging method according to claim 1, wherein the non-diffracted beam is a Bessel beam, an Airy beam, a Mathieu beam or a Weber beam.
5. The super-resolution microscopy imaging method as claimed in claim 1, wherein the forming of the structured light illuminated slide using the non-diffracted beam, the obtaining of the super-resolution single image of the structured light illuminated slide comprises:
modulating the non-diffracted beam into a structured light sheet;
irradiating the biological sample slice by using the structured light slide to form a structured light slide; acquiring a super-resolution single image of the structured light illuminating light sheet;
and moving the biological sample slice to acquire super-resolution single images corresponding to all the structured light illuminating polished sections.
6. The super resolution microscopy imaging method of claim 5, wherein said moving said biological sample section comprises translating said biological sample section and/or rotating said biological sample section about an axis perpendicular to a planar direction of said biological sample section.
7. A super-resolution microscope is characterized by comprising a sample preprocessing module, a microscopic imaging module and a data processing module;
the sample pretreatment module is used for performing expansion treatment on a biological sample and slicing the expanded biological sample to form a biological sample slice;
the microscopic imaging module comprises an object stage and at least one pair of imaging units, each pair of imaging units comprises a first imaging unit and a second imaging unit, the first imaging unit comprises a first light source, a first light modulation unit, a first objective lens and a first camera which are arranged along a first optical axis, the second imaging unit comprises a second light source, a second light modulation unit, a second objective lens and a second camera which are arranged along a second optical axis, and the first optical axis is vertical to the second optical axis;
the first light modulation unit is used for modulating the light emitted by the first light source into a first diffraction-free light beam and modulating the first diffraction-free light beam into a first structure light sheet, the first structure light sheet is used for providing illumination for the second objective, and the second camera is used for acquiring a super-resolution single image formed by the second objective;
the second light modulation unit is used for modulating the light emitted by the second light source into a second diffraction-free light beam and modulating the second diffraction-free light beam into a second structured light sheet, the second structured light sheet is used for providing illumination for the first objective lens, and the first camera is used for acquiring a super-resolution single image formed by the first objective lens;
the data processing module is used for fusing all the super-resolution single images to obtain a super-resolution image of the biological sample.
8. The super resolution microscope of claim 7, wherein the microscopic imaging module comprises two pairs of imaging units, and two planes of the first optical axis and the second optical axis in the two pairs of imaging units have an included angle different from zero.
9. The super resolution microscope of claim 7, wherein the first light modulation unit comprises an axicon and a rotating galvanometer, a phase template and a rotating galvanometer or a spatial light modulator;
the second light modulation unit comprises a cone lens, a rotary galvanometer, a phase template and a rotary galvanometer or a spatial light modulator.
10. The super resolution microscope of claim 7, further comprising a third light source, a third objective lens, and a third camera;
the third light source is used for providing illumination for the third objective lens;
the third camera is used for obtaining the imaging of the third objective lens and observing the biological sample.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113325563B (en) * | 2021-04-21 | 2022-06-10 | 浙江大学 | Multicolor three-dimensional super-resolution expansion microscope system with large view field |
| CN113450259A (en) * | 2021-08-31 | 2021-09-28 | 深圳百胜扬工业电子商务平台发展有限公司 | Micro-image observation processing method and device |
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