CN112213343A - Method, system and device for quickly imaging ultrathin biological section borne by plastic strip - Google Patents
Method, system and device for quickly imaging ultrathin biological section borne by plastic strip Download PDFInfo
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- 238000002474 experimental method Methods 0.000 claims description 69
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 17
- 229910052799 carbon Inorganic materials 0.000 claims description 17
- 238000005259 measurement Methods 0.000 claims description 11
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- G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
- G01N23/22—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material
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Abstract
The invention belongs to the technical field of characterization of surface structures of biological materials, and particularly relates to a method, a system and a device for quickly imaging a plastic strip-supported biological ultrathin section, aiming at solving the problems that in the prior art, the speed of a plastic strip-surface biological ultrathin section is low in the imaging process, the imaging contrast is poor, and the plastic strip-surface biological ultrathin section is easily damaged by electron irradiation. Preparing a care before imaging of an electron microscope by carrying a biological ultrathin section on a plastic strip; the electron irradiation damage on the surface of the imaging biological slice material under the high beam density of the electron microscope is avoided, and the electron microscope image is rapidly obtained. According to the method, the relatively high imaging beam current density is obtained by adjusting the aperture size of an objective lens diaphragm of an electron microscope, the injection speed and action time of specifically metered electrons on the surface of a sample are controlled, and an electron permanent protective layer is formed between a biological ultrathin section and a bearing plastic strip, so that the electron irradiation damage on the surface of the biological section under high beam current density during formal imaging is avoided, and the imaging speed of the electron microscope is improved.
Description
Technical Field
The invention belongs to the technical field of characterization of surface structures of biological materials, and particularly relates to a method, a system and a device for quickly imaging a plastic strip-loaded biological ultrathin slice.
Background
Scanning electron microscope imaging is a mainstream technology in three-dimensional reconstruction, and when the method for acquiring the fine microstructure on the surface of the biological ultrathin slice is used, imaging signals of the method comprise two signals, namely secondary electrons and backscattered electrons. The secondary electron signal source and the depth of a sample surface which is more than ten nanometers are sensitive to surface information, some micron-sized pollutants are obviously represented under the secondary electron imaging, the back scattering electrons come from the depth of a sample which is hundreds nanometers, and the back scattering electrons are not sensitive to the micro-particle discharge and pollution on the surface, the imaging effect is fine and smooth, and the image quality is clear. For the biological ultrathin section, the material composition is a high molecular product of cross-linking polymerization of epoxy resin and acid anhydride, and a few heavy metal elements are introduced for enhancing the structural imaging contrast of the biological tissue membrane in the sample preparation.
The main factors influencing the imaging signal are the beam density of the electron beam, the acceleration voltage, the working distance and the like, wherein the beam density of the electron beam has the greatest influence. The beam density is large, which indicates that the electron energy which interacts with the sample is large, the imaging speed of the electron microscope is influenced by the electron energy and the quantity, when the conventional scanning electron microscope is used for shooting biological ultrathin sections, the beam density is about 800pA or less, and the available single-pixel acquisition time is about 0.5-2 according to different sample states
. The beam density of a high-speed scanning electron microscope (hereinafter referred to as an electron microscope) in an ultrahigh-speed three-dimensional imaging system used in the experiment is about 8nA or more, and the available single-pixel acquisition time is about 10-50 ns. The penetration depth of the electron beam varies from a few nanometers to a few micrometers, depending mainly on the magnitude of the energy of the electron beam. With increased imaging speedMeanwhile, a new problem, namely electron beam irradiation damage, also appears, because most of the components of the biological slice are polymer resin materials, the electrical conductivity and the thermal conductivity of the biological slice are relatively poor, back scattering electrons used in imaging have obvious difference according to the element components of sample materials, the electron penetration depth of the biological ultrathin slice is more than hundred nanometers level in imaging, because the electrical conductivity and the thermal conductivity of a plastic strip are also relatively poor, when electrons with high beam density interact with the sample, heat converted by the kinetic energy of the high-speed moving electron beam is gathered on the surface of the strip and can not be conducted away in time, the heat can be diffused towards the periphery of an action layer, when the heat is diffused to the bottom surface of the ultrathin slice, because the thermal conductivity is also poor, the irradiation damage can be formed from the bottom surface of the slice to the surface, and because the thickness of the slice is extremely thin, when the surface of the ultrathin slice is imaged, showing significant radiation damage, i.e., blistering. Causing the loss of fine details and affecting the reconstruction effect and accuracy.
Disclosure of Invention
The method aims to solve the problems in the prior art, namely the problems that in the prior art, the speed of the biological ultrathin section on the surface of the plastic strip is low, the imaging contrast is poor and the biological ultrathin section is easy to damage by electron irradiation in the imaging process. The application provides a plastic strip bearing biological ultrathin section rapid imaging method in a first aspect, which comprises the following steps: and S100, acquiring a biological ultrathin section, collecting the biological ultrathin section on a plastic strip, fixing the plastic strip to the surface of the wafer, and carrying out carbon plating treatment on the surface of the biological ultrathin section.
And S200, performing an electron beam irradiation experiment on an irradiation area set in the biological ultrathin section according to a preset first electron beam density.
Step S300, selecting a region to be imaged from the irradiation region; and acquiring images of the biological ultrathin section in the area to be imaged according to the preset second electron beam current density to obtain an image of the biological ultrathin section.
In some preferred technical solutions, the method of performing the electron beam irradiation experiment on the irradiation region set in the biological ultrathin section in step S200 is to perform the electron beam irradiation experiment on the irradiation region set in the biological ultrathin section through a scanning electron microscope; the step S200 further includes: and judging whether the irradiation area is larger than the projection area of the scanning electron microscope, if so, segmenting the irradiation area to obtain a plurality of irradiation subregions with the same resolution and the area not larger than the projection area of the scanning electron microscope.
And performing an electron beam irradiation experiment on the biological ultrathin section in each irradiation sub-area according to the size and the resolution of each irradiation sub-area, the preset electron beam irradiation retention time and the preset first electron beam density.
In some preferred technical solutions, the electron injection measurement per unit area required by the electron beam irradiation experiment is obtained based on the area of the irradiation region, the size of each irradiation sub-region, the resolution of each irradiation sub-region, the preset electron beam irradiation residence time of each irradiation sub-region, and the preset first electron beam density.
In some preferred technical solutions, the method for calculating the electron injection measurement per unit area includes:
wherein: deNumber of electrons per unit area, IPIs the magnitude of the main electron beam current, e is the electron charge amount, τDFor single pixel dwell time, AxyThe area of a single pixel, A is a constant related to the beam density, C is the beam density when the electron microscope works, pixel size is the size of each pixel when the electron microscope works, t is image acquisition time, and size is the number of pixels of one image. It is understood that, for a single pixel, that is, pixelsize, the irradiation region size/region to be imaged size is an area value obtained by multiplying the number of pixels and the size of the single pixel.
In some preferred embodiments, step S200 further includes: carrying out an automatic focusing experiment on the biological ultrathin section in the irradiation area after the electron beam irradiation experiment so as to determine whether irradiation damage occurs in the irradiation area; if the irradiation damage occurs, returning to the step S100; if no radiation damage occurs, go to step S300.
In some preferred technical solutions, the plastic strip is adhered to the wafer with a flat surface by a conductive adhesive tape.
In some preferred embodiments, the preset second electron beam current density is smaller than the preset first electron beam current density.
The application second aspect provides a plastics strip bears quick imaging system of biological ultrathin section electron microscope, and this system is including obtaining module, carbon-plated module, irradiation experiment module, image acquisition module.
The acquisition module is configured to acquire a biological ultrathin section.
The carbon plating module is configured to perform carbon plating treatment on the surface of the biological ultrathin section.
The irradiation experiment module is configured to perform an electron beam irradiation experiment on an irradiation area set in the biological ultrathin section based on a preset first electron beam density.
The image acquisition module is configured to acquire an image of the biological ultrathin section in the region to be imaged in the irradiation region based on a preset second electron beam current density, and acquire an image of the biological ultrathin section.
A third aspect of the present application provides a storage device, wherein a plurality of programs are stored, the programs are suitable for being loaded and executed by a processor to realize the method for rapidly imaging the plastic strip-bearing ultrathin biological section in any one of the above technical schemes.
A fourth aspect of the present application provides a processing arrangement comprising a processor, a storage device; a processor adapted to execute various programs; a storage device adapted to store a plurality of programs; the program is suitable for being loaded and executed by a processor to realize the plastic strip bearing biological ultrathin section rapid imaging method in any one of the technical schemes.
The invention has the beneficial effects.
According to the method, the relatively high imaging beam current density is obtained by adjusting the aperture size of the objective lens diaphragm of the electron microscope, the injection speed and action time of specifically metered electrons on the surface of a sample are controlled, and an electron permanent protective layer is formed between the biological ultrathin section and the bearing plastic strip, so that the electron irradiation damage on the surface of the biological section under high beam current density during formal imaging is avoided, and the imaging speed of the electron microscope is improved. The invention has the characteristics of simple implementation, low implementation cost, strong technical reliability and the like, and is suitable for wide application in the field of biological ultrathin slice surface research.
Drawings
Other features, objects and advantages of the present application will become more apparent upon reading of the following detailed description of non-limiting embodiments thereof, made with reference to the accompanying drawings.
Fig. 1 is a schematic flow chart of a plastic strip-supported biological ultrathin section rapid imaging method according to an embodiment of the invention.
FIG. 2 is a schematic diagram of a frame of an electron microscope rapid imaging system for a plastic strip-supported biological ultrathin section according to an embodiment of the present invention.
FIG. 3 is a schematic view of a biological ultrathin section sample in one embodiment of the invention.
FIG. 4 is an image of a sample taken without an irradiation experiment of an ultrathin section of an object in an embodiment of the invention.
FIG. 5 is an image of a specimen taken after irradiation experiments on ultra-thin biological sections in an embodiment of the present invention.
List of reference numerals: 1-a carbon film; 2-biological ultrathin section; 3-plastic strip.
Detailed Description
In order to make the embodiments, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it is apparent that the described embodiments are some, but not all embodiments of the present invention. It should be understood by those skilled in the art that these embodiments are only for explaining the technical principle of the present invention, and are not intended to limit the scope of the present invention.
The invention discloses a plastic strip bearing biological ultrathin section rapid imaging method, which comprises the following steps.
And S100, acquiring a biological ultrathin section, collecting the biological ultrathin section on a plastic strip, fixing the plastic strip to the surface of the wafer, and carrying out carbon plating treatment on the surface of the biological ultrathin section.
And S200, performing an electron beam irradiation experiment on an irradiation area set in the biological ultrathin section according to a preset first electron beam density.
Step S300, selecting a region to be imaged from the irradiation region; and acquiring images of the biological ultrathin section in the area to be imaged according to the preset second electron beam current density to obtain an image of the biological ultrathin section.
In order to more clearly explain the method for rapidly imaging the ultrathin biological section carried by the plastic strip, a preferred embodiment of the invention is described in detail below with reference to the accompanying drawings.
As a preferred embodiment of the present invention, the method for rapidly imaging a plastic strip-supported ultrathin biological section of the present invention is shown in FIG. 1 and comprises the following steps.
Step S100, obtaining a biological ultrathin section 2, collecting the biological ultrathin section 2 on a plastic strip 3, transferring and adhering the plastic strip 3 carrying the biological ultrathin section 2 to a wafer with a smooth surface through a conductive adhesive tape, and performing carbon plating treatment on the surface of the biological ultrathin section to obtain a biological ultrathin section sample. Referring specifically to FIG. 3, in the preferred embodiment of the present application, the ultrathin biological section 2 has a thickness of 30nm to 50nm and is a plastic strip carrying the ultrathin biological section with a thickness of 50nm
. In some preferred embodiments, the wafer with the plastic strips 3 fixed thereon is placed in a high vacuum coater to evaporate a carbon film 1 with a thickness of 10nm on the surface thereof to obtain a biological ultrathin section sample. The method can improve the conductivity of the surface of the biological ultrathin section sample. It is understood that the conductive tape is only one preferred embodiment, and those skilled in the art can mold the conductive tape in other waysThe strip is transferred to a wafer or metal substrate. And after the carbon film evaporation is finished, transferring the biological ultrathin section sample to a sample table special for a scanning electron microscope in a laboratory, putting the sample into an electron microscope cabin, and preparing to start an experiment.
And S200, performing an electron beam irradiation experiment on an irradiation area set in the biological ultrathin section according to a preset first electron beam density. It can be understood that the selection method of the irradiation region can be flexibly selected by a person skilled in the art, or the region to be imaged of the biological ultrathin cutting edge can be determined first, the region is taken as the center of a circle, the distance is set as the radius, and a circular region or a specific shape region is set as the set irradiation region.
Preferably, the location in the image acquisition software where each slice needs to be taken is selected. And then adjusting the size of an electron microscope diaphragm, and adjusting the beam current density of the electron beam to be a first beam current density of the electron beam, wherein the electron beam is a pre-irradiation electron beam. In a preferred embodiment of the present application the first electron beam has a beam current density of
，
. Preferably, the first electron beam current density can be set within the range by those skilled in the art as desired to be 18nA, 28nA, 30 nA, 38 nA, etc., which are not listed herein. The beam current density of the electron beam can be obtained by adjusting the size of the diaphragm of the electron microscope.
Further, the size of the electron beam irradiation region is selected according to the size of the photographing region, that is, the set irradiation region is determined. And judging whether the irradiation area is larger than the projection area of the scanning electron microscope, and when the irradiation area is larger than the projection area of the scanning electron microscope, realizing large-area electronic irradiation and image shooting in a multi-image splicing mode, namely segmenting the irradiation area to obtain a plurality of irradiation subareas with the same resolution ratio and the area smaller than or equal to the projection area of the scanning electron microscope. And adjusting the size and resolution of a single image of the irradiation sub-area and the preset electron beam irradiation retention time of the single image of the irradiation sub-area. After the parameter setting is completed, the electron beam irradiation experiment is started. According to the size and the resolution of each irradiation subarea, the preset electron beam irradiation retention time and the preset first electron beam density, the electron beam irradiation experiment is carried out on the biological ultrathin slice in each irradiation subarea.
Specifically, the size and resolution of a single image determine the area size of a picture; the preset electron beam irradiation time of a single image is related to the size and resolution of the single image, and can be flexibly set by a person skilled in the art. The preset electron beam irradiation time of a single image can be converted to accurately calculate the electron injection measurement in unit area within the irradiation time. Meanwhile, the electron injection measurement required by the irradiation experiment can be judged in advance through the final image shooting resolution.
After the irradiation experiment is finished, performing an automatic focusing experiment on the biological ultrathin section in the irradiation area after the electron beam irradiation experiment to determine whether irradiation damage occurs in the irradiation area; if the irradiation damage occurs, returning to the step S100 to obtain the biological ultrathin section again for experiment; if no radiation damage occurs, go to step S300. If the irradiation experiment is verified to be successful, the electron injection dosage is proved to be sufficient.
The reason for selecting the automatic focusing experiment to verify the success of the irradiation experiment is that when the image is photographed, the automatic focusing area is small, the photographing resolution is high, the electron injection metering is much higher than that of the non-automatic focusing area, and once the automatic focusing area has no irradiation damage, the irradiation experiment is judged to be successful. And if the irradiation damage occurs, the biological ultrathin section sample is discarded, the unit area electron injection measurement of the irradiation experiment is calculated and used as the experiment data reference so as to improve the subsequent experiment data. Specifically, the unit area electron injection measurement calculation method includes: and obtaining the beam current based on the area of the irradiation region, the size of each irradiation sub-region, the resolution of each irradiation sub-region, the preset electron beam irradiation retention time of each irradiation sub-region and the preset first electron beam current density.
The unit area electron injection metering calculation formula is as follows:
wherein: deNumber of electrons per unit area, IPIs the magnitude of the main electron beam current, e is the electron charge amount, τDFor single pixel dwell time, AxyThe area of a single pixel, A is a constant related to the beam density, C is the beam density when the electron microscope works, pixel size is the size of each pixel when the electron microscope works, t is image acquisition time, and size is the number of pixels of one image. It is understood that, for a single pixel, that is, pixelsize, the irradiation region size/region to be imaged size is an area value obtained by multiplying the number of pixels and the size of the single pixel.
Step S300, selecting a region to be imaged from the irradiation region; and acquiring images of the biological ultrathin section in the area to be imaged according to the preset second electron beam current density to obtain an image of the biological ultrathin section.
Specifically, after the irradiation experiment verification is completed, the region to be imaged is selected from the irradiation region, and the region to be imaged can be flexibly selected by a person skilled in the art. In step S300, the image acquisition method of "acquiring an image of a biological ultrathin section in the region to be imaged to obtain an image of the biological ultrathin section" is the same as the method in step S200, and the image acquisition is performed by a scanning electron microscope. And shooting a high-resolution image according to the irradiated area, adjusting the aperture of an electron microscope diaphragm, and setting the size of the shooting beam current to be a second electron beam current density, wherein the second electron beam current density is required to be smaller than the first electron beam current density. In a preferred embodiment of the present application the second electron beam has a beam current density of
，
. Preferably, the first electron beam current density can be set within the range of 5nA, 8nA, 10 nA, 13 nA, etc. by those skilled in the art at willThey are not listed here.
The reason why the second electron beam current density needs to be smaller than the first electron beam current density is as follows: the size of the beam is mainly adjusted by switching diaphragms with different apertures, the smaller the aperture of the diaphragm is, the smaller the obtainable beam density is, the larger the aperture of the diaphragm is, the larger the obtainable beam density is, and the highest resolution of an image obtained by an instrument under the corresponding large-aperture diaphragm is reduced. A large number of experiments show that the irradiation experiment is carried out in the experimental state of the process
The irradiation experiment and the formal image acquisition experiment can be rapidly carried out by selecting 28nA
Selecting 8nA can obtain a better quality image.
Further, in the step S200, it is determined whether the area to be imaged is larger than the projection area of the scanning electron microscope (i.e. the field of view of the scanning electron microscope), and if so, the area to be imaged is segmented to obtain a plurality of sub-areas to be imaged with the same resolution; and carrying out image acquisition on the biological ultrathin section in each sub-area to be imaged according to the size and the resolution of each sub-area to be imaged and the preset second electron beam irradiation retention time of each sub-area to be imaged and the preset second electron beam current density to obtain an image of the biological ultrathin section. Namely, the size and the resolution of a single sub-image to be imaged and the preset second electron beam irradiation retention time of the sub-area to be imaged are adjusted. And after the parameter setting is finished, starting to perform a high-resolution image acquisition experiment on the biological ultrathin section in the area to be imaged so as to obtain a biological ultrathin section image.
And after the image acquisition is finished, checking the image quality, checking whether damage caused by electron beam irradiation exists, and if not, ending the experiment. Referring to fig. 4 and 5, fig. 4 is a state of a sample directly photographed under the condition that the biological ultrathin section is not subjected to an irradiation experiment, and it can be clearly known from fig. 4 that the sample is damaged by electron beam irradiation. Fig. 5 shows the state of the sample photographed after the irradiation experiment is performed on the ultrathin biological section by the method of the present application, and the damage phenomenon shown in fig. 4 does not occur.
It should be noted that, in the method for performing the experiment in the preferred embodiment of the present application, an irradiation region is selected first, and then a region to be imaged is selected from the irradiation region, it can be understood that, a person skilled in the art may also determine the region to be imaged first, then set a circular region or a region of any shape with the region to be imaged as a center of a circle and a preset distance as a radius, perform the irradiation experiment on the region, then perform image acquisition on the region to be imaged, and obtain the biological ultrathin section image.
The method for rapidly imaging the plastic strip-supported biological ultrathin section is described in detail with reference to specific embodiments.
The first embodiment of the invention: the beam current density of the first electron beam is set to be 28nA, and the area of an irradiation region is 150
x150
The size of an image of the irradiation subarea is 1024x1024, the size of an image pixel of the irradiation subarea is 50nm, the irradiation time of a single image of the irradiation subarea is 3s, and the number of multi-region irradiation images is 3x 3.
Preparing a biological ultrathin section sample, binding the biological ultrathin section sample to a wafer, placing the wafer in a high vacuum coating instrument, and evaporating a carbon film with the thickness of about 10nm on the surface of the biological ultrathin section.
And transferring the coated biological ultrathin section sample to a special sample table for observing an electron microscope sample by using a conductive adhesive tape, and then transferring the sample to a cavity of the electron microscope sample for imaging observation.
The region to be observed was located using image acquisition software, with an observation area of 150
x150
。
After positioning is finished, the size of an electron microscope diaphragm is adjusted to enable the beam current density to be about 28nA, the size of an irradiation image is 1024x1024, the pixel size is 50nm, and the irradiation time of a single image is 3 s.
After the irradiation experiment is finished, randomly selecting a plurality of places in the irradiation finished area to carry out automatic focusing experiment verification, wherein the focusing parameters are set as follows: the beam current density of the second electron beam is set to be 8nA, the image size is 512x512, the pixel size is 3nm, and the residence time is 25 ns.
The focusing area has no irradiation damage, the irradiation experiment is successful, the formal image acquisition can be started, and the irradiation time is increased from 500ns to 25ns while the sample is protected.
Irradiation experiment electron injection metering:
。
focusing experiment electron injection measurement:
。
second embodiment of the invention: the beam density of the first electron beam is set to be 28nA, and the area of an irradiation region is 200
x200
The size of an image of the irradiation subarea is 1024x1024, the size of an image pixel of the irradiation subarea is 100nm, the irradiation time of a single image of the irradiation subarea is 5s, and the number of multi-region irradiation images is 2x 2.
Preparing a biological ultrathin section sample, binding the biological ultrathin section sample to a wafer, placing the wafer in a high vacuum coating instrument, and evaporating a carbon film with the thickness of about 10nm on the surface of the biological ultrathin section.
And transferring the coated biological ultrathin section sample to a special sample table for observing an electron microscope sample by using a conductive adhesive tape, and then transferring the sample to a cavity of the electron microscope sample for imaging observation.
Using image acquisition software to locate and view the area to be viewedObservation area is 200
x200
。
After positioning is finished, the size of an electron microscope diaphragm is adjusted to enable the beam current density to be about 28nA, the size of an irradiation image is 1024x1024, the size of a pixel is 100nm, and the irradiation time of a single image is 5 s.
After the irradiation experiment is finished, randomly selecting a plurality of places in the irradiation finished area to carry out automatic focusing experiment verification, wherein the focusing parameters are set as follows: the 8nA beam current is photographed, the image size is 512x512, the pixel size is 6nm, and the residence time is 20 ns.
The focusing area has no irradiation damage, the irradiation experiment is successful, the formal image acquisition can be started, and the irradiation time is increased from 500ns to 20ns while the sample is protected.
Irradiation experiment electron injection metering:
。
focusing experiment electron injection measurement:
。
third embodiment of the invention: the beam density of the first electron beam is set to be 18nA, and the area of an irradiation region is 200
x200
Image size of irradiation subarea 1024x1024, image pixel size of irradiation subarea 40nm, image irradiation time of single irradiation subarea 2.5s, and multi-region irradiation image quantity 5x 5.
Preparing a biological ultrathin section sample, binding the biological ultrathin section sample to a wafer, placing the wafer in a high vacuum coating instrument, and evaporating a carbon film with the thickness of about 10nm on the surface of the biological ultrathin section.
And transferring the coated biological ultrathin section sample to a special sample table for observing an electron microscope sample by using a conductive adhesive tape, and then transferring the sample to a cavity of the electron microscope sample for imaging observation.
Using image acquisition software, the region to be observed is located with an observation area of 200
x200
。
After positioning is finished, the size of an electron microscope diaphragm is adjusted to enable the beam current density to be about 18nA, the size of an irradiation image is 1024x1024, the size of a pixel is 40nm, and the irradiation time of a single image is 2.5 s.
After the irradiation experiment is finished, randomly selecting a plurality of places in the irradiation finished area to carry out automatic focusing experiment verification, wherein the focusing parameters are set as follows: the 8nA beam current is photographed, the image size is 512x512, the pixel size is 2.5nm, and the residence time is 15 ns.
The focusing area has no irradiation damage, the irradiation experiment is successful, the formal image acquisition can be started, and the irradiation time is increased from 500ns to 15ns while the sample is protected.
Irradiation experiment electron injection metering:
。
focusing experiment electron injection measurement:
。
as can be seen from the above embodiments, the technical solutions of the present application have at least the following technical effects and advantages.
According to the method, the relatively high imaging beam current density is obtained by adjusting the aperture size of the objective lens diaphragm of the electron microscope, the injection speed and action time of specifically metered electrons on the surface of the biological ultrathin section are controlled, and an electron permanent protective layer is formed between the biological ultrathin section and the plastic strip bearing the biological ultrathin section, so that the electron irradiation damage on the surface of the biological section under high beam current density during formal imaging is avoided, and the imaging speed of the electron microscope is improved. The protective layer is permanently effective upon one successful exposure. The imaging method provided by the invention is simple to operate, low in implementation cost and strong in technical reliability, and is suitable for wide application in the field of biological ultrathin slice surface research.
The invention provides a preferred embodiment of a plastic strip bearing biological ultrathin section rapid imaging system, which comprises an acquisition module, a carbon-coated module, an irradiation experiment module and an image acquisition module. Wherein the acquisition module is configured to acquire a biological ultrathin section; the carbon plating module is configured to perform carbon plating treatment on the surface of the biological ultrathin section; the irradiation experiment module is configured to perform an electron beam irradiation experiment on an irradiation area set in the biological ultrathin section based on a preset first electron beam density; and the image acquisition module is configured to acquire an image of the biological ultrathin section in the region to be imaged in the irradiation region based on the preset second electron beam current density, so as to acquire an image of the biological ultrathin section.
It can be clearly understood by those skilled in the art that, for convenience and brevity of description, the specific working process and related description of the system described above may refer to the corresponding process in the foregoing method embodiment, and details are not described herein again.
It should be noted that the plastic strip-carrying ultrathin biological section rapid imaging system provided in the above embodiment is only exemplified by the division of the above functional modules, and in practical applications, the above functions may be allocated to different functional modules according to needs, that is, the modules or steps in the embodiment of the present invention are further decomposed or combined, for example, the modules in the above embodiment may be combined into one module, or may be further split into multiple sub-modules, so as to complete all or part of the above described functions. The names of the modules and steps involved in the embodiments of the present invention are only for distinguishing the modules or steps, and are not to be construed as unduly limiting the present invention.
A storage device according to a fourth embodiment of the present invention stores a plurality of programs, and the programs are suitable for being loaded by a processor and implementing the above-mentioned method for rapidly imaging ultrathin slices of a plastic strip-supported organism.
A processing apparatus according to a fifth embodiment of the present invention includes a processor, a storage device; a processor adapted to execute various programs; a storage device adapted to store a plurality of programs; the program is suitable to be loaded and executed by a processor to realize the plastic strip bearing biological ultrathin section rapid imaging method.
It is clear to those skilled in the art that, for convenience and brevity, the specific working processes and descriptions of the storage device and the processing device described above may refer to the corresponding processes in the example of the signing method, and are not described herein again.
Those of skill in the art would appreciate that the various illustrative modules, method steps, and modules described in connection with the embodiments disclosed herein may be implemented as electronic hardware, computer software, or combinations of both, and that programs corresponding to the software modules, method steps may be located in Random Access Memory (RAM), memory, Read Only Memory (ROM), electrically programmable ROM, electrically erasable programmable ROM, registers, hard disk, a removable disk, a CD-ROM, or any other form of storage medium known in the art. To clearly illustrate this interchangeability of electronic hardware and software, various illustrative components and steps have been described above generally in terms of their functionality. Whether such functionality is implemented as electronic hardware or software depends upon the particular application and design constraints imposed on the solution. Skilled artisans may implement the described functionality in varying ways for each particular application, but such implementation decisions should not be interpreted as causing a departure from the scope of the present invention.
It should be noted that in the description of the present invention, the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. indicating the directions or positional relationships are based on the directions or positional relationships shown in the drawings, which are only for convenience of description, and do not indicate or imply that the device or element must have a specific orientation, be constructed and operated in a specific orientation, and thus, should not be construed as limiting the present invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.
Furthermore, it should be noted that, in the description of the present invention, unless otherwise explicitly specified or limited, the terms "mounted," "connected," and "connected" are to be construed broadly, and may be, for example, fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
The terms "comprises," "comprising," or any other similar term are intended to cover a non-exclusive inclusion, such that a process, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, article, or apparatus.
So far, the technical solutions of the present invention have been described in connection with the preferred embodiments shown in the drawings, but it is easily understood by those skilled in the art that the scope of the present invention is obviously not limited to these specific embodiments. Equivalent changes or substitutions of related technical features can be made by those skilled in the art without departing from the principle of the invention, and the technical scheme after the changes or substitutions can fall into the protection scope of the invention.
Claims (10)
1. A plastic strip bearing biological ultrathin section rapid imaging method is characterized by comprising the following steps: s100, obtaining a biological ultrathin slice, collecting the biological ultrathin slice on a plastic strip, fixing the plastic strip to the surface of a wafer, and carrying out carbon plating treatment on the surface of the biological ultrathin slice; step S200, performing an electron beam irradiation experiment on an irradiation area set in the biological ultrathin section according to a preset first electron beam density; step S300, selecting a region to be imaged from the irradiation region; and acquiring images of the biological ultrathin section in the area to be imaged according to the preset second electron beam current density to obtain an image of the biological ultrathin section.
2. The method for rapidly imaging a plastic strip-supported biological ultrathin section as claimed in claim 1, wherein the step S200 of performing the electron beam irradiation experiment on the set irradiation region in the biological ultrathin section is to perform the electron beam irradiation experiment on the set irradiation region in the biological ultrathin section through a scanning electron microscope; the step S200 further includes: judging whether the irradiation area is larger than the projection area of the scanning electron microscope, if so, dividing the irradiation area to obtain a plurality of irradiation sub-areas with the same resolution; and performing an electron beam irradiation experiment on the biological ultrathin section in each irradiation sub-area according to the size and the resolution of each irradiation sub-area, the preset electron beam irradiation retention time and the preset first electron beam density.
3. The plastic strip supported biological ultrathin slice rapid imaging method as claimed in claim 2, characterized in that electron injection measurement per unit area required by electron beam irradiation experiment is obtained based on the area of the irradiation region, the size of each irradiation sub-region, the resolution of each irradiation sub-region, the preset electron beam irradiation dwell time of each irradiation sub-region and the preset first electron beam current density.
4. The plastic strip bearing biological ultrathin section rapid imaging method as claimed in claim 3, characterized in that the unit area electron injection measurement calculation method is as follows:
wherein: deNumber of electrons per unit area, IPIs a primary electron beam currentMagnitude, e is the electron charge amount, τDFor single pixel dwell time, AxyThe area of a single pixel, A is a constant related to the beam density, C is the beam density when the electron microscope works, pixel size is the size of each pixel when the electron microscope works, t is image acquisition time, and size is the number of pixels of one image.
5. The plastic strip supported biological ultrathin section rapid imaging method as claimed in claim 1, wherein the step S200 further comprises: carrying out an automatic focusing experiment on the biological ultrathin section in the irradiation area after the electron beam irradiation experiment so as to determine whether irradiation damage occurs in the irradiation area; if the irradiation damage occurs, returning to the step S100; if no radiation damage occurs, go to step S300.
6. The plastic tape-supported biological ultrathin section rapid imaging method as claimed in claim 1, characterized in that the plastic tape is adhered to a wafer with a flat surface by a conductive adhesive tape.
7. The method of claim 1, wherein the second predetermined beam current density is less than the first predetermined beam current density.
8. A plastic strip bearing biological ultrathin section rapid imaging system is characterized by comprising an acquisition module, a carbon-plated module, an irradiation experiment module and an image acquisition module; the acquisition module is configured to acquire a biological ultrathin section; the carbon plating module is configured to perform carbon plating treatment on the surface of the biological ultrathin section; the irradiation experiment module is configured to perform an electron beam irradiation experiment on an irradiation area set in the biological ultrathin section based on a preset first electron beam density; the image acquisition module is configured to acquire an image of the biological ultrathin section in the region to be imaged in the irradiation region based on a preset second electron beam current density, and acquire an image of the biological ultrathin section.
9. A storage device having stored therein a plurality of programs, wherein the programs are adapted to be loaded and executed by a processor to implement the method for rapid imaging of plastic strip-bearing ultrathin biological sections as claimed in any one of claims 1 to 7.
10. A processing arrangement comprising a processor, a storage device; a processor adapted to execute various programs; a storage device adapted to store a plurality of programs; characterized in that the program is adapted to be loaded and executed by a processor to implement the plastic strip supported biological ultrathin section rapid imaging method of any one of claims 1 to 7.
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