CN112611775B - Method for observing biological tissue and electron microscope - Google Patents
Method for observing biological tissue and electron microscope Download PDFInfo
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- CN112611775B CN112611775B CN202011484757.4A CN202011484757A CN112611775B CN 112611775 B CN112611775 B CN 112611775B CN 202011484757 A CN202011484757 A CN 202011484757A CN 112611775 B CN112611775 B CN 112611775B
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
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- G01N23/2206—Combination of two or more measurements, at least one measurement being that of secondary emission, e.g. combination of secondary electron [SE] measurement and back-scattered electron [BSE] measurement
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N23/20—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 using diffraction of the radiation by the materials, e.g. for investigating crystal structure; by using scattering of the radiation by the materials, e.g. for investigating non-crystalline materials; by using reflection of the radiation by the materials
- G01N23/20008—Constructional details of analysers, e.g. characterised by X-ray source, detector or optical system; Accessories therefor; Preparing specimens therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N23/20—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 using diffraction of the radiation by the materials, e.g. for investigating crystal structure; by using scattering of the radiation by the materials, e.g. for investigating non-crystalline materials; by using reflection of the radiation by the materials
- G01N23/20083—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 using diffraction of the radiation by the materials, e.g. for investigating crystal structure; by using scattering of the radiation by the materials, e.g. for investigating non-crystalline materials; by using reflection of the radiation by the materials by using a combination of at least two measurements at least one being a transmission measurement and one a scatter measurement
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N23/20—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 using diffraction of the radiation by the materials, e.g. for investigating crystal structure; by using scattering of the radiation by the materials, e.g. for investigating non-crystalline materials; by using reflection of the radiation by the materials
- G01N23/203—Measuring back scattering
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N23/2202—Preparing specimens therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
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- G01N23/227—Measuring photoelectric effect, e.g. photoelectron emission microscopy [PEEM]
- G01N23/2276—Measuring photoelectric effect, e.g. photoelectron emission microscopy [PEEM] using the Auger effect, e.g. Auger electron spectroscopy [AES]
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J37/00—Discharge tubes with provision for introducing objects or material to be exposed to the discharge, e.g. for the purpose of examination or processing thereof
- H01J37/02—Details
- H01J37/04—Arrangements of electrodes and associated parts for generating or controlling the discharge, e.g. electron-optical arrangement, ion-optical arrangement
- H01J37/06—Electron sources; Electron guns
- H01J37/065—Construction of guns or parts thereof
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J37/00—Discharge tubes with provision for introducing objects or material to be exposed to the discharge, e.g. for the purpose of examination or processing thereof
- H01J37/26—Electron or ion microscopes; Electron or ion diffraction tubes
- H01J37/261—Details
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Abstract
The invention discloses a method for observing biological tissues and an electron microscope, wherein the method comprises the following steps: slicing and sampling biological tissues to obtain samples; irradiating the sample by adopting a first electron source system to obtain a sample to be detected containing polymer components; and observing the sample to be detected by adopting an electron microscope. The invention can ensure that the imaging resolution ratio is high, the imaging speed is high, the imaging is clear, and the observation accuracy is high when the electron microscope observes biological tissues.
Description
Technical Field
The invention belongs to the technical field of biological sample observation, and particularly relates to a method for observing biological tissues and an electron microscope.
Background
In the prior art, in biology, animals and plants are mainly composed of cells, and in order to better study life science, the cells are often required to be observed through an electron microscope for understanding.
When observing the structure of biological cells by using an electron microscope, firstly, slicing and sampling biological tissues, cutting biological tissues with proper sizes on a living body by means of dissection, operation and the like, then putting the biological tissues into a fixing liquid, fixing the biological tissues put into the fixing liquid by a physical method or a chemical method, rinsing, dehydrating and dyeing the biological tissues after fixing, embedding and polymerizing the biological tissues into a solid sample by adding water-soluble resin, and finally slicing the sample polymerized into the solid sample to obtain the sample after slicing and sampling. The sliced sample was placed in an electron microscope and observed. However, due to the resolution requirement for electron microscope imaging and the imaging speed requirement, when a high-speed, high-density electron beam current is applied to such a sliced sample, damages such as deformation, decomposition and destruction of fine structures occur. In particular, in the electron beam focusing area, the large-dose electrons act in the surface of the small-area sample, so that the sample is damaged in a honeycomb shape, reconstruction information is affected due to the damage of the sample, and the image cannot be reconstructed later. Resulting in failure to properly analyze biological tissue structures and reduced accuracy of observation.
The present invention has been made in view of this.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for observing biological tissues and an electron microscope.
In order to solve the technical problems, the invention adopts the basic conception of the technical scheme that:
a method for viewing biological tissue, comprising:
slicing and sampling biological tissues to obtain samples;
irradiating the sample by adopting a first electron source system to obtain a sample to be detected containing polymer components;
and observing the sample to be detected by adopting an electron microscope.
Further, the irradiating the sample with the first electron source system to obtain a sample to be measured containing a polymer component includes:
the irradiation is performedThe dose rate of (2) is 1.12X10 13 e - /mm 2 ·s-6.25×10 15 e - /mm 2 S, the heat radiation is 0.025W/mm 2 -3W/mm 2 。
In some alternative embodiments, irradiating the sample with the first electron source system to obtain a sample to be measured comprising a polymer component comprises:
the current introduced by the first electron source system is 1A-9A;
the distance between the first electron source system and the sample surface is 5mm-150mm;
the potential difference between the first electron source system and the sample is between 0.5kv and 12kv.
In some alternative embodiments, irradiating the sample with the first electron source system to obtain a sample to be measured comprising a polymer component comprises:
the current introduced by the first electron source system is 3A;
the distance between the first electron source system and the sample surface is 64mm;
the potential difference between the first electron source system and the sample was 2kv.
In some alternative embodiments, irradiating the sample with the first electron source system to obtain a sample to be measured comprising a polymer component comprises:
the area of the first electron source system irradiating the sample is 1mm 2 -100mm 2 ；
The first electron source system irradiates the sample for 1s-60s.
In some alternative embodiments, the potential difference between the first electron source system and the sample is 2kv volts comprises:
the voltage of the first electron source system is 0kv, and the voltage of the sample is 2kv;
alternatively, the voltage of the first electron source system is-2 kv, and the voltage of the sample is 0kv.
The present invention also provides an electron microscope for observing biological tissue, comprising:
the first electron source system is connected with a first vacuum chamber at the lower end and irradiates a sample placed in the first vacuum chamber;
the electron optical lens barrel is connected with a second vacuum chamber at the lower end of the electron optical lens barrel, and electron beams emitted by the electron optical lens barrel act on a sample to be measured placed in the second vacuum chamber.
Further, the first electron source system comprises a filament, a first winding component and a second winding component which are arranged in parallel at intervals, wherein the first winding component and the second winding component comprise at least one winding unit, the winding units in the first winding component and the winding units in the second winding component are arranged in a staggered mode, the filament enters from the winding units at one end of the first winding component or the second winding component and winds on the winding units staggered in the second winding component or the first winding component, and the filament sequentially winds around until the winding units at the other end of the first winding component or the second winding component extend out.
In some alternative embodiments, the first electron source system comprises a mount and a filament, the filament being spirally coiled;
the lamp filaments are at least one, and a plurality of lamp filaments are arranged on the mounting seat at intervals.
In some alternative embodiments, the first electron source system comprises a spring wire filament, the filament being arranged in a line segment, or the filament being arranged in a ring with an opening.
By adopting the technical scheme, compared with the prior art, the invention has the following beneficial effects.
According to the method for observing biological tissues and the electron microscope, the first electron source system irradiates the sliced sample, and the sample generates physical reaction and chemical reaction under the action of heat radiation and electron beams emitted by the first electron source system, so that the cross-linking polymerization reaction of the sample is initiated, and the sample to be detected containing polymer components is formed by modification. The irradiated sample to be measured containing the polymer component can resist high temperature and bear the action of high-speed and high-density electron beam current without damage, the electron microscope can adopt the high-speed and high-density electron beam current to act on the sample to be measured, the imaging resolution of the electron microscope is high, the imaging speed is high, the imaging is clear, and the observation accuracy is high.
The following describes the embodiments of the present invention in further detail with reference to the accompanying drawings.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention. It is evident that the drawings in the following description are only examples, from which other drawings can be obtained by a person skilled in the art without the inventive effort. In the drawings:
FIG. 1 is a flow chart of a method for viewing biological tissue provided by the present invention;
FIG. 2 is a schematic view of an electron microscope for observing biological tissue according to the present invention;
FIG. 3 is a schematic view of an embodiment of a mounting base and a filament according to the present invention;
FIG. 4 is a schematic view of another embodiment of the mounting base and filament according to the present invention;
FIG. 5 is a schematic view of a structure of a mounting base and a filament according to another embodiment of the present invention;
fig. 6 is a schematic structural diagram of a mounting base and a filament according to another embodiment of the present invention.
In the figure: 1. an electron optical lens barrel; 101. a second electron source system; 102. an electron acceleration structure; 103. a deflection device; 104. an objective lens; 2. a first electron source system; 201. a connection terminal; 202. a mounting base; 203. a filament; 3. a sample injection door body; 4. a first vacuum chamber; 5. a telescopic bracket; 6. a tray; 7. a sample; 8. an isolation door body; 9. a second vacuum chamber; 10. a sample stage; 11. and (5) testing a sample.
It should be noted that these drawings and the written description are not intended to limit the scope of the inventive concept in any way, but to illustrate the inventive concept to those skilled in the art by referring to the specific embodiments.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments will be clearly and completely described with reference to the accompanying drawings in the embodiments of the present invention, and the following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.
In the description of the present invention, it should be noted that the directions or positional relationships indicated by the terms "upper", "lower", "front", "rear", "left", "right", "vertical", "inner", "outer", etc. are based on the directions or positional relationships shown in the drawings, are merely for convenience of describing the present invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific orientation, be configured and operated in a specific orientation, and thus should not be construed as limiting the present invention.
In the description of the present invention, it should be noted that, unless explicitly specified and limited otherwise, the terms "mounted," "connected," and "connected" are to be construed broadly, and may be either fixedly connected, detachably connected, or integrally connected, for example; can be mechanically or electrically connected; can be directly connected or indirectly connected through an intermediate medium. The specific meaning of the above terms in the present invention will be understood in specific cases by those of ordinary skill in the art.
As shown in fig. 1 to 6, the present invention provides a method for observing biological tissue, the method comprising the steps of:
s110, slicing and sampling biological tissues to obtain a sample 7;
s120, irradiating the sample 7 by adopting a first electron source system 2 to obtain a sample 11 to be detected containing polymer components;
and S130, observing the sample 11 to be detected by adopting an electron microscope.
Specific:
s110, slicing and sampling biological tissues to obtain a sample 7;
in detail, after a biological tissue of a proper size is cut out by dissection, surgery or the like on a living body, the biological tissue is put into a fixing solution, and a chemical fixing method can be adopted for fixing, for example, the fixing solution can be used for fixing a cell structure by using 2.5% glutaraldehyde and 4% paraformaldehyde mixed buffer fixing solution. Physical fixation methods, such as freezing, microwave irradiation, critical point drying, etc., may also be used to preserve cell structure. After fixing, rinsing, dehydrating and dyeing, embedding and polymerizing into a solid sample by adding water-soluble resin, and slicing the solid sample by using a diamond knife automatic strip slicing machine to obtain a sample 7 after slicing and sample preparation. Biological tissue section preparation is a common process means in the art and is not described here.
S120, irradiating the sample 7 by adopting a first electron source system 2 to obtain a sample 11 to be detected containing polymer components;
in detail, the dose rate of irradiation was 1.12X10 13 e - /mm 2 ·s-6.25×10 15 e - /mm 2 S, the heat radiation is 0.025W/mm 2 -3W/mm 2 。
The first electron source system 2 comprises a filament 203 and a terminal 201, a power supply is connected with the terminal 201, the terminal 201 is connected with the filament 203, and the power supply supplies power to the filament 203 through the terminal 201. The filament 203 emits electron beams and heat radiation when current is applied. When the first electron source system 2 is energized, the electron beam and the thermal radiation emitted from the first electron source system 2 act on the sample 7, and the sample 7 is subjected to the electron beam irradiation at a dose rate of 1.12X10 13 e - /mm 2 ·s-6.25×10 15 e - /mm 2 S, the heat radiation to which the sample 7 was subjected was 0.025W/mm 2 -3W/mm 2 Sample 7 undergoes physical and chemical reactions, triggeringSample 7 was crosslinked and modified to form sample 11 to be tested containing the polymer component. The irradiated sample 11 to be measured containing the polymer component can resist high temperature and bear the action of high-speed and high-density electron beam current without damage.
Preferably, the first electron source system 2 irradiates the sample 7 by controlling the current supplied to the first electron source system 2 at a dose rate of 1.875×10 13 e - /mm 2 S, the heat radiation is 0.21W/mm 2 . To obtain a sample to be measured 11 containing a polymer component.
In some alternative embodiments, irradiating the sample 7 with the first electron source system 2 to obtain a sample 11 to be measured containing a polymer component includes:
the current introduced by the first electron source system 2 is 1A-9A;
the distance between the first electron source system 2 and the surface of the sample 7 is 5mm-150mm;
the potential difference between the first electron source system 2 and the sample 7 is 0.5kv-12kv.
When the first electron source system 2 is electrified, electron beams and heat radiation emitted by the first electron source system 2 act on the sample 7, the electrified current of the first electron source system 2 is 1A-9A, the distance between the first electron source system 2 and the surface of the sample 7 is 5mm-150mm, and the potential difference between the first electron source system 2 and the sample 7 is 0.5kv-12kv. By irradiating the sample 7 in the parameter range, the sample 7 can generate physical reaction and chemical reaction, so as to trigger the cross-linking polymerization reaction of the sample 7, and the sample 11 to be detected containing polymer components is formed by modification. The irradiated sample 11 to be measured containing the polymer component can resist high temperature and bear the action of high-speed and high-density electron beam current without damage.
It should be noted that, the specific value of the current flowing into the first electron source system 2, the specific value of the distance between the first electron source system 2 and the surface of the sample 7, the specific value of the potential difference between the first electron source system 2 and the sample 7, which can be selected by those skilled in the art within the numerical range of the above embodiments, can all initiate the cross-linking polymerization reaction of the sample 7, and the sample 11 to be tested containing the polymer component is formed by modification.
Preferably, irradiating the sample 7 with the first electron source system 2 to obtain a sample 11 to be measured containing a polymer component includes:
the current introduced by the first electron source system 2 is 3A;
the distance between the first electron source system 2 and the surface of the sample 7 is 64mm;
the potential difference between the first electron source system 2 and the sample 7 is 2kv.
When the first electron source system 2 is supplied with current, the electron beam and the heat radiation emitted by the first electron source system 2 act on the sample 7, the current supplied to the first electron source system 2 is 3A, the distance between the first electron source system 2 and the surface of the sample 7 is 64mm, and the potential difference between the first electron source system 2 and the sample 7 is 2kv. By adopting the specific parameters to irradiate the sample 7, the physical reaction and the chemical reaction of the sample 7 can be more completely and thoroughly caused, the cross-linking polymerization reaction of the sample 7 is initiated, and the sample 11 to be tested containing the polymer component is formed by modification.
Further, irradiating the sample 7 with the first electron source system 2 to obtain a sample 11 to be measured containing a polymer component includes:
the area of the first electron source system 2 irradiating the sample 7 was 1mm 2 -100mm 2 ；
The first electron source system 2 irradiates the sample 7 for a time of 1s-60s.
Specific:
when the first electron source system 2 is electrified, the electron beam and the thermal radiation emitted by the first electron source system 2 act on the sample 7, and the area of the first electron source system 2 irradiating the sample 7 is 1mm 2 -100mm 2 The first electron source system 2 irradiates the sample 7 for a time of 1s to 60s.
Optionally, the first electron source system 2 scans and irradiates the sample 7, and during the scanning process, the irradiation area of the electron beam spot is 1mm 2 -100mm 2 The irradiation time is 1s-60s, after one area is irradiated, the electron beam spot is moved to irradiate the next area, and the large-area sample is sequentially completed7.
It should be noted that, the specific value of the irradiation area of the sample 7 by the first electron source system 2, the specific value of the irradiation time of the sample 7 by the first electron source system 2, and the specific value of the irradiation time of the sample 7 may be selected by those skilled in the art within the numerical ranges of the above embodiments, and may all initiate the cross-linking polymerization reaction of the sample 7, so as to modify the sample to be tested 11 containing the polymer component.
Preferably, irradiating the sample 7 with the first electron source system 2 to obtain a sample 11 to be measured containing a polymer component includes:
the area of the first electron source system 2 irradiating the sample 7 was 75mm 2 ；
The first electron source system 2 irradiates the sample 7 for 10s;
by adopting the specific parameters to irradiate the sample 7, the physical reaction and the chemical reaction of the sample 7 can be more completely and thoroughly caused, the cross-linking polymerization reaction of the sample 7 is initiated, and the sample 11 to be tested containing the polymer component is formed by modification.
Still further, the potential difference between the first electron source system 2 and the sample 7 being 2kv volts comprises:
the voltage of the first electron source system 2 was 0kv and the voltage of the sample 7 was 2kv;
specifically, the voltage of the first electron source system 2 was 0kv and the voltage of the sample 7 was 2kv, so that the potential difference between the first electron source system 2 and the sample 7 was 2kv. An accelerating electric field is formed between the first electron source system 2 and the sample 7, and the electron beam emitted by the first electron source system 2 acts on the sample 7 after being accelerated by the accelerating electric field.
Alternatively, the voltage of the first electron source system 2 is-2 kv and the voltage of the sample 7 is 0kv;
specifically, the voltage of the first electron source system 2 was-2 kv and the voltage of the sample 7 was 0kv, so that the potential difference between the first electron source system 2 and the sample 7 was 2kv. An accelerating electric field is formed between the first electron source system 2 and the sample 7, and the electron beam emitted by the first electron source system 2 acts on the sample 7 after being accelerated by the accelerating electric field.
And S130, observing the sample 11 to be detected by adopting an electron microscope.
The sample 7 generates physical reaction and chemical reaction under the action of the heat radiation and the electron beam emitted by the first electron source system 2, and initiates the cross-linking polymerization reaction of the sample 7, so as to form the sample 11 to be tested containing the polymer component.
The sample 11 to be measured is observed by an electron microscope. The irradiated sample 11 to be measured contains polymer components, so that the irradiation sample can bear the action of high-speed and high-density electron beams without damage, the electron microscope can select larger electron beams to act on the sample 11 to be measured, the electron microscope adopts the electron beams with large beam to act on the sample 11 to be measured, the imaging resolution of the electron microscope is higher, the imaging speed is high, the imaging is clear, and the observation accuracy is high.
As shown in fig. 2 to 6, the present invention provides an electron microscope for observing biological tissue, which includes a first electron source system 2, a first vacuum chamber 4, an electron optical column 1, and a second vacuum chamber 9.
The lower end of the first electron source system 2 is connected with a first vacuum chamber 4, and the first electron source system 2 irradiates a sample 7 placed in the first vacuum chamber 4;
the lower end of the electron optical lens barrel 1 is connected with a second vacuum chamber 9, and electron beams emitted by the electron optical lens barrel 1 act on a sample 11 to be measured placed in the second vacuum chamber 9.
Specifically, the lower extreme of first electron source system 2 is connected with first vacuum chamber 4, and first vacuum chamber 4 is including the sampling door body 3 of openable and closable, and sliding connection has flexible bracket 5 on the sampling door body 3, and flexible bracket 5 can go up and down to slide on sampling door body 3, height-adjusting. The telescopic bracket 5 is provided with a tray 6, and the sample 7 is placed on the tray 6. The first electron source system 2 includes a filament 203, a connection terminal 201, and a mounting base 202, wherein the material of the mounting base 202 is preferably ceramic, the material of the filament 203 may be tungsten, or tungsten-rhenium alloy, or yttrium iridium oxide, or lanthanum hexaboride, which is a common filament 203 material, and one of the materials may be selected as the filament 203 by those skilled in the art. The filament 203 is installed on the mount pad 202, and the power is connected with binding post 201, and binding post 201 is connected with filament 203, and the power passes through binding post 201 and supplies power for filament 203. The filament 203 emits electron beams and heat radiation when current is applied. That is, when a current is applied to the first electron source system 2, the electron beam and the heat radiation emitted from the first electron source system 2 act on the sample 7 placed in the first vacuum chamber 4, and the sample 7 undergoes a physical reaction and a chemical reaction under irradiation to induce a cross-linking polymerization reaction of the sample 7, thereby modifying the sample to be measured 11 containing a polymer component.
The lower end of the electron optical lens barrel 1 is connected with a second vacuum chamber 9, and electron beams emitted by the electron optical lens barrel 1 act on a sample 11 to be measured placed in the second vacuum chamber 9. A sample stage 10 is arranged in the second vacuum chamber 9, the sample stage 10 being capable of five degrees of freedom of movement including: three-dimensional translation (X, Y and Z translation), rotation about a central axis (R), and tilt (T). The second vacuum chamber 9 is connected to the first vacuum chamber 4 through an openable and closable isolation door 8.
The sample 7 is irradiated in the first vacuum chamber 4 by the first electron source system 2 to obtain a sample 11 to be measured. The isolation door body 8 is opened, at the moment, the first vacuum chamber 4 is communicated with the second vacuum chamber 9, the telescopic bracket 5 performs stretching movement to drive the tray 6 and the sample 11 to be measured placed on the tray 6, and the first vacuum chamber 4 stretches into the second vacuum chamber 9. When the tray 6 is extended above the sample stage 10, the extending movement is stopped. The sample stage 10 lifts up the jack-up tray 6, the telescopic bracket 5 makes a contraction movement, the telescopic bracket 5 retracts the first vacuum chamber 4, and the isolation door 8 is closed.
The tray 6 and the sample 11 to be measured placed on the tray 6 are placed on the sample stage 10 of the second vacuum chamber 9. The sample 11 to be measured is driven to move to a proper working position by adjusting the five-degree-of-freedom movement of the sample table 10, so that the sample 11 to be measured can be conveniently observed. The electron optical column 1 is used for generating an electron beam and focusing the electron beam on a sample 11 to be measured, and the electron optical column 1 comprises a second electron source system 101, an electron acceleration structure 102 and an objective lens system.
Specifically, the second electron source system 101 is used for generating an electron beam.
The electron acceleration structure 102 is an anode, and is used for forming an electric field along the emission direction of the electron beam to increase the movement speed of the electron beam.
The objective lens system is used to control the beam size and the beam advancing direction of the electron beam emitted by the second electron source system 101. The objective system focuses the electron beam onto the sample 7 and scans it.
The objective system comprises an objective lens 104 and a deflection device 103, the objective lens 104 may be a magnetic lens, or an electric lens, or an electromagnetic compound lens. The deflection means 103 may be magnetic deflection means or electrical deflection means.
The deflection device 103 is used for changing the movement direction of the electron beam before entering the sample 11 to be measured, and can generate a scanning field with any deflection direction.
The electron beam acts on the sample 11 to be measured to generate signal electrons such as secondary electrons, backscattered electrons, auger electrons, etc. The electron microscope for observing biological tissues provided by the invention further comprises a detector for receiving signal electrons generated by the electron beam acting on the sample 11 to be measured.
Taking the received signal electrons as secondary electrons and back scattered electrons as an example, the detector may be a secondary electron detector that receives secondary electrons alone, or a back scattered electron detector that receives back scattered electrons alone, or a detector that receives both secondary electrons and back scattered electrons simultaneously. In the present invention, the person skilled in the art can select the detector type according to his own actual needs to receive the corresponding electronic type. The detector can be optionally integrated in the electron optical lens barrel 1 or the detector and the electron optical lens barrel 1 are mutually independent.
As shown in fig. 1 to 6, a specific example will be described below, in which a biological tissue is sliced to obtain a sample 7, and the sample 7 is placed on a tray 6. The sample injection door 3 is opened, the tray 6 and the sample 7 placed on the tray 6 are placed on the telescopic bracket 5, the sample injection door 3 is closed, and the telescopic bracket 5 is adjusted so that the sample 7 is located below the first electron source system 2. Adjustment ofThe degree of vacuum of the first vacuum chamber 4 is less than 1×10 -4 Torr. Preferably, the first vacuum chamber 4 has a vacuum degree of 5×10 - 5 Torr。
The telescopic length and height of the telescopic bracket 5 were adjusted so that the sample 7 was located below the first electron source system 2, and the distance between the first electron source system 2 and the surface of the sample 7 was 64mm, the current flowing to the first electron source system 2 was 3A, the voltage of the first electron source system 2 was-2 kv, and the voltage of the sample 7 was 0kv. The potential difference between the first electron source system 2 and the sample 7 is 2kv. The electron beam and the thermal radiation emitted from the first electron source system 2 act on the sample 7, and the area of the sample 7 irradiated by the first electron source system 2 is 75mm 2 The first electron source system 2 irradiates the sample 7 for 10s.
By adopting the specific parameters to irradiate the sample 7, the physical reaction and the chemical reaction of the sample 7 can be more completely and thoroughly caused, the cross-linking polymerization reaction of the sample 7 is initiated, and the sample 11 to be tested containing the polymer component is formed by modification. The sample 7 is irradiated in the first vacuum chamber 4 by the first electron source system 2 to obtain a sample 11 to be measured. The isolation door body 8 is opened, at the moment, the first vacuum chamber 4 is communicated with the second vacuum chamber 9, the telescopic bracket 5 performs stretching movement to drive the tray 6 and the sample 11 to be measured placed on the tray 6, and the first vacuum chamber 4 stretches into the second vacuum chamber 9. When the tray 6 is extended above the sample stage 10, the extending movement is stopped. The sample stage 10 lifts up the jack-up tray 6, the telescopic bracket 5 makes a contraction movement, the telescopic bracket 5 retracts the first vacuum chamber 4, and the isolation door 8 is closed.
The degree of vacuum of the second vacuum chamber 9 is adjusted to be lower than 5×10 -5 Torr. Preferably, the second vacuum chamber 9 has a vacuum degree of 5×10 -6 Torr. The tray 6 and the sample 11 to be measured placed on the tray 6 are placed on the sample stage 10 in the second vacuum chamber 9. The sample 11 to be measured is driven to move to a proper working position by adjusting the five-degree-of-freedom movement of the sample table 10, so that the sample 11 to be measured can be conveniently observed. The electron optical lens barrel 1 is used for generating an electron beam and focusing the electron beam onto a sample 11 to be measured, and the electron beam acts on the sample 11 to be measured to generate secondary electrons and back scatteringElectrons, auger electrons, etc. The detector is arranged to receive signal electrons generated by the electron beam acting on the sample 11 to be measured.
The sample 11 to be measured is observed by an electron microscope. The irradiated sample 11 to be measured contains polymer components, so that the irradiation sample can bear the action of high-speed and high-density electron beams without damage, the electron microscope can select larger electron beams to act on the sample 11 to be measured, the electron microscope adopts the electron beams with large beam to act on the sample 11 to be measured, the imaging resolution of the electron microscope is higher, the imaging speed is high, the imaging is clear, and the observation accuracy is high.
In some alternative embodiments, as shown in fig. 3, the present invention provides an electron microscope for viewing biological tissue. The first electron source system 2 comprises a filament 203, a first winding component and a second winding component which are arranged in parallel at intervals, wherein the first winding component and the second winding component comprise at least one winding unit, the winding units in the first winding component and the winding units in the second winding component are arranged in a staggered mode, the filament 203 enters from the winding units at one end of the first winding component or the winding units at one end of the second winding component and winds on the winding units staggered in the second winding component or the first winding component, and the filament 203 sequentially circulates and is arranged until the winding units at the other end of the first winding component or the second winding component extend. The first winding assembly and the second winding assembly are mounted on the mounting base 202, and both ends of the filament 203 are connected with a power supply through the connection terminals 201.
With the filament 203 of this embodiment, the first electron source system 2 can emit a large-area and large-beam electron beam, so that the irradiation area is large, the heat radiation is high, the time is saved, the sample 7 can be more completely and thoroughly subjected to physical reaction and chemical reaction, the cross-linking polymerization reaction of the sample 7 is initiated, and the sample 11 to be measured containing polymer components is formed by modification on the basis of ensuring the irradiation dose rate and the heat radiation.
In some alternative embodiments, as shown in fig. 4, the present invention provides an electron microscope for viewing biological tissue. The first electron source system 2 includes a mounting base 202 and filaments 203, the filaments 203 are spirally wound, at least one filament 203, and a plurality of filaments 203 are arranged on the mounting base 202 at intervals.
Preferably, 4 filaments 203 spirally wound are provided, the mounting seat 202 is disc-shaped, the 4 filaments 203 are uniformly distributed on the mounting seat 202 at intervals by taking the center of the mounting seat 202 as the center of a circle, and each filament 203 is connected with a power supply through a wiring terminal 201.
With the shape and arrangement of the filament 203 in this embodiment, the first electron source system 2 can emit a large-area and large-beam electron beam, so that the irradiation area is large, the heat radiation is high, the time is saved, the physical reaction and chemical reaction of the sample 7 can be more completely and thoroughly caused, and the cross-linking polymerization reaction of the sample 7 is initiated, so that the sample 11 to be measured containing the polymer component is formed by modification.
It should be noted that, with the specific number of filaments 203 in this embodiment, the spacing distance between the filaments 203, and the arrangement of the filaments 203, those skilled in the art may choose the number according to the actual situation.
In some alternative embodiments, as shown in fig. 5, the present invention provides an electron microscope for viewing biological tissue. The first electron source system 2 comprises a spring wire filament 203, the spring wire filament 203 being in a ring-shaped arrangement with an opening. The filament 203 is connected to a power supply through a connection terminal 201. The filament 203 is connected with the mounting base 202 through a filament post, and is fixedly mounted on the mounting base 202.
With the shape and arrangement of the filament 203 in this embodiment, the first electron source system 2 can emit a large-area and large-beam electron beam, so that the irradiation area is large, the heat radiation is high, the time is saved, the physical reaction and chemical reaction of the sample 7 can be more completely and thoroughly caused, and the cross-linking polymerization reaction of the sample 7 is initiated, so that the sample 11 to be measured containing the polymer component is formed by modification.
In some alternative embodiments, as shown in fig. 6, the present invention provides an electron microscope for viewing biological tissue. The first electron source system 2 includes a spring wire filament 203, and the spring wire filament 203 is arranged in a line segment. The filament 203 is connected to a power supply through a connection terminal 201. The filament 203 is connected with the mounting base 202 through a filament post, and is fixedly mounted on the mounting base 202.
With the shape and arrangement of the filament 203 in this embodiment, the first electron source system 2 can emit a large-area and large-beam electron beam, so that the irradiation area is large, the heat radiation is high, the time is saved, the physical reaction and chemical reaction of the sample 7 can be more completely and thoroughly caused, and the cross-linking polymerization reaction of the sample 7 is initiated, so that the sample 11 to be measured containing the polymer component is formed by modification.
The foregoing description is only illustrative of the preferred embodiment of the present invention, and is not to be construed as limiting the invention, but is to be construed as limiting the invention to any and all simple modifications, equivalent variations and adaptations of the embodiments described above, which are within the scope of the invention, may be made by those skilled in the art without departing from the scope of the invention.
Claims (9)
1. A method for viewing biological tissue, comprising:
slicing and sampling biological tissues to obtain samples;
irradiating the sample by adopting a first electron source system to obtain a sample to be detected containing a polymer component, wherein the polymer is obtained by performing cross-linking polymerization reaction on the sample under the irradiation of the first electron source system, and the irradiation dose rate is 1.12 multiplied by 10 13 e - /mm 2 ·s-6.25×10 15 e - /mm 2 S, the heat radiation is 0.025W/mm 2 -3W/mm 2 ；
And observing the sample to be detected by adopting an electron microscope.
2. The method for observing biological tissue of claim 1 wherein irradiating the sample with the first electron source system to obtain a sample to be measured comprising a polymer component comprises:
the current introduced by the first electron source system is 1A-9A;
the distance between the first electron source system and the sample surface is 5mm-150mm;
the potential difference between the first electron source system and the sample is between 0.5kv and 12kv.
3. The method for observing biological tissue of claim 1 wherein irradiating the sample with the first electron source system to obtain a sample to be measured comprising a polymer component comprises:
the current introduced by the first electron source system is 3A;
the distance between the first electron source system and the sample surface is 64mm;
the potential difference between the first electron source system and the sample was 2kv.
4. The method for observing biological tissue of claim 1 wherein irradiating the sample with the first electron source system to obtain a sample to be measured comprising a polymer component comprises:
the area of the first electron source system irradiating the sample is 1mm 2 -100mm 2 ；
The first electron source system irradiates the sample for 1s-60s.
5. A method for viewing biological tissue according to claim 3, wherein the potential difference between the first electron source system and the sample is 2kv comprises:
the voltage of the first electron source system is 0kv, and the voltage of the sample is 2kv;
alternatively, the voltage of the first electron source system is-2 kv, and the voltage of the sample is 0kv.
6. An electron microscope for viewing biological tissue, comprising:
the first electron source system is connected with a first vacuum chamber at the lower end and irradiates a sample placed in the first vacuum chamber, the first vacuum chamber comprises an openable sample injection door body, a telescopic bracket is connected onto the sample injection door body in a sliding manner, a tray is arranged on the telescopic bracket, and the telescopic bracket can lift and slide on the sample injection door body;
the electron optical lens cone, electron optical lens cone lower extreme is connected with the second vacuum chamber, be provided with the sample platform in the second vacuum chamber, flexible bracket can drive the tray removes to the top of sample platform, just the sample platform rises and jack-up the tray, in order to with the tray removes to on the sample platform, electron beam that electron optical lens cone transmitted is on placing the interior sample that awaits measuring of second vacuum chamber.
7. The electron microscope for observing biological tissues according to claim 6, wherein the first electron source system comprises a filament, a first winding assembly and a second winding assembly which are arranged in parallel at intervals, wherein the first winding assembly and the second winding assembly comprise at least one winding unit, the winding units in the first winding assembly and the winding units in the second winding assembly are arranged in a staggered manner, the filament enters from the winding units at one end of the first winding assembly or the second winding assembly and winds on the winding units staggered in the second winding assembly or the first winding assembly, and the filament sequentially circulates and extends until the winding units at the other end of the first winding assembly or the second winding assembly.
8. The electron microscope for viewing biological tissue of claim 6, wherein the first electron source system comprises a mount and a filament, the filament being helically coiled;
the lamp filaments are at least one, and a plurality of lamp filaments are arranged on the mounting seat at intervals.
9. The electron microscope for viewing biological tissue of claim 6, wherein the first electron source system comprises a spring wire filament, the filament being arranged in a line segment or the filament being arranged in a ring having an opening.
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