CN112611775A - Method for observing biological tissue and electron microscope - Google Patents

Method for observing biological tissue and electron microscope Download PDF

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CN112611775A
CN112611775A CN202011484757.4A CN202011484757A CN112611775A CN 112611775 A CN112611775 A CN 112611775A CN 202011484757 A CN202011484757 A CN 202011484757A CN 112611775 A CN112611775 A CN 112611775A
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sample
source system
electron source
electron
filament
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CN112611775B (en
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杨润潇
何伟
李帅
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Focus eBeam Technology Beijing Co Ltd
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Focus eBeam Technology Beijing Co Ltd
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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
    • G01N23/22—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
    • G01N23/2206—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
    • 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
    • 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
    • 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
    • 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
    • G01N23/22—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
    • 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
    • G01N23/22—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
    • G01N23/227—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]
    • 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
    • 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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  • Analytical Chemistry (AREA)
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  • Life Sciences & Earth Sciences (AREA)
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  • Health & Medical Sciences (AREA)
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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 a sample; 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 is used for observing the biological tissue.

Description

Method for observing biological tissue and electron microscope
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 sciences, the cells are observed through an electron microscope for understanding and observation.
When observing the structure of biological cells by using an electron microscope, firstly, biological tissues are sliced and sampled, the biological tissues with proper sizes are cut from organisms through dissection, surgery and the like and then are put into a fixing solution, the biological tissues put into the fixing solution are fixed by a physical method or a chemical method, then rinsing, dehydrating and dyeing are carried out after the fixation, then water-soluble resin is added for embedding and polymerization to form a solid sample, and finally the solid sample is sliced to obtain the sample after the slicing and sampling. The sliced sample was observed in an electron microscope. However, due to the resolution requirements for electron microscopy imaging and the imaging speed requirements, when a high velocity, high density electron beam stream is applied to such sliced samples, damage such as deformation, decomposition and destruction of fine structures can occur. Especially in an electron beam focusing area, a sample is subjected to cellular damage due to the action of large-dose electrons in a small-area sample surface, reconstruction information is influenced due to the fact that the sample is damaged, and subsequent reconstruction of an image cannot be carried out. Resulting in failure to correctly analyze the biological tissue structure and reduced accuracy of observation.
The present invention has been made in view of this situation.
Disclosure of Invention
The technical problem to be solved by the invention is 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 technical scheme that:
a method for viewing biological tissue, comprising:
slicing and sampling biological tissues to obtain a sample;
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 by using the first electron source system to obtain the sample to be measured containing the polymer component includes:
the dose rate of the irradiation is 1.12X 1013e-/mm2·s-6.25×1015e-/mm2S, heat radiation of 0.025W/mm2-3W/mm2。
In some alternative embodiments, said irradiating said sample with a first electron source system to obtain a sample to be tested containing 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 surface of the sample is 5mm-150 mm;
the potential difference between the first electron source system and the sample is 0.5kv-12 kv.
In some alternative embodiments, said irradiating said sample with a first electron source system to obtain a sample to be tested containing 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 was 64 mm;
the potential difference between the first electron source system and the sample was 2 kv.
In some alternative embodiments, said irradiating said sample with a first electron source system to obtain a sample to be tested containing a polymer component comprises:
the irradiation area of the first electron source system to the sample is 1mm2-100mm2；
The irradiation time of the first electron source system to the sample is 1-60 s.
In some optional embodiments, the potential difference between the first electron source system and the sample of 2kv volts comprises:
the voltage of the first electron source system is 0kv, and the voltage of the sample is 2 kv;
or the voltage of the first electron source system is-2 kv, and the voltage of the sample is 0 kv.
The present invention also provides an electron microscope for observing biological tissues, comprising:
the lower end of the first electron source system is connected with a first vacuum chamber, and the first electron source system irradiates a sample placed in the first vacuum chamber;
the lower end of the electron optical lens barrel is connected with a second vacuum chamber, and an electron beam emitted by the electron optical lens barrel acts on a sample to be measured placed in the second vacuum chamber.
Further, first electron source system includes filament and interval parallel arrangement's first winding subassembly and second winding subassembly, first winding subassembly with the second winding subassembly all includes at least one winding unit, wherein winding unit in the first winding subassembly with winding unit staggered arrangement in the second winding subassembly, the filament certainly first winding subassembly or the winding unit of second winding subassembly one end gets into and around winding on second winding subassembly or staggered winding unit in the first winding subassembly, the filament circulates in proper order around establishing until first winding subassembly or the winding unit of the other end of second winding subassembly stretches out.
In some alternative embodiments, the first electron source system comprises a mount and a filament, the filament being helically coiled;
the filament is at least one, and a plurality of filament intervals set up on the mount pad.
In some alternative embodiments, the first electron source system comprises a spring wire filament, the filament being arranged in a wire segment, or the filament being arranged in a ring with an opening.
After the technical scheme is adopted, 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 sample after being sliced and sampled is irradiated by the first electron source system, 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 sample crosslinking polymerization reaction is initiated, and the sample to be detected containing polymer components is formed through modification. The irradiated sample to be detected containing the polymer components can resist high temperature, bear high speed and high density electron beam action without being damaged, the electron microscope can adopt high speed and high density electron beam action on the sample to be detected, the imaging resolution ratio of the electron microscope is high, the imaging speed is high, the imaging is clear, and the observation accuracy is high.
The following describes 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, 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 without limiting the invention to the right. It is obvious that the drawings in the following description are only some embodiments, and that for a person skilled in the art, other drawings can be derived from them without inventive effort. In the drawings:
FIG. 1 is a flow chart of a method for viewing biological tissue according to the present invention;
FIG. 2 is a schematic structural diagram of an electron microscope for observing biological tissues according to the present invention;
FIG. 3 is a schematic structural diagram of one embodiment of a mounting base and a filament provided by the present invention;
FIG. 4 is a schematic structural diagram of another embodiment of the mounting base and the filament provided by the present invention;
FIG. 5 is a schematic structural diagram of another embodiment of a mounting base and a filament provided in the present invention;
fig. 6 is a schematic structural diagram of another embodiment of the mounting base and the filament provided by the invention.
In the figure: 1. an electron optical lens barrel; 101. a second electron source system; 102. an electron accelerating structure; 103. a deflection device; 104. an objective lens; 2. a first electron source system; 201. a wiring terminal; 202. a mounting seat; 203. a filament; 3. a sample introduction door body; 4. a first vacuum chamber; 5. a telescopic bracket; 6. a tray; 7. a sample; 8. isolating the door body; 9. a second vacuum chamber; 10. a sample stage; 11. and (5) testing the sample to be tested.
It should be noted that the drawings and the description are not intended to limit the scope of the inventive concept in any way, but to illustrate it by a person skilled in the art with reference to specific embodiments.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and the following embodiments are used for illustrating the present invention and are not intended to limit the scope of the present invention.
In the description of the present invention, it should be noted that the terms "upper", "lower", "front", "rear", "left", "right", "vertical", "inner", "outer", etc., indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, and are only for convenience of description and simplicity of description, but do not indicate or imply that the device or element being referred to must have a particular orientation, be constructed and operated in a particular 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 otherwise explicitly specified or limited, the terms "mounted," "connected," and "connected" are to be construed broadly, e.g., as meaning either a fixed connection, a removable connection, or an integral connection; can be mechanically or electrically connected; may be directly connected or indirectly connected through an intermediate. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.
As shown in fig. 1 to 6, the present invention provides a method for observing biological tissues, the method comprising the steps of:
s110, slicing and sampling biological tissues to obtain a sample 7;
s120, irradiating the sample 7 by using a first electron source system 2 to obtain a sample 11 to be measured containing polymer components;
and S130, observing the sample 11 to be detected by adopting an electron microscope.
Specifically, the method comprises the following steps:
s110, slicing and sampling biological tissues to obtain a sample 7;
in detail, biological tissues with proper sizes are cut from organisms through dissection, surgery and the like and then put into a fixing solution, and a chemical fixing method can be adopted for fixing, for example, a fixing solution can be selected to fix cell structures by using a mixed buffer fixing solution of 2.5% glutaraldehyde and 4% paraformaldehyde. Physical fixation methods, such as freezing, microwave irradiation, critical point drying, etc., may also be used to preserve the cellular structure. And after fixation, rinsing, dehydrating and dyeing, adding water-soluble resin, embedding and polymerizing to obtain a solid sample, and slicing the solid sample by using a diamond knife automatic strip slicer to obtain a sample 7 after slicing and sample preparation. The preparation of biological tissue slices is a conventional process in the art and is not described herein.
S120, irradiating the sample 7 by using a first electron source system 2 to obtain a sample 11 to be measured containing polymer components;
in detail, the dose rate of irradiation is 1.12X 1013e-/mm2·s-6.25×1015e-/mm2S, heat radiation of 0.025W/mm2-3W/mm2。
The first electron source system 2 comprises a filament 203 and a connection terminal 201, a power supply is connected with the connection terminal 201, the connection terminal 201 is connected with the filament 203, and the power supply supplies power to the filament 203 through the connection terminal 201. Filament 203 throughAfter current is input, electron beams and heat radiation are emitted. When the first electron source system 2 is electrified, the electron beam and the heat emitted by the first electron source system 2 act on the sample 7, and the irradiation dose rate of the electron beam on the sample 7 is 1.12 multiplied by 1013e-/mm2·s-6.25×1015e-/mm2S, sample 7 received a heat radiation of 0.025W/mm2-3W/mm2The sample 7 will undergo physical and chemical reactions to initiate the cross-linking polymerization of the sample 7, and the sample 11 to be tested containing the polymer component is formed by modification. The irradiated sample 11 to be measured containing the polymer components can resist high temperature and bear the action of high-speed and high-density electron beams without being damaged.
Preferably, the current passing through the first electron source system 2 is controlled, the first electron source system 2 irradiates the sample 7, and the irradiation dose rate is 1.875 × 1013e-/mm2S, heat radiation is 0.21W/mm2. To obtain a sample 11 to be tested containing a polymer component.
In some alternative embodiments, irradiating the sample 7 with the first electron source system 2 to obtain the sample 11 to be measured containing the polymer component comprises:
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-150 mm;
the potential difference between the first electron source system 2 and the sample 7 was 0.5kv-12 kv.
When the first electron source system 2 is electrified, the electron beam and the heat radiation emitted by the first electron source system 2 act on the sample 7, the current electrified to 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-12 kv. When the sample 7 is irradiated within the parameter range, the sample 7 can generate physical reaction and chemical reaction to initiate the cross-linking polymerization reaction of the sample 7, and the sample 11 to be detected containing polymer components is formed through modification. The irradiated sample 11 to be measured containing the polymer components can resist high temperature and bear the action of high-speed and high-density electron beams without being damaged.
It should be noted that, a specific value of the current passed through the first electron source system 2, a specific value of the distance between the first electron source system 2 and the surface of the sample 7, and a specific value of the potential difference between the first electron source system 2 and the sample 7, which can be optionally selected by those skilled in the art within the value range of the above embodiment, can all initiate the cross-linking polymerization reaction of the sample 7, and modify to form the sample 11 to be tested, which contains the polymer component.
Preferably, the irradiation of the sample 7 with the first electron source system 2 to obtain the sample 11 to be measured containing the polymer component comprises:
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 was 64 mm;
the potential difference between the first electron source system 2 and the sample 7 was 2 kv.
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 2 kv. By adopting the specific parameters to irradiate the sample 7, the sample 7 can be subjected to physical reaction and chemical reaction more completely, the cross-linking polymerization reaction of the sample 7 is initiated, and the sample 11 to be detected containing polymer components is formed through modification.
Further, irradiating the sample 7 with the first electron source system 2 to obtain the sample 11 to be measured containing the polymer component comprises:
the area of the sample 7 irradiated by the first electron source system 2 is 1mm2-100mm2；
The time for irradiating the sample 7 by the first electron source system 2 is 1s-60 s.
Specifically, the method comprises the following steps:
when the first electron source system 2 is energized, the electron beam and the heat radiation emitted by the first electron source system 2 act on the sample 7, and the first electron source system 2 aligns the sampleThe area of the product 7 irradiated is 1mm2-100mm2The irradiation time of the sample 7 by the first electron source system 2 is 1s-60 s.
Optionally, the first electron source system 2 performs scanning irradiation on the sample 7, and in the scanning process, the irradiation area of the electron beam spot is 1mm2-100mm2The irradiation time is 1s-60s, after one area is irradiated, the electron beam spot is moved to irradiate the next area, and the irradiation of the large-area sample 7 is sequentially completed.
It should be noted that, a specific value of the area irradiated by the first electron source system 2 on the sample 7 and a specific value of the time irradiated by the first electron source system 2 and the sample 7 may be selected by those skilled in the art within the range of the above-mentioned embodiments, and both may initiate the cross-linking polymerization reaction of the sample 7, so as to modify and form the sample 11 to be tested, which contains the polymer component.
Preferably, the irradiation of the sample 7 with the first electron source system 2 to obtain the sample 11 to be measured containing the polymer component comprises:
the area irradiated by the first electron source system 2 to the sample 7 was 75mm2；
The irradiation time of the sample 7 by the first electron source system 2 is 10 s;
by adopting the specific parameters to irradiate the sample 7, the sample 7 can be subjected to physical reaction and chemical reaction more completely, the cross-linking polymerization reaction of the sample 7 is initiated, and the sample 11 to be detected containing polymer components is formed through modification.
Further, the potential difference between the first electron source system 2 and the sample 7 of 2kv volts comprises:
the voltage of the first electron source system 2 was 0kv, and the voltage of the sample 7 was 2 kv;
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 2 kv. 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 is accelerated by the accelerating electric field and then acts on the sample 7.
Alternatively, the voltage of the first electron source system 2 is-2 kv, and the voltage of the sample 7 is 0 kv;
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 2 kv. 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 is accelerated by the accelerating electric field and then acts on the sample 7.
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, the cross-linking polymerization reaction of the sample 7 is initiated, and the sample 11 to be measured containing polymer components is formed through modification.
The sample 11 to be measured is observed with an electron microscope. The irradiated sample 11 to be measured contains polymer components, so that the sample can bear the action of high-speed and high-density electron beams without being damaged, the electron microscope can select larger electron beam flow to act on the sample 11 to be measured, the electron microscope acts on the sample 11 to be measured by adopting the electron beam with large beam flow, 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 tissues, which includes a first electron source system 2, a first vacuum chamber 4, an electron optical barrel 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 tube 1 is connected with a second vacuum chamber 9, and the electron beam emitted by the electron optical tube 1 acts on a sample 11 to be measured placed in the second vacuum chamber 9.
Specifically, the lower end of the first electron source system 2 is connected with a first vacuum chamber 4, the first vacuum chamber 4 comprises an openable and closable sample introduction door body 3, a telescopic bracket 5 is connected to the sample introduction door body 3 in a sliding manner, and the telescopic bracket 5 can slide up and down on the sample introduction door body 3 to adjust the height. A tray 6 is arranged on the telescopic bracket 5, and a 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, 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, and other common filament 203 materials, and one of the materials may be selected by those skilled in the art as the filament 203. Filament 203 is installed on 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 thermal radiation when energized. When current is applied to the first electron source system 2, electron beams and heat emitted by the first electron source system 2 act on a sample 7 placed in the first vacuum chamber 4, and the sample 7 is irradiated to generate physical reaction and chemical reaction, so that the cross-linking polymerization reaction of the sample 7 is initiated, and the sample 11 to be measured containing polymer components is formed through modification.
The lower end of the electron optical tube 1 is connected with a second vacuum chamber 9, and the electron beam emitted by the electron optical tube 1 acts 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 can perform five-degree-of-freedom motion, and the five-degree-of-freedom motion comprises: three-dimensional translation (X, Y and translation in the three Z directions), 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 by the first electron source system 2 in the first vacuum chamber 4 to obtain a sample 11 to be measured. Open and keep apart door body 8, first vacuum chamber 4 and second vacuum chamber 9 UNICOM this moment, telescopic bracket 5 makes and stretches out the motion and drives tray 6 and place the sample 11 that awaits measuring on tray 6, stretches into in the second vacuum chamber 9 by first vacuum chamber 4. When the tray 6 is inserted above the sample stage 10, the extending movement is stopped. The sample stage 10 raises the jack-up tray 6, the telescopic bracket 5 makes a contraction motion, the telescopic bracket 5 retracts into the first vacuum chamber 4, and the isolation door body 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 stage 10, so that the sample 11 to be measured can be observed conveniently. 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 includes a second electron source system 101, an electron acceleration structure 102 and an objective lens system.
In particular, the second electron source system 101 is used to generate an electron beam.
The electron accelerating structure 102 is an anode along the emission direction of the electron beam for forming an electric field to increase the moving speed of the electron beam.
The objective system is used for controlling the beam current size and the electron 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 to change the moving direction of the electron beam before it is incident on the sample 11 to be measured, and can generate a scanning field with any deflection direction.
The electron beam acting on the sample 11 to be measured can generate signal electrons such as secondary electrons, backscattered electrons, auger electrons, and the like. The electron microscope for observing biological tissues further comprises a detector for receiving signal electrons generated by the action of the electron beams on the sample 11 to be detected.
Taking the example of receiving signal electrons as secondary electrons and backscattered electrons, the detector may be a secondary electron detector which receives secondary electrons separately, or the detector may be a backscattered electron detector which receives backscattered electrons separately, or the detector may receive both secondary electrons and backscattered electrons simultaneously. In the present invention, the skilled person can select the detector type to receive the corresponding electronic type according to his actual needs. The detector can be integrated in the electron optical tube 1, or the detector and the electron optical tube 1 can be independent.
As shown in FIGS. 1 to 6, in one embodiment, a biological tissue is sliced and sampled to obtain a sample 7, and the sample 7 is placed on a tray 6. Opening the sample introduction door body 3, placing the tray 6 and the sample 7 placed on the tray 6 on the telescopic bracket 5, closing the sample introduction door body 3, and adjusting the telescopic bracket 5, so that the sample 7 is positioned below the first electron source system 2. The degree of vacuum of the first vacuum chamber 4 is adjusted to be less than 1X 10-4And (5) Torr. Preferably, the degree of vacuum of the first vacuum chamber 4 is 5X 10- 5Torr。
The telescopic length and height of the telescopic carriage 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 applied 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 0 kv. The potential difference between the first electron source system 2 and the sample 7 was 2 kv. The electron beam and the heat 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 75mm2The irradiation time of the sample 7 by the first electron source system 2 was 10 s.
By adopting the specific parameters to irradiate the sample 7, the sample 7 can be subjected to physical reaction and chemical reaction more completely, the cross-linking polymerization reaction of the sample 7 is initiated, and the sample 11 to be detected containing polymer components is formed through modification. The sample 7 is irradiated by the first electron source system 2 in the first vacuum chamber 4 to obtain a sample 11 to be measured. Open and keep apart door body 8, first vacuum chamber 4 communicates with second vacuum chamber 9 this moment, and telescopic bracket 5 makes and stretches out the motion and drives tray 6 and place the sample 11 that awaits measuring on tray 6, stretches into in the second vacuum chamber 9 by first vacuum chamber 4. When the tray 6 is inserted above the sample stage 10, the extending movement is stopped. The sample stage 10 raises the jack-up tray 6, the telescopic bracket 5 makes a contraction motion, the telescopic bracket 5 retracts into the first vacuum chamber 4, and the isolation door body 8 is closed.
The degree of vacuum of the second vacuum chamber 9 is adjusted to be less than 5X 10-5And (5) Torr. Preferably, the firstThe vacuum degree of the two vacuum chambers 9 is 5X 10-6And (5) 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 stage 10, so that the sample 11 to be measured can be observed conveniently. The electron optical tube 1 is used for generating an electron beam and focusing the electron beam on a sample 11 to be measured, and the electron beam acting on the sample 11 to be measured can generate signal electrons such as secondary electrons, backscattered electrons, auger electrons and the like. The detector is used for receiving signal electrons generated by the action of the electron beams on the sample 11 to be measured.
The sample 11 to be measured is observed with an electron microscope. The irradiated sample 11 to be measured contains polymer components, so that the sample can bear the action of high-speed and high-density electron beams without being damaged, the electron microscope can select larger electron beam flow to act on the sample 11 to be measured, the electron microscope acts on the sample 11 to be measured by adopting the electron beam with large beam flow, 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 observing biological tissue. The first electron source system 2 comprises a filament 203, and 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 respectively 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 203 enters from the winding unit at one end of the first winding assembly or the second winding assembly and winds on the staggered winding units in the second winding assembly or the first winding assembly, and the filament 203 sequentially and circularly winds until the winding units at the other end of the first winding assembly or the second winding assembly stretch out. The first winding assembly and the second winding assembly are installed on the installation base 202, and two ends of the filament 203 are connected with a power supply through the connection terminals 201.
By adopting the shape of the filament 203 in this embodiment, the first electron source system 2 can emit electron beams with large area and large beam current, and on the basis of ensuring the irradiation dose rate and the heat radiation, the irradiation area is large, the heat radiation is high, the time is saved, the sample 7 can be subjected to physical reaction and chemical reaction more completely, and the cross-linking polymerization reaction of the sample 7 is initiated, and the sample 11 to be detected containing polymer components is formed by modification.
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 mount 202 and a filament 203, the filament 203 is spirally wound, at least one filament 203 is provided, and a plurality of filaments 203 are arranged on the mount 202 at intervals.
Preferably, 4 filaments 203 spirally wound are arranged, the mounting seat 202 is disc-shaped, the 4 filaments 203 are arranged on the mounting seat 202 with the center of the mounting seat 202 as the center, the circumferences are evenly distributed on the mounting seat 202 at intervals, and each filament 203 is connected with a power supply through a connecting terminal 201.
By adopting the shape and arrangement of the filament 203 in this embodiment, the first electron source system 2 can emit electron beam with large area and large beam current, and can ensure that the irradiation dose rate and the heat radiation are ensured, the irradiation area is large, the heat radiation is high, the time is saved, the sample 7 can be subjected to physical reaction and chemical reaction more completely, the sample 7 can be subjected to cross-linking polymerization reaction, and the sample 11 to be detected containing polymer components is formed by modification.
It should be noted that, with the specific number of the filaments 203, the spacing distance of each filament 203, and the arrangement of each filament 203 in this embodiment, those skilled in the art can select the filament according to the actual needs.
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 includes a spring wire filament 203, and the spring wire filament 203 is disposed in a ring shape having an opening. The filament 203 is connected to a power supply through a terminal 201. The filament 203 is connected with the mounting base 202 through a filament column and is fixedly mounted on the mounting base 202.
By adopting the shape and arrangement of the filament 203 in this embodiment, the first electron source system 2 can emit electron beam with large area and large beam current, and can ensure that the irradiation dose rate and the heat radiation are ensured, the irradiation area is large, the heat radiation is high, the time is saved, the sample 7 can be subjected to physical reaction and chemical reaction more completely, the sample 7 can be subjected to cross-linking polymerization reaction, and the sample 11 to be detected containing polymer components is formed by modification.
In some alternative embodiments, as shown in fig. 6, the present invention provides an electron microscope for observing 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 terminal 201. The filament 203 is connected with the mounting base 202 through a filament column and is fixedly mounted on the mounting base 202.
By adopting the shape and arrangement of the filament 203 in this embodiment, the first electron source system 2 can emit electron beam with large area and large beam current, and can ensure that the irradiation dose rate and the heat radiation are ensured, the irradiation area is large, the heat radiation is high, the time is saved, the sample 7 can be subjected to physical reaction and chemical reaction more completely, the sample 7 can be subjected to cross-linking polymerization reaction, and the sample 11 to be detected containing polymer components is formed by modification.
Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (10)

1. A method for viewing biological tissue, comprising:
slicing and sampling biological tissues to obtain a sample;
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.
2. The method of claim 1, wherein irradiating the sample with a first electron source system to obtain a test sample comprising a polymer component comprises:
the dose rate of the irradiation is 1.12X 1013e-/mm2·s-6.25×1015e-/mm2S, heat radiation of 0.025W/mm2-3W/mm2。
3. The method of claim 1, wherein irradiating the sample with a first electron source system to obtain a test sample 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 surface of the sample is 5mm-150 mm;
the potential difference between the first electron source system and the sample is 0.5kv-12 kv.
4. The method of claim 1, wherein irradiating the sample with a first electron source system to obtain a test sample 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 was 64 mm;
the potential difference between the first electron source system and the sample was 2 kv.
5. The method of claim 1, wherein irradiating the sample with a first electron source system to obtain a test sample comprising a polymer component comprises:
the irradiation area of the first electron source system to the sample is 1mm2-100mm2；
The irradiation time of the first electron source system to the sample is 1-60 s.
6. The method for observing biological tissue of claim 4, wherein the potential difference between the first electron source system and the sample being 2kv volts comprises:
the voltage of the first electron source system is 0kv, and the voltage of the sample is 2 kv;
or the voltage of the first electron source system is-2 kv, and the voltage of the sample is 0 kv.
7. An electron microscope for observing biological tissue, comprising:
the lower end of the first electron source system is connected with a first vacuum chamber, and the first electron source system irradiates a sample placed in the first vacuum chamber;
the lower end of the electron optical lens barrel is connected with a second vacuum chamber, and an electron beam emitted by the electron optical lens barrel acts on a sample to be measured placed in the second vacuum chamber.
8. The electron microscope of claim 7, wherein the first electron source system comprises a filament and a first winding assembly and a second winding assembly arranged in parallel at intervals, the first winding assembly and the second winding assembly each comprise at least one winding unit, wherein the winding units in the first winding assembly are arranged alternately with the winding units in the second winding assembly, the filament enters from the winding unit at one end of the first winding assembly or the second winding assembly and winds around the second winding assembly or the winding units alternately arranged in the first winding assembly, and the filament sequentially and circularly winds around the winding units arranged at the other end of the first winding assembly or the second winding assembly.
9. The electron microscope for observing biological tissue of claim 7, wherein the first electron source system comprises a mount and a filament, the filament being helically coiled;
the filament is at least one, and a plurality of filament intervals set up on the mount pad.
10. The electron microscope for observing biological tissues according to claim 7, 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 shape having an opening.
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