CN1138977C - Equipment and method for directly observing water-contained biologic sample in ambient scanning electronic microscope - Google Patents
Equipment and method for directly observing water-contained biologic sample in ambient scanning electronic microscope Download PDFInfo
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- CN1138977C CN1138977C CNB001327046A CN00132704A CN1138977C CN 1138977 C CN1138977 C CN 1138977C CN B001327046 A CNB001327046 A CN B001327046A CN 00132704 A CN00132704 A CN 00132704A CN 1138977 C CN1138977 C CN 1138977C
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Abstract
The present invention relates to a device and a method for directly observing a water-contained biologic sample in an ambient scanning electron microscope. The device comprises a tridimensional sample moving part, a pre-drawing chamber, a sample feeding part and a sample cooling part, wherein the tridimensional sample moving part is installed in a door cover of a sample chamber, and the pre-drawing chamber and the sample feeding part are installed outside the door cover. When the device is used for observing the water-contained biologic sample, the sample can be quickly put in a sample stage via the pre-drawing chamber under the condition that the door cover of the sample chamber is not opened; adjustment can be carried out in the X direction, the Y direction and the Z direction so that the sample quickly comes to the observed position of the electron microscope, and a natural state image of the biologic sample without losing water can be obtained in about 1 minute.
Description
The present invention relates to the biological sample table apparatus that a kind of environmental scanning electron microscope is used, particularly relate to a kind of biological sample table apparatus and method thereof that is used for the environmental scanning electron microscope of Direct observation hydrated biological material.
Scanning electron microscope (hereinafter to be referred as scanning electron microscope or Electronic Speculum) is modern large-scale precision instrument, and as the strong instrument of research microworld, it has a wide range of applications in fields such as physics, chemistry, material science and life sciences.Environmental scanning electron microscope (Enviromental Scanning Electron Microscope-ESEM, hereinafter to be referred as environmental scanning electronic microscope) be a kind of novel scanning electron microscope, it is better than conventional sweep Electronic Speculum (Scanning Electron Microscope-SEM) part and is: can be under the ventilation environment Direct observation without the nonconductor and the aqueous specimen of surface treatment (metal spraying or freezing sample preparation etc.).
If with conventional scanning electron microscope (sample vacuum chamber degree 10
-2～10
-3Holder, 1 holder=1mmHg=1/760 atmospheric pressure) the nonconductor sample is observed, because the effect of incident electron, electronics can constantly be assembled on the nonconductor sample surfaces, causes charge and discharge phenomena, can not clearly observe surface image.So, must carry out metalized (metal spraying etc.) to idioelectric surface before observing.At present, in the scanning electron microscope of routine, observe moisture biological sample two kinds of methods are arranged: put into scanning electron microscopic observation behind (1) sample drying and the metal spraying, for example: document [1] (" scanning electron microscope analysis technology " Du Zili, Xu Ziang, Chemical Industry Press, 1984): can be in order to make moisture biological sample at metal spraying under the high vacuum, and in the high vacuum sample chamber, observe, the necessary skeleton of using glutaraldehyde and osmic acid fixed cell earlier is again with ability metal spraying behind dehydration of ethanol gradient and the carbon dioxide critical point drying.Such preparation method not only time-consuming (generally prepare a sample and need 2-3 days), expensive, and can make sample lose original natural appearance (we will provide its difference with the contrast photo in the decline of this instructions).(2) use freezing sample stage to put into scanning electron microscopic observation after the sample cryogenic refrigeration.Make low-temperature receiver for the freezing sample table device use of the commodity of conventional sweep Electronic Speculum configuration liquid nitrogen, sample temperature is dropped to about-150 ℃.Because sample must prepare in cryogenic refrigeration, the liquid in the biological sample partly is frozen into solid, and sample will lose original state.And this sample table device complex structure, for the sample that makes deep cooling is unlikely to frosting when the sample introduction, opened a sample introduction window in addition in the electron microscopic sample chamber, increased the freezing sampling device of a cover again, therefore cost an arm and a leg complicated operation.
When adopting environmental scanning electronic microscope to observe, basic variation takes place in situation.The sample vacuum chamber degree is 0.1～20 holder, the environmental gas that feeds produces a large amount of electronics and positive ion under the collision of incident electron and signal electron, these positive ions can act on rapidly with the electronics of sample surfaces accumulation, eliminated charge and discharge phenomena, Direct observation becomes possibility without the surface metalation processing to make nonconducting sample (comprising biological sample).
In environmental scanning electronic microscope, though the vacuum tightness of sample chamber can be regulated.Yet the vacuum tightness of sample chamber affects the resolution of Electronic Speculum significantly, and vacuum tightness is low more, and resolution is low more, and image is fuzzy more.In environmental scanning electronic microscope used, the vacuum tightness of sample chamber generally all can not too low (being no more than 5 holders).We can know from the physical data of water: the water saturation vapour pressure is 4.6 holders in the time of 0 ℃; The water saturation vapour pressure is 17.5 holders during 20 ℃ of room temperatures.Therefore, when observing moisture biological sample down, water still is in gaseous state with being not less than water saturation vapour pressure (17.5 holder) in room temperature, and moisture biological sample still exists water loss problem.
When in environmental scanning electronic microscope, using common sample platform sample introduction, must open the gate, sample chamber, put sample well, close the sample chamber, open the vacuum system of Electronic Speculum again, after the sample chamber reaches certain condition of high vacuum degree, could feed gas, make the sample chamber reach predetermined low vacuum, open electron gun at last, observation sample, this process approximately needs more than ten minute, and moisture biological sample is dehydration soon in such process.
The method of the solution water loss problem that we taked is: suitably reduce temperature, reduce vacuum tightness and shorten the observing time of sample in the sample chamber of low vacuum, to reduce the fluid loss of moisture biological sample.
The objective of the invention is to: the problem that solves the slow and aqueous specimen dehydration that room temperature observation post causes of existing environmental scanning electronic microscope sample introduction speed in the general low vacuum of sample chamber; A kind of low price, simple in structure is provided, and mechanical part cooperates compact, and is easy to operate, be exclusively used in the biological sample table apparatus of the environmental scanning electronic microscope of Direct observation hydrated biological material flexibly and accurately; And use this device to shoot at short notice not by the method for the state of nature image of the biological sample of dehydration.
The object of the present invention is achieved like this: on the gate of scanning electron microscope example chamber, not only having can be at three directions of XYZ parts of mobile example (just as the parts on the electron microscopic sample platform of routine) promptly; But also the pre-vacuum chamber that has the sample introduction parts is housed; Also has refrigerating function simultaneously.Thereby can be under the situation of not opening gate, electron microscopic sample chamber, make sample promptly enter in the Electron Microscopy Room (can be cooled simultaneously or enter Electron Microscopy Room then be cooled) by pre-vacuum chamber, just make biological sample without high vacuum stage of Fig, directly enter the ambient condition of low vacuum, thereby avoided the quick dehydration of sample in high vacuum, reach the shortening time and reduce the requirement of temperature, realize at last shooting at short notice not by the purpose of the state of nature image of the biological sample of dehydration.
A kind of biological sample table apparatus that is used for the environmental scanning electronic microscope of Direct observation hydrated biological material provided by the invention comprises: (1) pre-vacuum chamber: pre-vacuum chamber is positioned at the door closure top of sample chamber, and a gland bonnet is arranged on the pre-vacuum chamber collar extension; Pre-vacuum chamber is inboard to be communicated with by a wicket with the sample chamber, and (the sample chamber door closure outside) is equipped with a gate valve on the window, and gate valve is the flashboard valve that separates sample chamber and pre-vacuum chamber; One vacuum passage is arranged at the pre-vacuum chamber top, communicates with the sample chamber, and a motorized valve is housed on the passage, control opening and closing of vacuum passage, when motorized valve was opened, what the air pressure in the pre-vacuum chamber just can be with the sample chamber was identical, complete to guarantee to exhaust in advance in the short time (a few second); An air release is equipped with in the pre-vacuum chamber side, when pre-vacuum chamber air pressure is vacuum, opens air release, can make the Electronic Speculum air outside flow into pre-vacuum chamber, and when the pre-vacuum chamber internal gas pressure was identical with on every side atmospheric pressure, the pre-vacuum chamber door closure of sealing just can be opened.(2) sample introduction parts: the sample introduction parts mainly comprise a sample transfer bar, and it is positioned at the O-ring seal at pre-vacuum chamber gland bonnet center, and it can freely be pumped and be air tight; The sample transfer bar head is threaded; Observed sample can be fixed on the top cover of difform sample cup with several different methods, and a little bar is arranged at the sample cup bottom, and little bar is inserted in the aperture at sample cup top, and fixes with Screw; Sample cup side has threaded hole, and it can be screwed into the screw thread that the sample transfer bar head has.When pre-vacuum chamber exhausts completely in advance, when gate valve was opened, the sample cup base that has sample and sample cup can pass the wicket of pre-vacuum chamber, was sent into the sample cup locating slot that is arranged on the sample stage by sample transfer bar.Like this, under the situation of not opening gate, electron microscopic sample chamber, by the sample introduction parts, sample has promptly been sent in the sample chamber of Electronic Speculum.(3) cooling-part: it can have various ways, and for example with the cooling sample cup of ice cube, semiconductor cooling-part etc., it can make sample be cooled to 0-4 ℃.(4) sample moving-member: on the door closure of sample chamber, have three and revolve handle, each revolves one towards the mobile slide block of single direction (directions X or Y direction or Z direction) by one group of gear and screw drive respectively, slide block drives the sample stage main body again and moves, sample is seated in the sample cup locating slot of sample stage main body central authorities, thereby three revolve can be at X, Y, three directions of Z mobile example promptly, so that sample can promptly enter observable position.
The sample table device that uses in environmental scanning electronic microscope provided by the invention carries out the Direct observation method of hydrated biological material, comprises that sample following steps such as sends into and observe:
1. earlier the sample chamber door closure is closed, open the Electronic Speculum vacuum system again, after the sample chamber reaches certain condition of high vacuum degree, feed gas, make the sample chamber reach predetermined low vacuum;
2. the pre-vacuum chamber door closure is covered on pre-vacuum chamber, open the motorized valve on the pre-vacuum chamber vacuum passage, pre-vacuum chamber just is communicated with the sample chamber of low vacuum, and pre-vacuum chamber reaches the low vacuum of sample chamber after several seconds;
3. open the lampet in gate valve and the sample chamber, the sample transfer bar that has sample, sample cup and sample cup is passed the window of sample chamber door closure, the sample cup is sent in the sample cup locating slot of sample stage, turn on sample transfer bar;
4. extraction sample transfer bar, closing gate valve, lampet extinguishes simultaneously;
5. open the parts such as electron gun, scanning yoke, stigmator of all observation samples of Electronic Speculum, prepare observation sample;
6. regulate the handle that revolves of X, Y, three directions of Z, but make sample can promptly enter the observation place, take down not by the state of nature image of the biological sample of dehydration;
7. in the time will taking out sample, can carry out the inverse operation of above program: open gate valve, stretch into sample transfer bar, the screw thread at sample transfer bar top is screwed into the sample cup, extracts the sample transfer bar of carry sample cup and sample cup out, closing gate valve, open the pre-vacuum chamber air release, when the air pressure of pre-vacuum chamber is identical with on every side atmospheric pressure, just can open the pre-vacuum chamber gland bonnet, take out sample.
Biological sample platform of the present invention utilizes the low vacuum sample chamber of pre-vacuum chamber JA(junction ambient) scanning electron microscope, add simple cooling sample cup and accurate physical construction, satisfy the requirement of vacuum tightness, temperature and observing time simultaneously, reached the purpose of Direct observation aqueous specimen.For moisture sample, under enough air pressure, evaporation of water speed reduces, and moist sample can not dry out and be out of shape soon, can be without freezing sample preparation its original natural pattern of Direct observation.This biological sample platform is because simple in structure, and price is half of general freezing sample stage.
This biological sample table apparatus sample presentation time, positioning time was less than 1 minute less than 20 seconds; The cooling sample cup can be put into ice cube, and the sample minimum temperature is 4～10 ℃.This biological sample platform can be used for fixing and cooling sample in the environmental scanning electronic microscope, for the pattern and the structure of Direct observation aqueous specimen under aeration status (animal and plant cells, axis undercut sheet, wet NaCl crystal etc.), also can be for observing and dehydration (animal, plant etc.) or dry (wet cement etc.) process of research aqueous specimen.
The invention has the advantages that: the biological sample table apparatus of scanning electron microscope is simple in structure, and mechanical part cooperates compact, and is easy to operate, flexible and accurate; After particularly having added the pre-vacuum chamber that communicates with the environmental sample chamber, can in the short time (about 1 minute), shoot not by the state of nature image of the biological sample of dehydration.
The present invention is described in detail below in conjunction with drawings and Examples.
Fig. 1 is the biological sample table apparatus structural representation of scanning electron microscope of the present invention.
Fig. 2 is nephrocyte (Vero cell) pattern of the cercopithecus aethiops of viewed process specimen preparation (drying and metal spraying) in the scanning electron microscope of routine.
Fig. 3 is to use the pattern of biological sample table apparatus of the present invention viewed Vero cell in environmental scanning electronic microscope.
The drawing of Fig. 1 is described as follows:
Pre-vacuum chamber-[1] sample stage main body-[2] sample cup-[3]
Sample cup-[4] pre-vacuum chamber door closure-[5] sample transfer bar-[6]
Gate valve-[7] vacuum passage-[8] pre-vacuum chamber motorized valve-[9] (not shown)
Pre-vacuum chamber air release-[10] sample pedestal-door closure-[12], [11] sample chamber
XYZ Directional rotating handle-[13], [14], [15] X, Y, Z direction slide block-[16], [17], [18]
Sample cup locating slot-[19] window-[20]
Embodiment 1
By the biological sample table apparatus that the structure fabrication one scan Electronic Speculum of Fig. 1 is used, below be described in detail to use the structure of cooling sample cup as the biological sample table apparatus of the scanning electron microscope of example:
Pre-vacuum chamber 1 is positioned at the top, the outside of the sample chamber door closure 12 of common scanning electron microscope, pre-vacuum chamber gland bonnet 5 has sample transfer bar 6, (promptly when pre-vacuum chamber is in vacuum state, all air tight when pumping before and after the sample transfer bar) can be pumped and seal to sample transfer bar before and after the gland bonnet center; Screw thread is arranged at the sample transfer bar top, above can being screwed in sample cup 4.Gate valve 7 is the flashboard valves that separate sample chamber and pre-vacuum chamber, is positioned at when closing on the window 20 of sample chamber door closure 12.The vacuum passage 8 of pre-vacuum chamber links to each other with the sample chamber, and the motorized valve 9 that control is opened and closed is housed on the passage.Cooling sample cup 3 usefulness copper or aluminium are made, and cylinder type has bowl cover, the interior ice cube of putting; , there is a little bar at the cup end, is inserted into the aperture of sample cup 4, is screwed.Sample can be fixed on the cooling sample cup bowl cover with double sticky tape, obtains cooling simultaneously.Sample stage main body 2 is contained on the sample chamber door closure 12, and the sample stage main body has sample pedestal 11, behind the track that the bottom of sample pedestal 11 is inserted in the sample chamber, just can push the sample chamber to the whole sample platform, and shut sample chamber door closure 12.Except that pre-vacuum chamber 1 is arranged, also have three to revolve handle on the door closure of sample chamber: directions X revolves 13, Y Directional rotating handle 14, Z Directional rotating handle 15, and they move three slide blocks-directions X slide block 16, Y direction slide block 17, Z direction slide block 18 by gear and screw rod.Slide block drives sample stage main body 2 again and moves, and sample is seated in the sample cup locating slot 19 of sample stage main body 2 central authorities, regulates, but promptly enters the observation place of Electronic Speculum in X, Y, three directions of Z thereby sample can be used to three groups of parts.
In the biological sample table apparatus of the dress cooling sample cup that embodiment 1 is manufactured, the concrete grammar and the step of carrying out moisture Vero cell observation and shooting are as follows:
1. earlier sample chamber door closure 12 is closed, open the Electronic Speculum vacuum system again, after the sample chamber reaches certain condition of high vacuum degree, feed gas, make the sample chamber reach predetermined low vacuum;
2. pre-vacuum chamber gland bonnet 5 is covered on pre-vacuum chamber 1, open the motorized valve 9 on the pre-vacuum chamber vacuum passage 8, pre-vacuum chamber just is communicated with the sample chamber of low vacuum, and pre-vacuum chamber reaches the low vacuum of sample chamber after several seconds;
3. open gate valve 7, at this moment, the lampet in the sample chamber is opened, and by the glass window of pre-vacuum chamber door closure 12, we can be clear that the sample chamber inner case.The sample transfer bar 6 that has sample, sample cup 3 and sample cup 4 is passed the window 20 of sample chamber door closure 12, and can immediately sample cup 4 be sent in the sample cup locating slot 19 on the indoor sample stage main body 2 of electron microscopic sample, turn on sample transfer bar 6, like this, sample cup 3 that has sample and sample cup 4 have been stayed in the sample cup locating slot 19 in the sample chamber;
4. extract sample transfer bar 6 out, closing gate valve 7, lampet extinguishes simultaneously.Like this, do not opening under sample chamber door closure 12 situations, sample has promptly been put into the sample cup locating slot 19 of sample stage via pre-vacuum chamber 1;
5. open each parts (electron gun, scanning yoke, stigmator etc.) of all observation samples of Electronic Speculum, prepare observation sample;
6. regulate revolving of X, Y, three directions of Z, but make sample can promptly enter inspecting position, take down not by the state of nature image of the biological sample of dehydration 13,14,15;
7. in the time will taking out sample, can carry out the inverse operation of above program: open gate valve 7, stretch into sample transfer bar 6, the screw thread at sample transfer bar top is screwed into sample cup 4, extracts the sample transfer bar 6 of carry sample cup 4 and sample cup 3 out, closing gate valve 7, open pre-vacuum chamber air release 10, when the air pressure of pre-vacuum chamber is identical with on every side atmospheric pressure, just can open pre-vacuum chamber door closure 5, take out sample.
Fig. 2 is the pattern image through the Vero cell after the conventional biological sample preparation (twice fixing, dehydration, drying and metal spraying).Fig. 3 is the image without the Vero cell of specimen preparation, and it is to use biological sample table apparatus of the present invention and method, the pattern of viewed Vero cell in environmental scanning electronic microscope.At this moment, sample does not need preparation: after the microcarrier of band cell can take out from nutrient solution, blot with filter paper, just directly be fixed on the sample cup of this biological sample platform, and can send into electron microscopic observation immediately.Comparison diagram 3 and Fig. 2, we can be clear that the not natural pattern of dehydration cell from Fig. 3, their cell skin is connected to each other, and attaches growth on microcarrier.Lost biological water through pretreated Vero cell (Fig. 2), each cell such as bead, they are deposited on the microcarrier, form the aggregate of bead one by one, and their pattern is very different with the natural pattern (Fig. 3) of dehydration cell not.So environmental scanning electronic microscope and biological sample table apparatus thereof are the essential tool of research hydrated biological material microworld in the research of life science.
Claims (3)
1. the device of a Direct observation hydrated biological material in environmental scanning electronic microscope, comprise: sample cup, sample moving-member, it has three to revolve [13] [14] [15] on the door closure of sample chamber, each revolves the slide block [16] [17] [18] that moves by one group of gear and single direction in X, Y, Z three directions of screw drive respectively, and slide block and sample stage main body [2] are combined; It is characterized in that: also comprise a pre-vacuum chamber [1], sample introduction parts and cooling-part, wherein said pre-vacuum chamber [1] is positioned on the door closure [12] of sample chamber, on pre-vacuum chamber [1] collar extension gland bonnet [5] is arranged; Pre-vacuum chamber [1] is inboard to be communicated with by a wicket [20] with the sample chamber, and window [20] inwall is equipped with a gate valve [7] that is used for separating sample chamber and pre-vacuum chamber [1]; One vacuum passage [8] is arranged at pre-vacuum chamber [1] top, communicates with the sample chamber, and the motorized valve of opening and cutting out [9] of a control vacuum passage [8] is housed on the vacuum passage [8], and an air release [10] is equipped with in pre-vacuum chamber [1] side; Described sample introduction parts comprise a sample transfer bar [6], and sample transfer bar [6] head has the screw thread that is used for fixing sample cup [11], and sample transfer bar [6] is inserted in the O-ring seal at gland bonnet [5] center of pre-vacuum chamber [1]; A little bar is arranged at the bottom of sample cup [3], and little bar is inserted in the aperture at sample cup [11] top, and fixes with Screw; Described cooling-part comprises the cooling sample cup or is fixed on the supercentral semiconductor cooling-part of sample stage main body [2]; Described sample transfer bar [6] passes the wicket [20] on the sample chamber door closure [12], when gate valve [7] when opening, between pre-vacuum chamber [1] and sample chamber, freely pump, can not open under sample chamber door closure [12] situation, sample promptly put into the sample cup locating slot [19] at sample stage center via pre-vacuum chamber.
2, by claim 1 described a kind of in environmental scanning electronic microscope the device of Direct observation hydrated biological material, it is characterized in that, place ice cube within the described cooling sample cup [3], be stamped bowl cover on the cooling sample cup [3].
3, a kind of application rights require 1 described in environmental scanning electronic microscope the Direct observation method of the device of Direct observation hydrated biological material, it is characterized in that: may further comprise the steps:
(1) make the sample chamber reach predetermined low vacuum earlier;
(2) pre-vacuum chamber gland bonnet [5] is covered on pre-vacuum chamber [1], open the motorized valve [9] on the pre-vacuum chamber vacuum passage [8], pre-vacuum chamber is communicated with the sample chamber of low vacuum, and pre-vacuum chamber reaches the low vacuum of sample chamber after several seconds;
(3) open lampet in gate valve [7] and the sample chamber, the sample transfer bar [6] that has sample, sample cup [3] and sample cup [4] is passed the window of sample chamber door closure [12], sample cup [4] is sent in the sample cup locating slot [19] of sample stage, turn on sample transfer bar; Extract sample transfer bar out, closing gate valve, lampet extinguishes simultaneously;
(4) all rule of opening Electronic Speculum are examined the parts such as electron gun, scanning yoke, stigmator of sample, prepare observation sample;
(5) regulate the handle that revolves of X, Y, three directions of Z, but make sample can promptly enter the observation place, take down not by the state of nature image of the biological sample of dehydration.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB001327046A CN1138977C (en) | 2000-11-13 | 2000-11-13 | Equipment and method for directly observing water-contained biologic sample in ambient scanning electronic microscope |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB001327046A CN1138977C (en) | 2000-11-13 | 2000-11-13 | Equipment and method for directly observing water-contained biologic sample in ambient scanning electronic microscope |
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| CN1353306A CN1353306A (en) | 2002-06-12 |
| CN1138977C true CN1138977C (en) | 2004-02-18 |
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Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1310283C (en) * | 2003-08-18 | 2007-04-11 | 旺宏电子股份有限公司 | Correcting method for scanning tool of semiconductor and standard correcting piece thereof |
| EP2245467B1 (en) * | 2008-02-05 | 2022-04-06 | Pocared Diagnostics Ltd. | System for conducting the identification of bacteria in biological samples |
| CN102323432B (en) * | 2011-05-27 | 2013-10-02 | 公安部第一研究所 | Full-automatic three-dimensional precision positioning motion sample injector |
| CN104374626A (en) * | 2014-12-08 | 2015-02-25 | 刘永英 | Moss scanning electron microscope observation material and preparation method thereof |
| CN105319397B (en) * | 2015-10-16 | 2018-11-16 | 武汉新芯集成电路制造有限公司 | A kind of device and method of sample transfer on nano-probe platform |
| CN106814095B (en) * | 2017-02-12 | 2019-05-24 | 马新军 | Freezing sample preparation device for scanning electron microscope |
| CN107860783B (en) * | 2017-10-23 | 2019-09-24 | 中国科学院化学研究所 | XPS sampling device |
| CN109632450B (en) * | 2018-11-19 | 2020-09-25 | 浙江大学 | Mechanism for cooling and transmitting sample in sealed cavity |
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