CN102692344A - Electrical stimulation and millisecond-class ultralow temperature rapid freezing method and device thereof - Google Patents
Electrical stimulation and millisecond-class ultralow temperature rapid freezing method and device thereof Download PDFInfo
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- CN102692344A CN102692344A CN2012101625699A CN201210162569A CN102692344A CN 102692344 A CN102692344 A CN 102692344A CN 2012101625699 A CN2012101625699 A CN 2012101625699A CN 201210162569 A CN201210162569 A CN 201210162569A CN 102692344 A CN102692344 A CN 102692344A
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Abstract
The invention relates to the technical field of an electron microscope and ultralow temperature rapid freezing. The invention aims to provide a method and special equipment which aim to ensure a biological tissue to be rapidly frozen and fixed at an ultralow temperature at the moment that the biological tissue is simulated and are convenient to research the morphological change of an ultrastructure when physiological functions of the biological tissue are changed in various specific states. The equipment disclosed by the invention comprises a sample stage (3), a vertical sliding plate (5), an ultralow temperature freezing chamber (7), an infrared sensor A (9), an infrared sensor B (10), a timer (11), a programmable stimulator (14), a computer (15) and the like. The electrical stimulation and millisecond-class ultralow temperature rapid freezing method and device provided by the invention solves the problems of ensuring the morphological research to be synchronized with the moment physiological function change of the biological tissue and obtaining an ultrastructure morphological image, the ion concentration and the locus change of the biological tissue in real time; when the physiological functions of the biological tissue are changed, more realer ultrastructure information can be obtained; the physiological functions and the morphological research can be synchronously carried out on the level of the millisecond class; and the method and the special equipment have important significance for developing the subjects such as the electron microscopy, the cell ultrastructure morphology, the physiology, the pathology and the like.
Description
Technical field
The present invention relates to electron microscopy and ultralow temperature snap frozen technical field; This technology is mainly used in basic medical research fields such as biological tissue-cell ultrastructure morphology, function assessment, Neurobiology, physiology, is specifically related to a kind of electro photoluminescence-Millisecond ultralow temperature quick-freezing method and device thereof that cooperates preparing electron microscopy specimen.
Background technology
In biomedical morphological analysis field, need under Electronic Speculum, observe the physiology ultrastructure structural change of biological tissue under extraneous spread effect.This just need make this biological tissue in environmental stimuli, fixed by Millisecond, can be ultralow temperature snap frozen technology at present with the technology of Millisecond fixed biological tissue.Physiological function changes required stimulation and reaction and is Millisecond unit, as: nerve impulse conduction, muscular tissue excitation contraction coupling etc.Therefore, the electro photoluminescence of Millisecond level and cryofixation simultaneous techniques are the instantaneous physiological functions of research when changing, the required condition of structural change.
Mostly the biological sample of Electronic Speculum is fixing at present is that chemical fixation, chemical fixation often make biological tissue, eucaryotic cell structure shrinkage, swelling or self-dissolving, the error that causes morphosis to analyze.Cryofixation is a physical process fully, can improve the physical strength of some soft biological tissue significantly.Be at present near the unique method of preserving mechanics of biological tissue and inner element under the condition of living organism.Though cryofixation can instantaneous (with millisecond meter) freeze ecological state; Comprise tissue morphology structure and cell interior element (particularly soluble ion); Avoid the change in biological tissue, eucaryotic cell structure and inner chemical constitution and site, but present cryofixation technology can't be carried out synchronously with electro photoluminescence biological tissue.
Still do not have any at present about the various states that adopt electro photoluminescence-when ultralow temperature snap frozen technology can keep biological tissue's physiological reaction and the bibliographical information of physiological characteristic.
The morphosis picture when though optical microscope can have been accomplished to obtain biological tissue's changes of function in real time, because the resolution of light microscopic and the restriction of enlargement ratio make the research level of light microscopic be difficult to satisfy the needs of exploring the ultrastructure field.Though and Electronic Speculum can be with the form of Ultrastructural Study on Level biological tissue, the ultrastructure form picture in the time of can not obtaining the instantaneous physiological function of biological tissue and change has limited the development of electron microscopy at biomedical sector.Therefore; How change synchronously with biological tissue instantaneous physiological function; (in real time) biological tissue ultrastructure form picture when obtaining the physiological function variation; The research of physiological function and morphology can be carried out on the level of Millisecond synchronously, will be significant to subject development such as electron microscopy, cell ultrastructure morphology, physiology and pathology.And solved biological tissue when the Millisecond changes of function; Obtain the problem of its ultrastructure form picture and ion concentration, site variation in real time; In the time of obtaining biological tissue's physiological function variation; Truer, more ultrastructure information, thus be that basic medical research, clinical research and diagnosis provide a brand-new technical method.
Summary of the invention
The object of the present invention is to provide a kind of for biological tissue was promptly fixed by the ultralow temperature snap frozen in the moment that is upset, the method and the specialized equipment of the ultrastructure metamorphosis when being convenient to study physiological function under the various particular states of this biological tissue and changing.
The invention provides a kind of electro photoluminescence-Millisecond ultralow temperature snap frozen device, can make the Millisecond of biological tissue after being upset in the time by snap frozen.
The present invention provides a kind of electro photoluminescence-Millisecond ultralow temperature snap frozen device; Comprise: sample stage, vertical slide, superfreeze chamber, infrared sensor A, infrared sensor B, timer, programmable stimulato and computing machine; Wherein vertical slide is installed on the superfreeze chamber, is equipped with on the vertical slide on sample stage, the sample stage stimulating electrode is installed; Vertical slide respectively is equipped with an infrared sensor in two ends up and down; Wherein infrared sensor A is installed on the sample stage upper end; Infrared sensor B is installed on vertical slide lower end and superfreeze appearance refrigerating chamber porch height such as grade, and infrared sensor A, B are connected with timer respectively, and timer is connected with computing machine; The computer control programmable stimulato, programmable stimulato links to each other with stimulating electrode.
On the described vertical slide sample stage is installed; Sample stage can vertically down be slided rapidly under the control of launching button; Sample stage is used to place sample; Particularly biological tissue samples such as myocardium myofilament, skeletal muscle myofilament, nerve, cardiac muscle cell, Skeletal Muscle Cell etc., sample stage selects for use copper material to process.
Described sample stage is slided rapidly downwards along vertical slide, is the button control of launching that is installed on the vertical slide.Sample stage can be launched downwards rapidly under the elastic force effect of launching button, makes the sample on the sample stage get into the superfreeze chamber fast.
The superfreeze chamber has refrigerating chamber inlet topmost, supplies the sample on the sample stage to get into.
Described programmable stimulato can carry out electro photoluminescence to sample through stimulating output lead to stimulating electrode in the process that sample stage glides.
The signal controlling timer of described infrared sensor A and B output is confirmed the time-histories of whole sample platform operation.Principle is following: there has been the anti-dazzling screen of interception at the sample stage rear portion, and anti-dazzling screen did not block sensors A when sample stage was launched downwards, makes its output high level.When beginning to launch, anti-dazzling screen leaves sensors A, because of photoelectric tube receives illumination, exports a low level signal, opens gating circuit, and standard clock signal (0.1mS pulse) gets into timer, starts the clock.When anti-dazzling screen moved to sensor B, anti-dazzling screen made the photoelectric tube of sensor B can not get illumination and export high level, closes gating circuit, cut off clock signal, and stopped timing.
Simultaneously, described infrared sensor A, the signal Synchronization programmable stimulato of output makes it stimulate biological tissue at the appropriate time that sample stage descends.
Described superfreeze chamber is available from the Australian Rechart KF80 of company type.
Described timer be used for the calculation sample platform from infrared sensor A to the needed time of infrared sensor B.
Described programmable stimulato, it is wide, at interval and parameter such as amplitude to set time-delay, square wave number, the ripple of programmable stimulato in advance through keyboard.After the trigger pip of receiving infrared sensor A, starting immediately stimulates it that biosome is stimulated.
Described computing machine also can connect display, is used for the screen mapping, shows experimental result; Described computing machine also can be stored experimental result in order to look into usefulness in the future; Described computing machine also can connect output device and print experimental result.
The present invention also provides a kind of electro photoluminescence-Millisecond ultralow temperature quick-freezing method that uses said apparatus, may further comprise the steps:
A. will need the sample of ultralow temperature snap frozen to be fixed on the sample stage, and stimulating electrode will be installed on the sample stage be connected with programmable stimulato through stimulating output line;
B. press and launch button, make the fast fast direction refrigerating chamber inlet of sample stage, and get into refrigerating chamber; Launch button simultaneously pressing, sample stage is left infrared sensor A, and the signal of infrared sensor A output is as the starting point of whole fall time, and the triggering timing device carries out timing; When sample got into the superfreeze chamber, the signal of infrared sensor B output made timer stop timing.The time that sample stage is moved between infrared sensor A, infrared sensor B, (being the timing of timer institute) was for always launching time T;
C. press launch button in; Infrared sensor A also produces commencing signal synchronous triggering computing machine starts working programmable stimulato; Behind suitable delay time t; Export to that standing wave is wide, the electrical stimulation signal of frequency and amplitude, stimulated samples (stimulation parameter can pre-set on computers).This process is carried out in the dynamic process that sample stage is launched fully.
D. the time-histories after electrical stimulation signal produces is S, and it with the relation of launching time T, delay time t is: S=T-t.
S also is that (biological tissue is fixed to the time that is fixed after by electro photoluminescence in biological tissue; Physiological function promptly stops); After having represented biological tissue by electro photoluminescence; The time that its physiological function changes is provided with different time delay time t, just can obtain the physiological function variation in the different time after the electro photoluminescence.
Each stimulation parameter of programmed electrical stimulation device output like time-delay, single/multiple, frequency, ripple is wide and amplitude all can preset through computing machine.
Beneficial effect of the present invention is: the instantaneous physiological function of solution and biological tissue changes synchronously; Obtain the problem of its ultrastructure form picture and ion concentration, site variation in real time; In the time of obtaining biological tissue's physiological function variation; Truer, more ultrastructure information can be carried out the research of physiological function and morphology synchronously on the level of Millisecond, will be significant to subject development such as electron microscopy, cell ultrastructure morphology, physiology and pathology.And, will bring breakthrough in many fields such as mechanism research of biomedical fundamental research, clinical pathology for basic medical research, clinical research and diagnosis provide a brand-new research method.The invention solves electro photoluminescence of biological sample Millisecond and the synchronous technology of cryofixation, is present unique in the world similar technology and instrument.The beneficial effect that the present invention obtains can be referring to the document of delivering early stages such as the inventor: 1. Yang Yong thoroughbred horse, Xing Xuan, Tang Ying; Shao Xiaoliang, Jiang Jian, Ye Xuting; Shen Yafeng " ultrastructural studies of sarcoplasmic reticulum during the flesh excitation contraction coupling. " " electron microscopic journal " 2006, Vol.25, p217-218.2. Yang Yong thoroughbred horse, Song Tianbin, Tang Ying, Wu Yue, Ye Xuting, Sha Jihong etc. " Millisecond of Skeletal Muscle Contraction sarcoplasmic reticulum structure in latent period changes research. " " electron microscopic journal " 2002, Vol.21, No.5, p485-486.3. Yang Yong thoroughbred horse, Kong Lingshan, Tang Ying, Song Tianbin, Su Jinlian, Wu Yue etc. " form-changes of function research that Ca2+ discharges in the sarcoplasmic reticulum during skeletal muscle excitation contraction coupling. " " electron microscopic journal " 2001, Vol.20, No.3, p192-198).
Description of drawings
Fig. 1 is the structural representation of apparatus of the present invention;
Fig. 2 is a fundamental diagram of the present invention;
Fig. 3 launches time-histories and stimulation time relation for sample stage;
Wherein: 1. sample stage fixed bar; 2. sample stage fixed screw; 3. sample stage; 4. sample; 5 vertical slides; 6. refrigerating chamber enters the mouth; 7. superfreeze chamber; 8. launch button; 9. infrared sensor A; 10. infrared sensor B; 11. timer; 12. display; 13. stimulation output lead; 14. programmable stimulato; 15. computing machine.
Embodiment
Combine embodiment and accompanying drawing at present, the present invention is further described, but enforcement of the present invention is not limited in this.
Embodiment 1:
A kind of electro photoluminescence-Millisecond ultralow temperature snap frozen device; Referring to Fig. 1; Comprise: sample stage 3, vertical slide 5, superfreeze chamber 7, infrared sensor A9, infrared sensor B10, timer 11, programmable stimulato 14 and computing machine 15, wherein vertical slide is installed on the superfreeze chamber, and sample stage is installed on the vertical slide; Sample stage is fixing with sample stage fixed screw 2 with sample stage fixed bar 1, and stimulating electrode is installed on the sample stage; Vertical slide respectively is equipped with an infrared sensor in two ends up and down; Wherein infrared sensor A is installed on the vertical slide upper end; Infrared sensor B is installed on vertical slide lower end (promptly with refrigerating chamber enter the mouth 6 height such as grade), and infrared sensor A, B are connected with timer respectively, and timer is connected with computing machine; The computer control programmable stimulato, programmable stimulato 14 is through stimulating output lead 13 to stimulating electrode.
As shown in Figure 2; The input termination standard clock signal of infrared gating circuit, output terminal is connected with counter, and this gating circuit receives the control of sensors A and B; This door is opened when object sland leaves sensors A; Allow rolling counters forward, when sliding to sensor B under the object sland, close this door, counting finishes.Count results (time) is fed to computing machine 15 mappings, and figure can show on display 12.
Embodiment 2:
The electro photoluminescence of mouse cardiac muscle-ultralow temperature snap frozen.After mouse heart takes out, adopt the glass minute hand that it is separated into myofilament, then:
A. the mouse cardiac muscle myofilament is fixed on the sample stage 3, and inserts mouse cardiac muscle myofilament two ends with stimulating electrode, stimulating electrode is connected with programmable stimulato 14 through stimulating output lead 13;
B. press and launch button 8, make the fast fast direction refrigerating chamber inlet 6 of sample stage, go forward side by side into the superfreeze chamber; Launch button simultaneously pressing, sample stage is left infrared sensor A9, and the signal of infrared sensor A output is as the starting point of whole fall time, and the triggering timing device carries out timing; When sample got into the superfreeze chamber, the signal of infrared sensor B10 output made timer stop timing, and the time that sample stage is moved between infrared sensor A, infrared sensor B, (being the timing of timer institute) was for always launching time T;
C. press launch button in; Infrared sensor A also produces commencing signal synchronous triggering computing machine starts working programmable stimulato; Behind suitable delay time (setting of available computers program), export to that standing wave is wide, the electrical stimulation signal of frequency and amplitude, stimulated samples.This process is carried out in the dynamic process that sample stage is launched fully.Time-histories behind the electrical stimulation signal is S, and it with the relation of launching time T, delay time t is: S=T-t (as shown in Figure 3).In the present embodiment: launch time T=12mS.Each stimulation parameter of programmed electrical stimulation device output: stimulate time-delay t=5mS, list/multiple=again, frequency=30Hz, ripple wide=0.3mS, amplitude=1.8V, all before experiment, preset through computing machine.
D. the time-histories behind the electrical stimulation signal is S=T-t=12mS-5mS=7mS, that is to say: biological tissue promptly is frozen fixing later on by electro photoluminescence 7mS, and the physiological function of biological tissue changes and stops.
Embodiment 3:
The electro photoluminescence of frog skeletal muscle-ultralow temperature snap frozen.Obtain the skeletal muscle muscle bundle from the separation of frog thigh, then:
A. frog skeletal muscle muscle bundle is fixed on the sample stage, and inserts frog skeletal muscle muscle bundle two ends with stimulating electrode, stimulating electrode is connected with programmable stimulato through stimulating output line;
B. with the step B of embodiment 2;
C. with the step C of embodiment 2
In the present embodiment: launch time T=12mS.Each stimulation parameter of programmed electrical stimulation device output: time-delay=10mS, list/answer=answer, frequency=50Hz, ripple be wide=and 0.2mS, amplitude=3.5V, all preset before experiment through computing machine.
D. the time-histories behind the electrical stimulation signal is S=T-t=12mS-10mS=2mS, that is to say: biological tissue promptly is frozen fixing later on by electro photoluminescence 2mS, and the physiological function of biological tissue changes and stops.
More than show and described ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; The present invention is not restricted to the described embodiments; That describes in the foregoing description and the instructions just explains principle of the present invention; The present invention also has various changes and modifications under the prerequisite that does not break away from spirit and scope of the invention, and these variations and improvement all fall in the scope of the invention that requires protection.The present invention requires protection domain to be defined by appending claims and equivalent thereof.
Claims (7)
1. electro photoluminescence-Millisecond ultralow temperature snap frozen device; It is characterized in that; This device comprises: sample stage (3), vertical slide (5), superfreeze chamber (7), infrared sensor A (9), infrared sensor B (10), timer (11), programmable stimulato (14) and computing machine (15); Vertical slide wherein is installed on the superfreeze chamber, is equipped with on the vertical slide on sample stage, the sample stage stimulating electrode is installed; Vertical slide respectively is equipped with an infrared sensor in two ends up and down; Wherein infrared sensor A is installed on the sample stage upper end; Infrared sensor B is installed on the height such as refrigerating chamber inlet (6) grade of vertical slide lower end and superfreeze chamber, and infrared sensor A, infrared sensor B are connected with timer respectively; Timer is connected with computing machine; The computer control programmable stimulato, programmable stimulato links to each other with stimulating electrode.
2. electro photoluminescence according to claim 1-Millisecond ultralow temperature snap frozen device is characterized in that, sample stage wherein (3) under the control of launching button (8) that is installed on the vertical slide (5), is down slided rapidly along vertical slide.
3. electro photoluminescence according to claim 1 and 2-Millisecond ultralow temperature snap frozen device is characterized in that, programmable stimulato (14) can carry out electro photoluminescence to sample through stimulating output lead (13) to stimulating electrode in the process that sample stage glides.
4. electro photoluminescence according to claim 1 and 2-Millisecond ultralow temperature snap frozen device is characterized in that, the signal controlling timer (11) of described infrared sensor A (9) and infrared sensor B (10) output.
5. electro photoluminescence according to claim 4-Millisecond ultralow temperature snap frozen device is characterized in that, the signal Synchronization programmable stimulato (14) of described infrared sensor A (9) output.
6. electro photoluminescence according to claim 1 and 2-Millisecond ultralow temperature snap frozen device is characterized in that described computing machine (15) also connects display (12).
7. electro photoluminescence-Millisecond ultralow temperature quick-freezing method that use is installed according to claim 1 is characterized in that this method may further comprise the steps:
A. will need the sample of ultralow temperature snap frozen to be fixed on the sample stage, and stimulating electrode will be installed on the sample stage be connected with programmable stimulato through stimulating output line;
B. press and launch button, make the fast fast direction refrigerating chamber inlet of sample stage, go forward side by side into the superfreeze chamber; Launch button simultaneously pressing, sample stage is left infrared sensor A, and the signal of infrared sensor A output is as the starting point of whole fall time, and the triggering timing device carries out timing; When sample got into the superfreeze chamber, the signal of infrared sensor B output made timer stop timing; The time that sample stage is moved between infrared sensor A, infrared sensor B is for always launching time T;
C. press launch button in; Infrared sensor A also produces commencing signal synchronous triggering computing machine starts working programmable stimulato; Behind suitable delay time t; Export to that standing wave is wide, the electrical stimulation signal of frequency and amplitude, stimulated samples, stimulation parameter can pre-set on computers;
D. the time-histories after electrical stimulation signal produces is S, and it with the relation of launching time T, delay time t is: S=T-t.
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| CN2012101625699A CN102692344A (en) | 2012-05-21 | 2012-05-21 | Electrical stimulation and millisecond-class ultralow temperature rapid freezing method and device thereof |
| PCT/CN2013/000588 WO2013174147A1 (en) | 2012-05-21 | 2013-05-17 | Electrical stimulation and millisecond-class ultralow temperature rapid freezing method and device thereof |
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| WO2013174147A1 (en) * | 2012-05-21 | 2013-11-28 | 中国人民解放军第二军医大学 | Electrical stimulation and millisecond-class ultralow temperature rapid freezing method and device thereof |
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| CN202614579U (en) * | 2012-05-21 | 2012-12-19 | 中国人民解放军第二军医大学 | Ultralow-temperature quick freezing device |
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| CN102692344A (en) * | 2012-05-21 | 2012-09-26 | 中国人民解放军第二军医大学 | Electrical stimulation and millisecond-class ultralow temperature rapid freezing method and device thereof |
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| CN202614579U (en) * | 2012-05-21 | 2012-12-19 | 中国人民解放军第二军医大学 | Ultralow-temperature quick freezing device |
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| WO2013174147A1 (en) * | 2012-05-21 | 2013-11-28 | 中国人民解放军第二军医大学 | Electrical stimulation and millisecond-class ultralow temperature rapid freezing method and device thereof |
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Application publication date: 20120926 |