WO2013174147A1

WO2013174147A1 - Electrical stimulation and millisecond-class ultralow temperature rapid freezing method and device thereof

Info

Publication number: WO2013174147A1
Application number: PCT/CN2013/000588
Authority: WO
Inventors: 杨勇骥; 夏金辉; 汤莹; 雷长海
Original assignee: 中国人民解放军第二军医大学
Priority date: 2012-05-21
Filing date: 2013-05-17
Publication date: 2013-11-28
Also published as: CN102692344A

Abstract

The present invention relates to the technical field of an electron microscope and ultralow temperature rapid freezing. Disclosed is an electrical stimulation and millisecond-class ultralow temperature rapid freezing device, comprising: a sampling stage (3), a vertical sliding plate (5), an ultralow temperature freezing room (7), an infrared sensor A (9), an infrared sensor B (10), a timer (11), a programmable stimulator (14) and a computer (15). The sampling stage (3) is arranged on the vertical sliding plate (5). The infrared sensor A (9) and the infrared sensor B (10) are arranged respectively at an upper end and a lower end of the vertical sliding plate (5). The infrared sensor A (9) is arranged at an upper end of the sampling stage (3). The infrared sensor B (10) is at an equal altitude to an inlet (6) of a freezing room of the ultralow temperature freezing room (7). The two infrared sensors (9, 10) are connected with the timer (11). The device can enable a biological tissue to be frozen and fixed rapidly by the ultralow temperature upon being stimulated, so that morphologic change of an ultrastructure of the biological tissue in various specific situations when a physiological function changes can be studied, and studies of the physiological function and the morphology can be performed simultaneously at a millisecond level, which is significant for the development of subjects such as the electron microscopy, the cell ultrastructure morphology, the physiology, and the pathology.

Description

Electric stimulation one millisecond ultra-low temperature rapid freezing method and device thereof

The invention relates to the field of electron microscopy technology and ultra-low temperature rapid freezing technology, which is mainly used in the field of basic medical research of biological tissue-cell ultrastructure morphology, functional science, neurobiology, physiology, etc., and specifically relates to preparation of a sample prepared by electron microscopy. Electrical stimulation of one millisecond ultra-low temperature rapid freezing method and device thereof. Background technique

In the field of biomedical morphological analysis, it is necessary to observe the physiological ultrastructural changes of biological tissues under external stimuli under electron microscope. This requires the biological tissue to be fixed in milliseconds while being stimulated by the outside world. The technology capable of fixing biological tissue in milliseconds is an ultra-low temperature rapid freezing technique. The stimuli and responses required for changes in physiological function are millisecond units, such as: nerve impulse conduction, muscle tissue excitation contraction and so on. Therefore, millisecond-level electrical stimulation and freeze-synchronization synchronization techniques are required to study structural changes when transient physiological functions change.

At present, the immobilization of biological samples by electron microscopy is mostly chemical fixation. Chemical fixation often causes shrinkage, swelling or autolysis of biological tissues and cell structures, resulting in errors in morphological structure analysis. Freezing fixation is a physical process that significantly increases the mechanical strength of certain soft biological tissues. It is the only way to preserve the structure and internal elements of the living organism closest to the living state. Although cryopreservation can instantaneously (in milliseconds) freeze ecological state, including tissue morphological structure and intracellular elements (especially soluble ions), avoid changes in biological tissues, cell structures and internal chemical components and sites, but current freeze fixation Technology cannot be synchronized with electrical stimulation of biological tissue.

There is currently no literature report on the various states and physiological characteristics of the physiological response of biological tissues using electrical stimulation and ultra-low temperature rapid freezing technology.

Although the optical microscope has been able to obtain the morphological structure image when the biological tissue function changes in real time, due to the limitation of the resolution and magnification of the light microscope, the research level of the light microscope is difficult to meet the needs of exploring the field of ultra-microstructure. While electron microscopy can study the morphology of biological tissues at the level of ultrastructural structure, it can not obtain the ultrastructural morphological image when the transient physiological function of biological tissues changes, which limits the SEM technology in biomedicine. The development of the field of study. Therefore, how to synchronize with the transient physiological function changes of biological tissues, obtain the (ie real-time) ultrastructural morphological images of biological tissues when physiological functions are changed, so that physiological functions and morphological studies can be synchronized at the millisecond level, and the electrons will be The development of microscopy, cell ultrastructural morphology, physiology and pathology is of great significance. It solves the problem that the biological tissue obtains its ultrastructural morphology and ion concentration and site change in real time when the millimeter-level function changes, and it will be able to obtain more real and more ultra-structural information when the biological tissue physiological function changes. , thus providing a new technical method for basic medical research, clinical research and diagnosis. Summary of the invention

It is an object of the present invention to provide a method and a special method for rapidly changing the ultrastructural morphology of a biological tissue in a specific state in which the biological tissue is rapidly frozen and fixed at the moment of being stimulated. device.

The present invention provides an electrical stimulation one millisecond ultra-low temperature rapid freezing device that allows biological tissue to be rapidly frozen in milliseconds after stimulation.

The invention provides an electric stimulation-microsecond ultra-low temperature rapid freezing device, comprising: a sample stage, a vertical sliding plate, an ultra-low temperature freezing chamber, an infrared sensor A, an infrared sensor B, a timer, a program-controlled stimulator and a computer, wherein the vertical sliding plate is installed on On the ultra-low temperature freezer compartment, a sample stage is mounted on the vertical slide plate, and a stimulation electrode is mounted on the sample stage; an infrared sensor is mounted on each of the upper and lower ends of the vertical slide plate, wherein the infrared sensor A is mounted on the upper end of the sample stage, and the infrared sensor B is mounted on the vertical slide plate. The lower end is at the same height as the entrance of the cryopreservation freezer compartment. The infrared sensors A and B are respectively connected to the timer, the timer is connected to the computer, the computer controlled program stimulator, and the programmable stimulator is connected to the stimulation electrode.

The vertical slide plate is mounted with a sample stage, and the sample stage can slide down vertically in the vertical direction under the control of the ejection button, and the sample stage is used for placing samples, especially biological tissue samples such as myocardial filaments, skeletal muscle filaments, The nerve, cardiomyocytes, skeletal muscle cells, etc., the sample stage is made of a copper material.

The sample stage slides down the vertical slide and is controlled by a pop-up button mounted on the vertical slide. The sample stage can be quickly ejected downwards under the elastic force of the ejection button, allowing the sample on the sample stage to quickly enter the ultra-low temperature freezer compartment.

The uppermost end of the ultra-low temperature freezer compartment has a freezer compartment inlet for sample entry on the sample stage.

The programmed stimulator can stimulate the sample to be electrically stimulated during the sliding down of the sample stage by stimulating the output lead to the stimulating electrode.

The signal control timer output by the infrared sensors A and B determines the operation of the entire sample stage Time course. The principle is as follows: The back of the sample stage has a light-shielding light-shielding sheet. When the sample stage is not ejected downward, the light-shielding sheet blocks the sensor A and outputs a high level. When the ejection starts, the visor leaves sensor A. Because the photocell is illuminated, a low level signal is output, the gate circuit is turned on, and the standard clock signal (O.lmS pulse) enters the timer to start calculating the time. When the visor runs to the sensor B, the visor causes the photocell of the sensor B to be unlit and outputs a high level, turns off the gating circuit, cuts off the clock signal, and terminates the timing.

At the same time, the infrared sensor A, the output signal synchronizes the program-controlled stimulator to stimulate the biological tissue at an appropriate time for the sample stage to fall.

The ultra-low temperature freezer was purchased from Rechart Company, Australia, Model KF80.

The timer is used to calculate the time required for the sample stage to go from infrared sensor A to infrared sensor B.

The program-controlled stimulator can set parameters such as delay, square wave number, wave width, interval and amplitude of the program-controlled stimulator in advance through the keyboard. When the trigger signal of infrared sensor A is received, it immediately activates the stimulus to stimulate the organism.

The computer can also be connected to a display for screen drawing to display experimental results; the computer can also store experimental results for later use; the computer can also be connected to an output device to print experimental results.

The present invention also provides an electrical stimulation one millisecond ultra-low temperature rapid freezing method using the above apparatus, comprising the steps of:

A. Fix the sample that needs ultra-low temperature and rapid freezing on the sample stage, and install the stimulating electrode on the sample stage through the stimulation output line to connect to the program-controlled stimulator;

B. Press the ejection button to make the sample stage quickly shoot at the entrance of the freezer compartment and enter the freezer compartment; while pressing the ejection button, the sample stage leaves the infrared sensor A, and the signal output by the infrared sensor A serves as the starting point of the entire fall time. The timer is triggered to count; when the sample enters the ultra-low temperature freezer, the signal from the infrared sensor B causes the timer to stop timing. The time during which the sample stage runs between infrared sensor A and infrared sensor B (ie, the timer is counted) is the total ejection time T;

C. While pressing the ejection button, the infrared sensor A also generates a start signal synchronization triggering computer to start the program stimulator, and after an appropriate delay time t, outputs an electrical stimulation signal of a given width, frequency and amplitude, Stimulation samples (stimulation parameters can be pre-set on the computer). This process is carried out completely during the dynamic process of the sample stage ejection.

D. The time history after the generation of the electrical stimulation signal is S, which is related to the ejection time T and the delay time t: S = Tt.

s is also the time after the biological tissue is electrically stimulated until it is fixed (the biological tissue is fixed, the physiological function is stopped), which represents the time when the biological tissue is changed after the electrical tissue is electrically stimulated, and the delay time t is set. Changes in physiological function at different times after electrical stimulation can be obtained.

The stimulus parameters output by the programmable electrostimulator such as delay, single/complex, frequency, wave width and amplitude can be preset by computer.

The invention has the beneficial effects of: solving the problem of the ultra-structural morphological image, ion concentration and site change in real time in synchronization with the change of the instantaneous physiological function of the biological tissue, and realizing more real and more when the physiological function of the biological tissue is obtained. The ultra-structural information enables physiological functions and morphological studies to be synchronized at the millisecond level, which will be important for the development of electron microscopy, cellular ultrastructure morphology, physiology and pathology. It also provides a new research method for basic medical research, clinical research and diagnosis, and will bring breakthroughs in many fields such as basic research in biomedicine and mechanism research of clinical lesions. The invention solves the technology of synchronizing millisecond-level electrical stimulation and freezing fixation of biological samples, and is the only similar technology and instrument in the world. The beneficial effects obtained by the present invention can be found in the literature published by the present inventors, etc.: 1. Yang Yongzhen, Xing Wei, Tang Ying, Shao Xiaoliang, Jiang Jian, Ye Yiting, Shen Yafeng. Ultrastructural study of sarcoplasmic reticulum during muscle excitation contraction coupling宄." 《Journal of Chinese Electron Microscopy》 2006, Vol.25, p217-218o 2. Yang Yongzheng, Song Tianbin, Tang Ying, Wu Yue, Ye Yiting, Sha Jihong et al. Changes in milliseconds of sarcoplasmic reticulum structure during skeletal muscle contraction latency Research." Journal of Electron Microscopy 2002, Vol. 21, No. 5, p485-486. 3. Yang Yongzhen, Kong Lingshan, Tang Ying, Song Tianbin, Su Jinlian, Wu Yue et al. "Study on the morphological-functional changes of Ca2+ release in sarcoplasmic reticulum during skeletal muscle excitation-contraction coupling." Journal of Electron Microscopy 2001, Vol.20, No. 3, pl 92-198).

Figure 1 is a schematic structural view of the device of the present invention;

Figure 2 is a working principle diagram of the present invention;

Figure 3 shows the relationship between the ejection time and the stimulation time of the sample stage;

Among them: 1. Sample stage fixing rod; 2. Sample stage fixing screw; 3. Sample stage; 4. Sample; 5 vertical slide; 6. Freezer inlet; 7. Ultra-low temperature freezer; 8. Eject button; A; 10. Infrared sensor B; 11. Timer; 12. Display; 13. Stimulus output lead; 14. Programmable stimulator; 15. Computer specific implementation

The invention will now be further described in connection with the embodiments and the drawings, but the invention is not limited thereto.

Example

An electric stimulation-microsecond ultra-low temperature rapid freezing device, as shown in Fig. 1, comprising: a sample stage 3, a vertical slide 5, an ultra-low temperature freezer 7, an infrared sensor A9, an infrared sensor B10, a timer 11, a program-controlled stimulator 14 and a computer 15. The vertical slide plate is mounted on the ultra-low temperature freezer compartment, and the sample slide table is mounted on the vertical slide plate. The sample stage is fixed by the sample stage fixing rod 1 and the sample stage fixing screw 2, and the sample stage is provided with the excitation electrode; Each is equipped with an infrared sensor, wherein the infrared sensor A is mounted on the upper end of the vertical slide, the infrared sensor B is mounted on the lower end of the vertical slide (ie, the same as the entrance of the freezer compartment 6), and the infrared sensors A and B are respectively connected with the timer, the timer and The computer is connected, the computer controls the programmed stimulator, and the programmed stimulator 14 stimulates the output lead 13 to the stimulating electrode.

As shown in Figure 2, the input terminal of the infrared gating circuit is connected to the standard clock signal, and the output terminal is connected to the counter. The gating circuit is controlled by the sensors A and B. When the specimen station leaves the sensor A, the door is opened, and the counter is allowed. Count, when the specimen stage slides down to sensor B, the door is closed and the counting ends. The count result (time) is sent to the computer 15 for drawing, and the picture can be displayed on the display 12. Example 2:

Electrical stimulation of mouse myocardium - ultra-low temperature rapid freezing. After the mouse heart is removed, it is separated into myofilaments using a glass minute hand, and then:

A. The mouse myocardium filament is fixed on the sample stage 3, and the stimulation electrode is inserted into both ends of the mouse myocardium, and the stimulation electrode is connected to the programmable stimulator 14 via the stimulation output lead 13;

B. Press the ejection button 8 to quickly shoot the sample stage to the freezer compartment inlet 6 and enter the ultra-low temperature freezer compartment; while pressing the ejection button, the sample stage leaves the infrared sensor A9, and the infrared sensor A outputs the signal as the entire fall time. Starting point, triggering timer to count; When the sample enters the ultra-low temperature freezer compartment, the signal output by the infrared sensor B10 causes the timer to stop timing, and the time that the sample stage runs between the infrared sensor A and the infrared sensor B (ie, the timer is counted) ) is the total ejection time T;

C. While pressing the ejection button, the infrared sensor A also generates a start signal to trigger the computer to start the program stimulator, and after the appropriate delay time (settable by computer program), the input An electrical stimulus signal with a given width, frequency and amplitude stimulates the sample. This process is carried out entirely in the dynamic process of the ejection of the sample stage. The time history after the electrical stimulation signal is S, which is related to the ejection time T and the delay time t: S 2 Tt (as shown in Figure 3). In this embodiment: the ejection time T=12 mS. The excitation parameters of the program-controlled electric stimulator output: stimuli delay t=5mS, single/complex=complex, frequency=30Hz, wavewidth=0.3mS, amplitude=1.8V, all preset by the computer before the experiment.

D. The time course after the electrical stimulation signal is S=T-t=12mS-5mS=7mS, that is to say: The biological tissue is frozen and fixed after 7mS, and the physiological function of the biological tissue changes. Example 3:

Electrical stimulation of skeletal muscles of frogs - ultra-low temperature rapid freezing. The skeletal muscle muscle bundle is separated from the frog's thigh, and then:

A. Fix the frog skeletal muscle muscle bundle on the sample stage, and insert the stimulating electrode into the frog skeletal muscle muscle bundle. The stimulating electrode is connected to the programmable stimulator via the stimulation output line;

B. Step B of the same embodiment 2;

C. Step C of the same embodiment 2

In this embodiment: the ejection time T = 12 mS. The stimulation parameters output by the programmable electrostimulator: delay = 10mS single / complex = complex, frequency = 50Hz, wave width = 0.2mS, amplitude = 3.5V, are preset by the computer before the experiment.

D. The time course after the electrical stimulation signal is S=T-t=12mS-10mS=2mS, that is to say: the biological tissue is frozen and fixed after 2mS of electrical stimulation, and the physiological function of the biological tissue changes. The basic principles, main features, and advantages of the present invention are shown and described above. It should be understood by those skilled in the art that the present invention is not limited by the foregoing embodiments, and that the present invention is described in the foregoing embodiments and the description of the present invention. Such changes and modifications are intended to fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and their equivalents.

Claims

1. An electrical stimulation one millisecond ultra-low temperature rapid freezing device, characterized in that the device comprises: a sample stage (3), a vertical slide (5), an ultra-low temperature freezer (7), an infrared sensor A (9), an infrared sensor B (10), a timer (11), a program-controlled stimulator (14), and a computer (15), wherein the vertical slide plate is mounted on the ultra-low temperature freezer compartment, the sample stage is mounted on the vertical slide plate, and the stimulation electrode is mounted on the sample stage; An infrared sensor is mounted on each of the upper and lower ends of the vertical slide, wherein the infrared sensor A is mounted on the upper end of the sample stage, and the infrared sensor B is mounted on the lower end of the vertical slide and the freezer inlet (6) of the ultra-low temperature freezer compartment, the infrared sensor A, the infrared sensor B is connected to the timer respectively; the timer is connected to the computer; the computer controlled program stimulator is connected to the stimulation electrode.

2. An electrical stimulation one millisecond ultra-low temperature rapid freezing apparatus according to claim 1, wherein the sample stage (3) is under the control of an ejection button (8) mounted on the vertical slide (5), Slide down the vertical slide down quickly.

3. The electrical stimulation one millisecond ultra-low temperature fast freezing apparatus according to claim 1 or 2, wherein the programmable excitation device (14) is slidable on the sample stage by exciting the output lead (13) to the stimulating electrode. The sample was electrically stimulated during the process.

The electric stimulation one millisecond ultra-low temperature fast freezing device according to claim 1 or 2, wherein the infrared sensor A (9) and the infrared sensor B (10) output a signal control timer (11) .

5. The electrical stimulation one millisecond ultra-low temperature fast freezing apparatus according to claim 4, wherein the signal output by the infrared sensor A (9) is synchronized with the programmable stimulator (14).

6. An electrical stimulation one millisecond ultra-low temperature fast freezing apparatus according to claim 1 or 2, characterized in that said computer (15) is further connected to a display (12).

7. An electrical stimulation one millisecond ultra-low temperature rapid freezing method using the apparatus of claim 1 wherein the method comprises the steps of:

A. Fix the sample that needs ultra-low temperature and rapid freezing on the sample stage. The sample stage is equipped with a stimulating electrode connected to the program-controlled stimulator via the stimulation output line;

B. Press the ejection button to make the sample stage quickly shoot at the entrance of the freezer compartment and enter the ultra-low temperature freezer compartment; while pressing the ejection button, the sample stage leaves the infrared sensor A, and the signal output by the infrared sensor A serves as the starting point of the entire fall time. , trigger the timer to time; when the sample enters ultra-low temperature freezing In the room, the signal output by the infrared sensor B causes the timer to stop timing; the time when the sample stage runs between the infrared sensor A and the infrared sensor B is the total ejection time T;

C. While pressing the ejection button, the infrared sensor A also generates a start signal synchronization triggering computer to start the program-controlled stimulator, and after an appropriate delay time t, outputs an electrical stimulation signal of a given width, frequency and amplitude, Stimulate the sample, the stimulation parameters can be pre-set on the computer;

D. The time history after the generation of the electrical stimulation signal is S, which is related to the ejection time T and the delay time t: S=T-t.