CN114184809A - Method and device for measuring three-dimensional dynamic morphology of single-beat cardiac muscle cell by atomic force probe - Google Patents
Method and device for measuring three-dimensional dynamic morphology of single-beat cardiac muscle cell by atomic force probe Download PDFInfo
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Abstract
The invention relates to a method and a device for measuring the three-dimensional dynamic morphology of a single-beat cardiac muscle cell by an atomic force probe, wherein the three-dimensional dynamic morphology of the single-beat cardiac muscle cell is measured by the atomic force probe, the atomic force probe is accurately positioned on the single-beat cardiac muscle cell to be measured according to an atomic force microscopic imaging system, a force feedback system and a closed-loop feedback system thereof, the atomic force probe is contacted with the surface of the single-beat cardiac muscle cell, a constant-force single-point multiple acquisition mode is adopted for the surface contact, the spatial position of one point on the surface of the cell at any moment is determined, and the single-beat cardiac muscle cell is scanned by the mode, so that the measurement of the three-dimensional dynamic morphology is realized. The measurement of the three-dimensional dynamic morphology of the single-beat cardiomyocyte can accurately position the position and the morphology of the single-beat cardiomyocyte at any moment in the beating period, realize dynamic measurement and further obtain the three-dimensional dynamic morphology of the single-beat cardiomyocyte on a nanometer scale.
Description
Technical Field
The invention relates to a method and a device for measuring the three-dimensional dynamic morphology of single-beat cardiac muscle cells by an atomic force probe, belonging to the technical field of engineering.
Background
Life sciences are one of the leading fields of this century, cells are the basic units of life bodies, and there are many different kinds of cells in the human body, each having its unique size, shape, structure and function. Recent studies have shown that cell shape affects cell fate in many physiological processes such as cell growth, differentiation, development, death and tumor growth. Therefore, the study on the morphology of the biological cells is helpful for people to know the physiological state and process of the biological cells more clearly, accurately and comprehensively, and can also make people know the individual difference of different cells in a cell population and the functions of some special cells better, thereby having important significance for revealing the secret of life. In the study of three-dimensional dynamic morphology of cells, a pulsating cell such as a cardiomyocyte is a major subject of study in this field. Studying the three-dimensional dynamics of such beating cells gives a deeper understanding of the heart.
With the rapid development of nanotechnology, ultrahigh resolution and non-invasive imaging detection technology will play an important role in the scientific fields of biology, materials and the like. The appearance of an Atomic Force Microscope (AFM) provides possibility for observing the ultrastructure on the surface of a living cell. The AFM performs raster scanning on a sample by using a sharp needle point integrated at the tail end of a cantilever beam to obtain the surface appearance of the sample. The AFM can work in a solution environment and has the nanometer-scale spatial resolution, and the unique advantage makes the AFM very suitable for researching the nanometer-scale physiological activities on the surfaces of living cells. The AFM is applied to research biological samples (living cells and natural membrane proteins) in a natural state, so that a great deal of new knowledge is brought to cell biology, including understanding tissues and dynamic behaviors of cell membranes at a nanometer scale, observing structures and activities of organelles, analyzing dynamic structures of protein molecules and the like. Due to its powerful function, AFM has become an important experimental tool in cell biology, and is an important supplement to conventional optical microscopy, electron microscopy and X-ray crystallography. However, optical imaging is mainly adopted in the imaging research of the beating myocardial cells at present, the imaging resolution is low, the accuracy is poor, the fine observation and analysis of the sample properties on the nanometer scale cannot be realized, and the biological research is not facilitated. The scanning probe microscope has real space resolution capability of nanometer or even atomic level, but the traditional scanning probe microscope technology can only observe the surface characteristics of a solid sample, and cannot obtain the information of a living cell sample.
In the previous research of the applicant, Zhan Miao et al utilized atomic force nano imaging technology in combination with electrophysiological property measurement method and mechanical property measurement method to measure electrophysiological properties and mechanical properties of a single pulsating cardiomyocyte during pulsation, measured and obtained action potential of a single pulsating cardiomyocyte of a newborn suckling mouse, successfully realized recording of action potential and contractility signals of the single pulsating cardiomyocyte in two measurement modes (constant force tracking measurement mode and non-constant force contact measurement mode), and applied for a patent named as a method for measuring action potential and pulsation force of the single pulsating cardiomyocyte by an atomic force microscope based on the experiment, which cannot accurately measure electrophysiological properties and mechanical properties of all points on the surface of a cell membrane and cannot measure three-dimensional dynamic morphology of the single pulsating cardiomyocyte, compared with the existing measurement method, the spatial position and the morphology of one point on the cell surface at any time in the beating period of the single-beating cardiomyocyte can be realized, and dynamic measurement is realized, so that the three-dimensional dynamic morphology of the single-beating cardiomyocyte is obtained on the nano scale. (see Zhang Si micro. robot nano-manipulation of living cells and investigation of electrical property detection technique [ D ]. university of vinpocetine, 2016).
In modern biomedical research and applications, three-dimensional dynamic imaging of single beating cardiomyocytes is often required. The cell morphology can reflect the information of cell surface molecules, cytoskeleton, organelle and the like, and reflect the physiological state of the cell, so that imaging observation and nano-structure research on single-beat cardiac muscle cells are possible to solve the pending problems of cell function, physiological behaviors of individual cells and groups, cell variation, biochemistry of cell communication, chemico-biology, medicine and the like. In 2007, Qihao et al studied the optimal imaging condition of the cell based on AFM, obtained the optimal imaging condition of the human liver cancer cell SMMC-7721 in the atmospheric environment and the solution environment by analyzing and studying AFM images of the human liver cancer cell SMMC-7721 under different treatment conditions and test conditions, and established an experimental method for observing the living cell by AFM (see Qihao, Liuying, Zhu le nan, Zhujie, Sunzhou Guangdong. study on the optimal imaging condition of atomic force microscope of the cell [ J ]. Photonic report, 2007). In 2005, Wang Li Juan et al applied AFM to study different imaging conditions in CHO cells slightly cured with 1% glutaraldehyde and physiological solution environment. Experimental results show that the probe with the elastic coefficient of 0.06N/m can obtain better effect when used for scanning cells in a physiological environment, and the scanning speed is preferably 2-3 Hz. The AFM has achieved the resolution capability of atomic level and molecular level, can work under natural condition or physiological condition, can dynamically observe the physiological activity of cells, and is the most suitable technique and method for imaging and observing single cells at present.
Disclosure of Invention
The invention solves the problems: the method and the device for measuring the three-dimensional dynamic morphology of the single beating cardiomyocyte by the atomic force probe are used for overcoming the defects of the prior art, obtaining feedback information of the acting force of the needle point of the atomic force probe and a sample according to an atomic force microscopic imaging system, a force feedback system and a closed-loop feedback system of the atomic force microscopic imaging system, accurately positioning the position of the cell, and determining the spatial position and the morphology of one point on the surface of the single beating cardiomyocyte at any moment in a beating period by using a constant force single point and a time sequence multi-acquisition mode to realize the measurement of the three-dimensional dynamic morphology, so that the three-dimensional dynamic morphology of the single beating cardiomyocyte is obtained on a nanometer scale.
The purpose of the invention can be realized by the following technical measures: a method and a device for measuring the three-dimensional dynamic morphology of single-beat cardiac muscle cells by an atomic force probe are characterized in that: the method is characterized in that the atomic force probe is contacted with the surface of a single-beating myocardial cell in a constant-force single-point multiple-acquisition mode according to time sequence through program control, the spatial position and the morphology of one point on the cell surface of the single-beating myocardial cell at any moment in a beating period are determined, the single-beating myocardial cell is scanned through the mode, the measurement of the three-dimensional dynamic morphology is realized, and the three-dimensional dynamic morphology of the single-beating myocardial cell is acquired on the nanometer scale, and the method comprises the following steps:
(1) controlling the nanometer displacement platform to move in the direction of an X, Y, Z axis, searching single-beat cardiac muscle cells, and moving the atomic force probe, the sample platform, the atomic force microscopic imaging system, the force feedback system and the closed-loop feedback system thereof to accurately position the atomic force probe on the single-beat cardiac muscle cells to be detected;
(2) observing the state of the single-beat myocardial cell under an optical microscope, and moving the atomic force probe to place the tip of the atomic force probe right above the single-beat myocardial cell to be detected;
(3) setting measurement parameters, enabling the atomic force probe to be in contact with the cell surface, adopting a constant-force single-point multiple-time acquisition mode according to a time sequence, determining the spatial position of one point on the cell surface at any moment, scanning the single-beat cardiac muscle cell through the acquisition mode, realizing the measurement of the three-dimensional dynamic morphology, accurately positioning the position and the morphology of the single-beat cardiac muscle cell at any moment in a beat period, realizing the dynamic measurement, and further obtaining the three-dimensional dynamic morphology of the single-beat cardiac muscle cell on a nanometer scale.
The atomic force probe is a silicon nitride probe, the micro-cantilever is triangular, the needle tip is conical, the radius of the tip of the needle tip is 5-10nm, the elastic constant of the micro-cantilever is 0.2-0.4N/m, and the length of the micro-cantilever is 90-100 microns.
The constant-force single-point multiple-acquisition mode is characterized in that a measurement parameter is set to enable an atomic force probe to measure surface height information of a single-beating cardiomyocyte on a Z axis on a cell surface point by a constant force according to a time sequence, so that the spatial position of the cell surface point of the single-beating cardiomyocyte at any moment in a beating period is obtained, the cell surface point is moved to the next position to repeat the above operation, the single-beating cardiomyocyte is scanned through the constant-force single-point multiple-acquisition mode, the position and the shape of the single-beating cardiomyocyte at any moment in the beating period are accurately positioned, and measurement of three-dimensional dynamic shapes is achieved, and the method specifically comprises the following steps:
(1) according to the measurement requirements, setting the surface height information measurement of the single-beat myocardial cell on the Z axis in time sequence at one point on the cell surface with constant force, and giving a set value of the measurement times at one point on the cell surface;
(2) judging whether the measurement frequency at one point on the cell surface reaches a set value or not through a closed-loop feedback system, if so, obtaining the spatial position of one point on the cell surface at any moment in a beating period of the single-beating myocardial cell through analysis, and moving the atomic force probe to the next position for continuous measurement;
(3) the single-beat cardiac muscle cell is scanned in the mode, the spatial position of the single-beat cardiac muscle cell at any moment in a beat period is accurately positioned, the three-dimensional dynamic morphology of the single-beat cardiac muscle cell is obtained through analysis, and the measurement of the three-dimensional dynamic morphology is realized.
Setting the measurement parameters in the step (3), and setting the needle inserting precision of the atomic force probe, wherein the specific implementation mode is that the coarse needle inserting of the atomic force probe gradually approaches to the single beating cardiomyocyte to be detected in a step length of 30nm, the coarse needle inserting is stopped when the distance between the needle point and the single beating cardiomyocyte membrane is 5nm, meanwhile, a constant force single point multiple acquisition mode of the atomic force micro-imaging system is started, the atomic force probe finely inserts in a step length of 0.02nm, and the spatial position and the shape of the single beating cardiomyocyte at any moment in the beating period are tracked and measured in real time in the constant force multiple acquisition mode in the time sequence, so that the measurement of the three-dimensional dynamic shape is realized, and the three-dimensional dynamic shape of the single beating cardiomyocyte is obtained on the nanometer scale.
In the step (3), by setting measurement parameters, according to the atomic force microscopic imaging system and the force feedback system and the closed-loop feedback system thereof, the atomic force probe is contacted with the single beating myocardial cell with constant force, when the single-beat myocardial cell contracts or expands, the atomic force probe moves along the Z-axis direction along with the single-beat myocardial cell, in the pulsation period of the single pulsation myocardial cell, the atomic force probe is always contacted with the single pulsation myocardial cell in a multiple acquisition mode with constant force according to the time sequence, so that the contact between the atomic force probe and the surface of the single pulsation myocardial cell membrane is stable, the measurement method is used for measuring and obtaining the spatial position and the morphology of the single-beat myocardial cell at any moment in the beat period, and a measurement mode with stable contact is constructed to realize the accurate measurement of the three-dimensional dynamic morphology, thereby obtaining the three-dimensional dynamic morphology of the single-beat myocardial cell on the nanometer scale.
The piezoelectric ceramic nanometer displacement platform controller drives the sample platform above the piezoelectric ceramic nanometer displacement platform to move in the direction of X, Y, Z axis, so that when the needle point of the atomic force probe moves on the surface of the sample, the atomic force probe deforms due to the interaction of the needle point and the force between single beating cardiac muscle cells, the laser is utilized to focus the laser beam on the back end of the atomic force probe, the laser beam is reflected to the four-quadrant photoelectric detector, the deformation of the atomic force probe causes the light spot to generate offset on the four-quadrant photoelectric detector, the four-quadrant photoelectric detector converts the light variation signal into an electrical signal, the electrical signal is taken as a feedback signal in a force feedback system to serve as an internal adjusting signal, and the piezoelectric ceramic nanometer displacement platform is driven to move properly to keep constant acting force between the sample and the needle point, the resulting system for adjusting the amount of force is called a force feedback system.
The method comprises the steps of collecting and amplifying electric signals obtained through measurement through a four-quadrant photoelectric detector voltage collection module, converting the electric signals through an A/D converter, transmitting the electric signals to a computer terminal control system, setting surface height information measurement of single beating myocardial cells on a Z axis at one point on the cell surface with constant force according to time sequence, giving a set value of measuring times at one point on the cell surface, judging whether the set value is reached, transmitting feedback signals compared with the set value to a D/A data converter, converting the feedback signals and transmitting the feedback signals to a piezoelectric ceramic nano displacement platform controller, and adjusting a sample platform above a piezoelectric ceramic nano displacement platform to move in the Z axis direction, so that a closed-loop control-involved system is formed and is called as a closed-loop feedback system.
Compared with the prior method and system, the invention has the following advantages:
(1) the resolution of optical imaging is limited by diffraction limit, and the resolution of the optical imaging cannot be less than half of the wavelength of incident light during measurement, so that compared with the optical imaging, the method and the device for measuring the three-dimensional dynamic morphology of the single beating myocardial cell have the advantages of nanoscale spatial resolution and good accuracy. Can realize the fine observation and analysis of the sample morphology on the nanometer scale, and is beneficial to the research on the cell size, shape, structure and function.
(2) In the electron microscope, the specimen must be observed under a vacuum environment, and thus living cells cannot be observed. The electrons of the electron microscope are extremely harmful to the biological sample, and the protection of the dye destroys the shape of the sample. The method and the device for measuring the three-dimensional dynamic morphology of the single-beat cardiac muscle cell can perform high-resolution imaging on living cells in a physiological environment, and the imaging resolution reaches an atomic level and a molecular level. The appearance of the sample obtained by the mode measurement is clearer, the nondestructive measurement of living cells can be realized, and the damage degree of the sample is greatly reduced.
(3) Compared with the traditional Atomic Force Microscope (AFM), the method and the device for measuring the three-dimensional dynamic morphology of the single-beat cardiomyocyte can accurately position the cell position, have high flexibility in operation, can determine the spatial position and the morphology of one point on the cell surface at any time in the beating period of the single-beat cardiomyocyte, realize dynamic measurement, and further obtain the three-dimensional dynamic morphology of the single-beat cardiomyocyte on a nanometer scale.
(4) In the previous research of the applicant, Zhan Miao et al utilized atomic force nano imaging technology in combination with electrophysiological property measurement method and mechanical property measurement method to measure electrophysiological properties and mechanical properties of a single beating cardiomyocyte during beating, and applied a patent named as a method for measuring action potential and beating force of a single beating cardiomyocyte by using an atomic force microscope based on the experiment, wherein a constant force/non-constant force tracking mode is adopted to measure electrophysiological properties and mechanical properties at a single point, the patent cannot accurately measure electrophysiological properties and mechanical properties of all points on the surface of a cell membrane, and cannot measure three-dimensional dynamic morphology of the single beating cardiomyocyte, compared with the existing measurement method, the method and device for measuring three-dimensional dynamic morphology of a single beating cardiomyocyte provided by the invention can realize spatial position and morphology of a point on the cell surface at any moment in the beating period of the single beating cardiomyocyte, dynamic measurement is realized, so that the three-dimensional dynamic morphology of the single-beat cardiac muscle cell is obtained on the nanometer scale.
Drawings
FIG. 1 is a schematic diagram of the method and apparatus for measuring the three-dimensional dynamic morphology of a single beating cardiomyocyte using an atomic force probe according to the present invention;
FIG. 2 is a schematic diagram of the measurement of a single beating cardiomyocyte in a tracking contraction state by an atomic force probe according to the present invention;
FIG. 3 is a schematic diagram of the atomic force probe tracking measurement of a single beating cardiomyocyte in a diastolic state according to the present invention;
FIG. 4 is a schematic diagram of an atomic force probe according to the present invention, in which (a) is a cantilever of the atomic force probe, and (b) is a tip of the atomic force probe.
Detailed Description
As shown in fig. 1, which is a schematic block diagram of the present invention, an atomic force probe cell measurement module 1 includes an optical lever system composed of an atomic force probe, a single beating cardiomyocyte, a sample stage, a laser and a four-quadrant photodetector, a piezoelectric ceramic nano-displacement platform 2, a four-quadrant photodetector voltage acquisition module 3, which includes a signal amplifier and an a/D converter, an optical microscope module 4, which includes an objective lens, an adapter and a zoom lens, a D/a data converter 5, a piezoelectric ceramic nano-displacement platform controller 6, a computer terminal control system 7, and a sample topography image display 8.
The computer terminal control system 7 controls the sample stage above the piezoelectric ceramic nano displacement platform 2 to move in the X, Y, Z axis direction, single beating cardiomyocyte is searched, the atomic force probe, the piezoelectric ceramic nano displacement platform 2, the sample stage are moved, and the atomic force probe is accurately positioned on the single beating cardiomyocyte on the sample stage above the target piezoelectric ceramic nano displacement platform 2 according to the force feedback system, the adapter and the zoom lens are adjusted through the optical microscope module 4, so that the state of the single beating cardiomyocyte is clearly observed, and the tip of the atomic force probe is placed right above the single beating cardiomyocyte to be detected. The atomic force probe in the atomic force probe cell measuring module 1 is used as a detection sensor capable of detecting weak force, when the needle point of the atomic force probe approaches the surface of a single beating myocardial cell to several nanometers, even smaller, the atomic force probe deforms due to the interaction of the needle point and the single beating myocardial cell, a laser is used for focusing a laser beam on the back end of the atomic force probe, the laser beam is reflected to a four-quadrant photoelectric detector, the deformation of the atomic force probe causes the displacement of a light spot on the four-quadrant photoelectric detector, the four-quadrant photoelectric detector converts the light variation signal into an electrical signal, the electrical signal is acquired and amplified by a four-quadrant photoelectric detector voltage acquisition module 3 and is transmitted to a computer terminal control system 7 after being converted by an A/D converter, and the surface height of the beating single myocardial cell on the Z axis is set to be sequentially performed on one point on the cell surface according to the time sequence according to the measurement requirement Measuring information, giving a set value of the number of times of measurement at one point on the cell surface, judging whether the set value is reached, sending a feedback signal to a D/A data converter 5 through a closed-loop feedback system, converting the feedback signal and then sending the converted feedback signal to a piezoelectric ceramic nano displacement platform controller 6, adjusting a sample platform above a piezoelectric ceramic nano displacement platform 2 to move in the Z-axis direction, obtaining the spatial position of one point on the cell surface of a single pulsating cardiomyocyte at any moment in a pulsating period through analysis if the set value is reached, moving an atomic force probe to the next position to continue measurement, scanning the single pulsating cardiomyocyte in the way, accurately positioning the spatial position of the single pulsating cardiomyocyte at any moment in the pulsating period, analyzing and obtaining the three-dimensional dynamic morphology of the single pulsating cardiomyocyte, sending all measurement data to a sample morphology image display 8 through a computer terminal control system 7 to be displayed, the system for realizing the three-dimensional dynamic topography measurement is called an atomic force microscopic imaging system.
As shown in FIGS. 2 and 3, the schematic diagram of the atomic force probe tracking the pulse of a single-beat cardiomyocyte is shown, wherein the laser 11, the four-quadrant photodetector 12, the atomic force probe 13, and the sample stage 14 are respectively a single-beat cardiomyocyte 15 in the contraction state and a single-beat cardiomyocyte 16 in the relaxation state. The atomic force probe 13 is used as a detection sensor capable of detecting weak force, and when the tip of the atomic force probe 13 approaches the surface of the single-beat cardiomyocyte 16 in the diastolic state or the single-beat cardiomyocyte 15 in the systolic state to several nanometers or less, the atomic force probe 13 is deformed due to the interaction of the tip with the force between the single-beat cardiomyocyte 16 in the diastolic state or the single-beat cardiomyocyte 15 in the systolic state. The laser 11 is used for focusing laser beams on the back end of the atomic force probe 13, the laser beams are reflected to the four-quadrant photoelectric detector 12, due to the fact that the atomic force probe 13 deforms, light spots generate displacement on the four-quadrant photoelectric detector 12, the four-quadrant photoelectric detector 12 converts light variation signals into electrical signals, and the morphology information of the sample is obtained after the electrical signals are amplified and calculated.
As shown in fig. 1, the present invention is implemented as:
(1) controlling a sample stage above a piezoelectric ceramic nano displacement platform 2 to move in the direction of an X, Y, Z axis, searching for single beating cardiomyocytes, moving an atomic force probe 1, the sample stage above the piezoelectric ceramic nano displacement platform 2, and accurately positioning the atomic force probe 1 on the single beating cardiomyocytes of the sample stage above the target piezoelectric ceramic nano displacement platform 2 according to an atomic force microscopic imaging system, a force feedback system and a closed-loop feedback system thereof;
(2) observing the state of the single-beat myocardial cell under the optical microscope module 5, moving the atomic force probe 1 and placing the tip of the atomic force probe right above the single-beat myocardial cell to be detected;
(3) setting measurement parameters, enabling the atomic force probe 1 to be in contact with the single-beat myocardial cell in a constant-force single-point multiple-acquisition mode according to the time sequence, and measuring the spatial position and the morphology of the single-beat myocardial cell at any moment in a beat period in real time in the constant-force multiple-acquisition mode according to the time sequence to realize measurement of three-dimensional dynamic morphology, so that the three-dimensional dynamic morphology of the single-beat myocardial cell is obtained on a nanometer scale.
As shown in FIG. 4, the atomic force probe used in the present invention is a silicon nitride probe, the micro-cantilever is triangular, the tip is conical, the radius of the tip is 7nm, the elastic constant of the micro-cantilever is 0.35N/m, and the length of the micro-cantilever is 95 μm. The conical design of the atomic force probe tip can be used to image cell surfaces with high resolution and low force under high humidity conditions. The damage to the sample is relatively less. Therefore, the probe can reduce the resistance of the probe in the solution during measurement, improve the moving speed of the probe in the solution during measurement, improve the contact stability of the probe tip and the single-beat cardiomyocyte, and improve the imaging precision, so that the more accurate spatial position and morphology of the single-beat cardiomyocyte at any moment in the beating period can be stably obtained, the measurement of the three-dimensional dynamic morphology is realized, and the three-dimensional dynamic morphology of the single-beat cardiomyocyte is obtained on the nanometer scale.
Claims (6)
1. A method for measuring the three-dimensional dynamic morphology of single-beat cardiac muscle cells by an atomic force probe is characterized by comprising the following steps: the method comprises the following steps of moving an atomic force probe, a sample stage, an atomic force microscopic imaging system, a force feedback system and a closed-loop feedback system of the atomic force microscopic imaging system to enable the atomic force probe to be accurately positioned on a single beating myocardial cell to be detected, measuring and determining the spatial position of one point on the cell surface at any moment in a mode of collecting the atomic force probe at a constant force single point for multiple times according to a time sequence, and scanning the single beating myocardial cell through the mode to realize the measurement of the three-dimensional dynamic morphology, wherein the method specifically comprises the following steps:
(1) controlling the nanometer displacement platform to move in the direction of an X, Y, Z axis, searching single-beat cardiac muscle cells, and moving the atomic force probe, the sample platform, the atomic force microscopic imaging system, the force feedback system and the closed-loop feedback system thereof to accurately position the atomic force probe on the single-beat cardiac muscle cells to be detected;
(2) observing the state of the single-beat myocardial cell under an optical microscope, and moving the atomic force probe to place the tip of the atomic force probe right above the single-beat myocardial cell to be detected;
(3) setting measurement parameters, enabling the atomic force probe to be in contact with the cell surface, adopting a constant-force single-point multiple-time acquisition mode according to a time sequence, determining the spatial position of one point on the cell surface at any moment, scanning the single-beat cardiac muscle cell through the acquisition mode, accurately positioning the position and the shape of the single-beat cardiac muscle cell at any moment in a beat period, and realizing the measurement of the three-dimensional dynamic shape, thereby obtaining the three-dimensional dynamic shape of the single-beat cardiac muscle cell on a nanometer scale.
2. The method for measuring the three-dimensional dynamic morphology of a single-beating cardiomyocyte with the atomic force probe according to claim 1, wherein: the atomic force probe is a silicon nitride probe, the micro-cantilever is triangular, the needle tip is conical, the radius of the tip of the needle tip is 5-10nm, the elastic constant of the micro-cantilever is 0.2-0.4N/m, and the length of the micro-cantilever is 90-100 microns.
3. The method for measuring the three-dimensional dynamic morphology of a single-beating cardiomyocyte with the atomic force probe according to claim 1, wherein: the constant-force single-point multi-acquisition mode is characterized in that a measurement parameter is set to enable an atomic force probe to measure surface height information of a single beating cardiomyocyte on a Z axis on a cell surface point by a constant force according to a time sequence, the spatial position of the cell surface point of the single beating cardiomyocyte at any moment in a beating period is obtained, the atomic force probe is moved to the next position to continue measurement, the single beating cardiomyocyte is scanned through the constant-force single-point multi-acquisition mode, the position and the shape of the single beating cardiomyocyte at any moment in the beating period are accurately positioned, and measurement of three-dimensional dynamic shapes is achieved, and the method specifically comprises the following steps:
(1) according to the measurement requirements, setting the surface height information measurement of the single-beat myocardial cell on the Z axis in time sequence at one point on the cell surface with constant force, and giving a set value of the measurement times at one point on the cell surface;
(2) judging whether the measurement frequency at one point on the cell surface reaches a set value or not through a closed-loop feedback system, if so, obtaining the spatial position of one point on the cell surface at any moment in a beating period of the single-beating myocardial cell through analysis, and moving the atomic force probe to the next position for continuous measurement;
(3) the single-beat cardiac muscle cell is scanned in the mode, the spatial position of the single-beat cardiac muscle cell at any moment in a beat period is accurately positioned, the three-dimensional dynamic morphology of the single-beat cardiac muscle cell is obtained through analysis, and the measurement of the three-dimensional dynamic morphology is realized.
4. The method for measuring the three-dimensional dynamic morphology of a single-beating cardiomyocyte with the atomic force probe according to claim 1, wherein: the measurement parameters in the step (3) are set as follows: setting the atomic force probe coarse needle to gradually approach to the single-beat myocardial cell to be detected in a step length of 30-40nm, stopping the coarse needle when the distance between the needle tip and the single-beat myocardial cell membrane is 5-10nm, simultaneously starting a constant force single-point multi-time acquisition mode of an atomic force microscopic imaging system, finely inserting the atomic force probe in the step length of 0.01-0.05nm, and tracking and measuring the spatial position and the morphology of the single-beat myocardial cell at any moment in a beating period in real time in the constant force multi-time acquisition mode.
5. The method for measuring the three-dimensional dynamic morphology of a single-beating cardiomyocyte with the atomic force probe according to claim 1, wherein: in the step (3), the atomic force probe is contacted with the single beating myocardial cell with constant force according to the atomic force microscopic imaging system, the force feedback system and the closed loop feedback system by setting the measurement parameters, when the single-beat myocardial cell contracts or expands, the atomic force probe moves along the Z-axis direction along with the single-beat myocardial cell, in the pulsation period of the single pulsation myocardial cell, the atomic force probe is always contacted with the single pulsation myocardial cell in a multiple acquisition mode with constant force according to the time sequence, so that the contact between the atomic force probe and the surface of the single pulsation myocardial cell membrane is stable, the measurement method is used for measuring and obtaining the spatial position and the morphology of the single-beat myocardial cell at any moment in the beat period, and a measurement mode with stable contact is constructed to realize the accurate measurement of the three-dimensional dynamic morphology, thereby obtaining the three-dimensional dynamic morphology of the single-beat myocardial cell on the nanometer scale.
6. An atomic force probe device for measuring the three-dimensional dynamic morphology of a single-beat myocardial cell is characterized by comprising: the device comprises an atomic force probe measuring cell module, a piezoelectric ceramic nano displacement platform, a four-quadrant photoelectric detector voltage acquisition module, an optical microscope module, a D/A data converter, a piezoelectric ceramic nano displacement platform controller, a computer terminal control system and a sample morphology image display; the atomic force probe measurement cell module comprises an atomic force probe, a single beating myocardial cell and a sample stage; the optical microscope module comprises an objective lens, an adapter and a zoom lens; the voltage acquisition module of the four-quadrant photoelectric detector comprises a four-quadrant photoelectric detector, a voltage acquisition module, a signal amplifier and an A/D converter; the computer control system controls the sample stage above the piezoelectric ceramic nano displacement platform to move in the direction of X, Y, Z axis, single beating cardiomyocyte is searched, the atomic force probe, the piezoelectric ceramic nano displacement platform, the sample stage and the force feedback system are moved, the atomic force probe is accurately positioned on the single beating cardiomyocyte on the sample stage above the target piezoelectric ceramic nano displacement platform, the state of the single beating cardiomyocyte is clearly observed by adjusting the adapter and the zoom lens through the optical microscope module, the atomic force probe is moved to place the tip of the atomic force probe right above the single beating cardiomyocyte to be detected, the atomic force probe in the atomic force probe cell measuring module is taken as a detecting sensor capable of detecting weak force, when the tip of the atomic force probe approaches the surface of the single beating cardiomyocyte to be detected to several nanometers or even smaller, the atomic force probe deforms due to the interaction of the tip and the single beating cardiomyocyte, focusing a laser beam on the back end of an atomic force probe by using a laser, reflecting the laser beam onto a four-quadrant photoelectric detector, generating deformation of the atomic force probe to cause light spots to generate offset on the four-quadrant photoelectric detector, converting the light variation signal into an electrical signal by the four-quadrant photoelectric detector, collecting the electrical signal by a collection module, amplifying the electrical signal by an amplifier, converting the electrical signal by an A/D converter, transmitting the electrical signal to a computer terminal control system, setting a constant force on one point on the cell surface by the computer control system according to measurement requirements, measuring surface height information of the single beating myocardial cell on a Z axis in time sequence, giving a set value of the number of times of measurement on one point on the cell surface, judging whether the set value is reached, transmitting a feedback signal to a D/A data converter by a closed-loop feedback system, converting the feedback signal and transmitting the feedback signal to a piezoelectric ceramic nano displacement platform controller, adjusting a sample stage above a piezoelectric ceramic nano displacement platform to move in the Z-axis direction, obtaining the spatial position of a point on the cell surface of a single-beat cardiomyocyte at any moment in a beat period through analysis if the sample stage reaches a set value, moving an atomic force probe to the next position to continue measurement, scanning the single-beat cardiomyocyte in the way, accurately positioning the spatial position of the single-beat cardiomyocyte at any moment in the beat period, obtaining the three-dimensional dynamic morphology of the single-beat cardiomyocyte through analysis, and transmitting all measurement data to a sample morphology image display through a computer terminal control system for display, so that a system for realizing the three-dimensional dynamic morphology measurement is called as an atomic force microscopic imaging system.
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