CN114184809B - Method and device for measuring three-dimensional dynamic morphology of single pulsating myocardial cell by atomic force probe - Google Patents
Method and device for measuring three-dimensional dynamic morphology of single pulsating myocardial cell by atomic force probe Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
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Abstract
The invention relates to a method and a device for measuring three-dimensional dynamic morphology of single-pulsation myocardial cells by an atomic force probe, wherein the atomic force probe is used for measuring the three-dimensional dynamic morphology of single-pulsation myocardial cells, the atomic force probe is precisely positioned on the single-pulsation myocardial cells to be measured 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, the atomic force probe is contacted with the surfaces of the single-pulsation myocardial cells, a constant force single-point time sequence multiple acquisition mode is adopted for determining the spatial position of one point on the surfaces of the cells at any moment, and the single-pulsation myocardial cells are scanned by the mode, so that the measurement of the three-dimensional dynamic morphology is realized. The invention can accurately position the position and the morphology of the single pulsating myocardial cell at any moment in the pulsating period to realize dynamic measurement so as to obtain the three-dimensional dynamic morphology of the single pulsating myocardial cell on the nanometer scale.
Description
Technical Field
The invention relates to a method and a device for measuring three-dimensional dynamic morphology of single pulsating myocardial cells by an atomic force probe, belonging to the technical field of engineering.
Background
Life sciences is one of the leading fields of this century, cells being the basic unit of life, there are many different kinds of cells in the human body, each cell 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 research on the morphology of the biological cells is helpful for people to understand the physiological state and process of the biological cells more clearly, accurately and comprehensively, and can also enable people to understand the individual differences of different cells in the cell population and the functions of some special cells better, so that the method has important significance for revealing the life mysterious. In the study of the three-dimensional dynamic morphology of cells, pulsating cells such as cardiomyocytes are the main subject of research in this field. The heart can be better understood by studying the three-dimensional dynamic morphology of the beating cells.
With rapid development of nano technology, ultra-high resolution and non-invasive imaging detection technology plays an important role in the scientific fields of biology, materials and the like. The advent of atomic force microscopy (Atomic Force Microscope, AFM for short) has provided the possibility to observe the ultrastructure of the surface of living cells. AFM uses a sharp tip integrated into the end of a cantilever beam to raster scan the sample to obtain the sample surface topography. The AFM can operate in a solution environment and has a spatial resolution on the order of nanometers, and this unique advantage makes it well suited for studying physiological activities on the nanoscale of living cell surfaces. The application of AFM to study biological samples (living cells, membrane proteins in natural state) in natural state brings a great deal of new knowledge to cell biology, including understanding the tissue and dynamic behavior of cell membranes, observing the structure and activity of organelles, and analyzing the dynamic structure of protein molecules, etc. on a nanometer scale. Because of the powerful functions of AFM, AFM has become an important experimental tool in cell biology, being an important supplement to traditional optical microscopy, electron microscopy and X-ray crystallography. However, optical imaging is mainly adopted in the current pulsating myocardial cell imaging research, the imaging resolution is low, the accuracy is poor, the fine observation and analysis of sample properties on the nanometer scale can not be realized, and the biological research is not facilitated. The scanning probe microscope has real space resolution capability of nanometer or even atomic scale, but the traditional scanning probe microscope technology can only observe the surface characteristics of a solid sample, but can not obtain the information of a living cell sample.
In the previous research of the applicant, zhang Saiwei et al utilize atomic force nano imaging technology to combine an electrophysiological property measurement method and a mechanical property measurement method to measure electrophysiological property and mechanical property of single-pulse myocardial cells when the single-pulse myocardial cells are pulsed, the single-pulse myocardial cell action potential of a newborn rat is obtained by measurement, the record of single-pulse myocardial cell action potential and contractile force signals under two measurement modes (constant force tracking measurement mode and non-constant force contact measurement mode) is successfully realized, a patent named as a method for measuring single-pulse myocardial cell action potential and pulse force by an atomic force microscope is applied on the basis of the experiment, the electrophysiological property and mechanical property of all points on the surface of a cell membrane cannot be accurately measured, and the measurement of the three-dimensional dynamic morphology of the single-pulse myocardial cells cannot be realized. (see Zhang Saiwei. Research on robotic nanomanipulation of living cells and electrical property detection techniques [ D ]. University of Changchun-tally, 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, organelles and the like and reflect the physiological state of cells, so that the imaging observation and the study of the nano structure are carried out on single pulsating myocardial cells, and the problems of pending cell functions, physiological behaviors of cell individuals and groups, cell variation, biochemistry, chemical biology, medicine and the like of cell communication can be solved. According to 2007 Ji Hao et al, based on the optimum imaging conditions of the cells under AFM research, the optimum imaging conditions of the liver cancer SMMC-7721 cells in the atmosphere and the solution environment are obtained through analysis and research of AFM images of the human liver cancer SMMC-7721 cells under different processing conditions and testing conditions, and an experimental method for observing living cells by AFM is established (see Ji Hao, liu Ying, zhuang Lena, zhu Jie, sun Runan. Atomic force microscope optimum imaging conditions research [ J ]. Photonics report, 2007). In 2005 Wang Lijuan et al AFM was used to study the different imaging conditions in a physiological solution environment of CHO cells slightly cured with 1% glutaraldehyde, respectively. Experimental results show that the probe with the elastic coefficient of 0.06N/m can be selected to scan cells in physiological environment to obtain good effect, and the scanning speed is preferably 2-3 Hz. The method provides a partial basis for the optimal imaging condition of living cells (see Wang Lijuan, zhang Yingge, zhang Sa, etc. the atomic force microscope is used for researching the imaging condition of living cells in physiological solution [ J ]. Electron microscopic report, 2005), AFM has reached atomic level and molecular level resolution capability, can work under natural conditions or physiological conditions, can dynamically observe the physiological activity of cells, and is the technology and method which are most suitable for imaging and observing single cells at present.
Disclosure of Invention
The invention solves the technical problems: the method and the device for measuring the three-dimensional dynamic morphology of the single pulsating myocardial cell by the atomic force probe are provided, the cell position is precisely positioned according to the atomic force microscopic imaging system, the force feedback system and the closed loop feedback system of the atomic force microscopic imaging system, the feedback information of the acting force of the atomic force probe tip and the acting force of a sample, the spatial position and morphology of a single pulsating myocardial cell at any moment on the cell surface in a pulsating period are determined by a constant force single point time sequence multiple acquisition mode, and the measurement of the three-dimensional dynamic morphology is realized, so that the three-dimensional dynamic morphology of the single pulsating myocardial cell is obtained on a nanometer scale.
The aim of the invention can be achieved by the following technical measures: a method and a device for measuring three-dimensional dynamic morphology of single pulsating myocardial cells by an atomic force probe are characterized in that: the atomic force probe is contacted with the surface of a single beating myocardial cell in a constant force single point time sequence multiple acquisition mode through program control, the spatial position and the morphology of the single beating myocardial cell at any time point on the surface of the single beating myocardial cell in a beating period are determined, the single beating myocardial cell is scanned in the mode, and the measurement of the three-dimensional dynamic morphology is realized, so that the three-dimensional dynamic morphology of the single beating myocardial cell is obtained on a nanometer scale, and the method comprises the following steps:
(1) Controlling the nano displacement platform to move in the X, Y, Z axial direction, searching single pulsating myocardial cells, and moving the atomic force probe, the sample stage, the atomic force microscopic imaging system, the force feedback system and the closed loop feedback system of the atomic force microscopic imaging system, so that the atomic force probe is accurately positioned on the single pulsating myocardial cells to be detected;
(2) Under an optical microscope, observing the state of single-pulse myocardial cells, and moving an atomic force probe to place the tip of the atomic force probe right above the single-pulse myocardial cells to be detected;
(3) The measurement parameters are set, so that the atomic force probe is in contact with the cell surface, a constant force single point is adopted for multiple time sequence acquisition modes, the spatial position of one point on the cell surface at any time is determined, single-pulse myocardial cells are scanned through the acquisition modes, the measurement of three-dimensional dynamic morphology is realized, the position and morphology of the single-pulse myocardial cells at any time in a pulse period are accurately positioned, the dynamic measurement is realized, and the three-dimensional dynamic morphology of the single-pulse myocardial cells is obtained on a nanometer scale.
The atomic force probe is a silicon nitride probe, the micro-cantilever is triangular, the tip of the micro-cantilever is conical, the radius of the 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 mu m.
The constant force single point time sequence multiple acquisition mode is characterized in that the atomic force probe is used for measuring the surface height information of single pulsating myocardial cells on a Z axis in time sequence at one point of the cell surface by setting measurement parameters, so that the spatial position of the single pulsating myocardial cells at any point of the cell surface in a pulsating period is obtained, then the single pulsating myocardial cells are moved to the next position to repeatedly perform the operation, the single pulsating myocardial cells are scanned in the mode, the position and the shape of the single pulsating myocardial cells at any point of the pulsating period are accurately positioned, and the measurement of the three-dimensional dynamic shape is realized, and the method specifically comprises the following steps:
(1) According to the measurement requirement, setting to measure the surface height information of single beating myocardial cells 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 times of a point on the cell surface reaches a set value or not through a closed loop feedback system, if so, analyzing to obtain the spatial position of a point on the cell surface of a single pulsating myocardial cell at any moment in a pulsating period, and moving an atomic force probe to the next position to continue measurement;
(3) By adopting the method, the single pulsating myocardial cells are scanned, the spatial position of the single pulsating myocardial cells at any moment in the pulsating period is accurately positioned, the three-dimensional dynamic morphology of the single pulsating myocardial cells is obtained by analysis, and the measurement of the three-dimensional dynamic morphology is realized.
Setting measurement parameters in the step (3), setting the needle inserting precision of the atomic force probe, wherein the specific implementation mode is that the atomic force probe coarse needle inserting gradually approaches to single pulsating myocardial cells to be measured at a step length of 30nm, stopping coarse needle inserting when the distance between a needle tip and a single pulsating myocardial cell membrane is 5nm, starting a constant force single point time sequence multiple acquisition mode of an atomic force microscopic imaging system, and the atomic force probe fine needle inserting at a step length of 0.02nm, and tracking and measuring the spatial position and the shape of the single pulsating myocardial cells at any moment in a pulsating period in real time in a constant force time sequence multiple acquisition mode, so as to realize the measurement of three-dimensional dynamic shapes, thereby obtaining the three-dimensional dynamic shapes of the single pulsating myocardial cells at a nanometer scale.
In the step (3), by setting measurement parameters, according to an atomic force microscopic imaging system, a force feedback system and a closed loop feedback system thereof, an atomic force probe is in constant force contact with a single pulsating myocardial cell, when the single pulsating myocardial cell contracts or expands, the atomic force probe moves along the Z axis direction along with the single pulsating myocardial cell, and in a pulsating period of the single pulsating myocardial cell, the atomic force probe is always in contact with the single pulsating myocardial cell in a constant force time sequence multiple acquisition mode, so that the contact between the atomic force probe and the surface of a single pulsating myocardial cell membrane is stable, the space position and the shape of the single pulsating myocardial cell at any moment in the pulsating period are obtained by measurement through the measurement method, a stable contact measurement mode is constructed, and the accurate measurement of the three-dimensional dynamic shape is realized, thereby obtaining the three-dimensional dynamic shape of the single pulsating myocardial cell on a nanometer scale.
The piezoelectric ceramic nano displacement platform controller drives a sample platform above the piezoelectric ceramic nano displacement platform to move in the X, Y, Z axial direction, so that when an atomic force probe tip moves on the surface of a sample, the atomic force probe deforms a micro cantilever due to the force interaction between the tip and single pulsating myocardial cells, a laser is utilized to focus a laser beam on the rear end of the atomic force probe, the laser beam is reflected to a four-quadrant photoelectric detector, a light spot generates an offset on the four-quadrant photoelectric detector due to the deformation of the atomic force probe, the four-quadrant photoelectric detector converts an optical change signal into an electrical signal, the electrical signal is used as a feedback signal in a force feedback system and is used as an internal adjusting signal, and the piezoelectric ceramic nano displacement platform is driven to move appropriately, so that a constant acting force is kept between the sample and the tip, and a system for adjusting the acting force is called a force feedback system.
The method comprises the steps of collecting and amplifying electrical signals obtained through measurement through a four-quadrant photoelectric detector voltage collecting module, converting the electrical signals through an A/D converter, transmitting the electrical signals to a computer terminal control system, setting to conduct surface height information measurement on single pulsating myocardial cells on a Z axis in time sequence at one point on the cell surface with constant force, giving a set value of measurement times at one point on the cell surface, judging whether the set value is reached or not, transmitting feedback signals which are 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 the piezoelectric ceramic nano displacement platform to move in the Z axis direction, so that a closed loop participation control system is formed.
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 is not less than half of the wavelength of incident light during measurement, so the method and the device for measuring the three-dimensional dynamic morphology of single pulsating myocardial cells have nanoscale spatial resolution and good accuracy compared with the optical imaging. Can realize the fine observation and analysis of the appearance of the sample on the nanometer scale, and is beneficial to the research of the size, shape, structure and function of cells.
(2) The sample in the electron microscope must be observed under a vacuum environment, and thus living cells cannot be observed. The electron of the electron microscope damages the biological sample greatly, and the protection of the dye damages the form of the sample. The method and the device for measuring the three-dimensional dynamic morphology of the single pulsating myocardial cells can carry out high-resolution imaging on living cells in a physiological environment, and the imaging resolution reaches atomic and molecular levels. 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 a traditional Atomic Force Microscope (AFM), the method and the device for measuring the three-dimensional dynamic morphology of the single-pulsation myocardial cell can accurately position the cell, have high flexibility in operation, can determine the spatial position and morphology of a point on the cell surface at any moment in the pulsation period of the single-pulsation myocardial cell, realize dynamic measurement, and acquire the three-dimensional dynamic morphology of the single-pulsation myocardial cell on a nanometer scale.
(4) In the previous research of the applicant, zhang Saiwei et al utilize atomic force nano imaging technology to combine an electrophysiological property measuring method and a mechanical property measuring method to measure electrophysiological property and mechanical property of single-pulse myocardial cells when in pulse, and apply for a patent named a method for measuring action potential and pulse force of single-pulse myocardial cells by an atomic force microscope based on the experiment, in the patent, a constant force/non-constant force tracking mode is adopted to measure electrophysiological property and mechanical property at a single point, the electrophysiological property and mechanical property of all points on the surface of a cell membrane cannot be accurately measured and obtained, and measurement of three-dimensional dynamic morphology of single-pulse myocardial cells cannot be realized.
Drawings
FIG. 1 is a diagram of a method and apparatus for measuring three-dimensional dynamic morphology of single pulsating cardiomyocytes using an atomic force probe according to the present invention;
FIG. 2 is a schematic diagram of a single beat cardiomyocyte measurement in the atomic force probe tracking contracted state of the present invention;
FIG. 3 is a schematic diagram of a single beat cardiomyocyte measurement in the atomic force probe tracking diastolic state of 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
Referring to fig. 1, a schematic block diagram of the present invention is shown, wherein an atomic force probe measurement cell module 1 comprises an atomic force probe, a single pulsating myocardial cell, a sample stage, a laser and a four-quadrant photoelectric detector, a piezoelectric ceramic nano displacement platform 2, a four-quadrant photoelectric detector voltage acquisition module 3 comprises a signal amplifier and an a/D converter, an optical microscope module 4 comprises 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 morphology 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 axial direction, searches single pulsating myocardial cells, moves the atomic force probe, the piezoelectric ceramic nano displacement platform 2, the sample stage and accurately positions the atomic force probe on the single pulsating myocardial cells on the sample stage above the target piezoelectric ceramic nano displacement platform 2 according to the force feedback system, and adjusts the adapter and the zoom lens through the optical microscope module 4 so as to clearly observe the states of the single pulsating myocardial cells, and moves the atomic force probe to place the tip of the single pulsating myocardial cells to be detected right above the single pulsating myocardial cells. The atomic force probe in the atomic force probe measuring cell module 1 is used as a weak force detection sensor, when the tip of the atomic force probe approaches the surface of a single pulsating myocardial cell to a plurality of nanometers or even smaller, the atomic force probe generates deformation due to the force interaction between the tip and the single pulsating myocardial cell, a laser is utilized to focus a laser beam on the back end of the atomic force probe, thereby the laser beam is reflected to a four-quadrant photoelectric detector, as 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 a light change signal into an electrical signal, then the electrical signal is acquired by a voltage acquisition module 3 of the four-quadrant photoelectric detector, amplified and converted by an A/D converter and then transmitted to a computer terminal control system 7, according to the measurement requirement, the surface height information of the single pulsating myocardial cell is measured on a Z axis in time sequence by a constant force at one point on the surface of the cell, whether the set value is reached or not is judged, a feedback signal is transmitted to a D/A data converter 5 through a closed loop feedback system to be converted and then transmitted to a ceramic controller, the ceramic nano-meter 6, the position of the single pulsating myocardial cell is moved to a single-pulse position in a space in a single-cycle mode, the single pulsating myocardial cell is obtained, the space is moved to a single pulsating position is moved in a single-pulse space position in a single-cycle, and the single pulsating myocardial cell is moved to a single-phase position is measured in a space position in a space cycle mode, and the single pulsating myocardial cell is measured by a single platform is moved in a single-phase position, if the space is moved in a single platform is in a space, if the space is moved in a space, if the position is measured by the single pulsating position is measured, and analyzing and obtaining the three-dimensional dynamic morphology of the single pulsating myocardial cells, and transmitting all measurement data to a sample morphology image display 8 for display through a computer terminal control system 7, so that the system for realizing the three-dimensional dynamic morphology measurement is called an atomic force microscopy imaging system.
As shown in fig. 2 and 3, the atomic force probe tracks the single-pulse cardiomyocyte pulse schematic diagram, wherein the laser 11, the four-quadrant photodetector 12, the atomic force probe 13 and the sample stage 14 are single-pulse cardiomyocyte 15 in a contracted state and single-pulse cardiomyocyte 16 in a relaxed state. The atomic force probe 13 serves as a weak force detection sensor capable of detecting weak force, and when the tip of the atomic force probe 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 by the force interaction between the tip and the single-beat cardiomyocyte 16 in the diastolic state or the single-beat cardiomyocyte 15 in the systolic state. The laser 11 is utilized to focus the laser beam on the tail end of the back surface of the atomic force probe 13, so that the laser beam is reflected to the four-quadrant photoelectric detector 12, the four-quadrant photoelectric detector 12 converts the optical change signal into an electrical signal due to the displacement of a light spot on the four-quadrant photoelectric detector 12 caused by deformation of the atomic force probe 13, and the morphological information of the sample is obtained after amplification and calculation processing.
As shown in fig. 1, the present invention is implemented as:
(1) Controlling a sample stage above the piezoelectric ceramic nano displacement platform 2 to move in the X, Y, Z axial direction, searching single pulsating myocardial cells, moving the atomic force probe 1, the sample stage above the piezoelectric ceramic nano displacement platform 2 and accurately positioning the atomic force probe 1 to the single pulsating myocardial cells 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 of the atomic force microscopic imaging system;
(2) Under the optical microscope module 5, observing the state of single-pulse myocardial cells, and moving the atomic force probe 1 to place the tip of the atomic force probe right above the single-pulse myocardial cells to be detected;
(3) The method comprises the steps of setting measurement parameters, enabling an atomic force probe 1 to be in contact with single pulsating myocardial cells in a constant force single-point time sequence multiple-time acquisition mode, measuring the spatial position and the shape of the single pulsating myocardial cells at any moment in a pulsating period in real time in the constant force time sequence multiple-time acquisition mode, and realizing measurement of three-dimensional dynamic shapes, so that the three-dimensional dynamic shapes of the single pulsating myocardial cells are obtained on a nanometer scale.
As shown in FIG. 4, the atomic force probe used in the invention is a silicon nitride probe, the micro-cantilever is triangular, the tip of the micro-cantilever 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 mu m. The conical design of the atomic force probe tip can be used to image the cell surface with less force under high humidity conditions with high resolution. The damage to the sample is relatively smaller. Therefore, the probe can reduce the resistance of the probe when the probe is measured in the solution, improve the moving speed of the probe when the probe is measured in the solution, improve the contact stability of the probe tip and single pulsating myocardial cells, and improve the imaging precision, thereby stably obtaining more accurate spatial position and morphology of the single pulsating myocardial cells at any moment in the pulsating period, realizing the measurement of three-dimensional dynamic morphology, and further obtaining the three-dimensional dynamic morphology of the single pulsating myocardial cells on the nanometer scale.
Claims (2)
1. A method for measuring three-dimensional dynamic morphology of single pulsating cardiomyocytes with an atomic force probe, the method using an apparatus comprising: the system comprises an atomic force probe measurement 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 pulsating myocardial cell and a sample stage; the optical microscope module comprises an objective lens, an adapter and a zoom lens; the four-quadrant photoelectric detector voltage acquisition module comprises a four-quadrant photoelectric detector, a voltage acquisition module, a signal amplifier and a D/A data converter; the piezoelectric ceramic nano displacement platform controller drives a sample platform above the piezoelectric ceramic nano displacement platform to move in the X, Y, Z axial direction, so that an atomic force probe tip moves on the surface of the sample, the atomic force probe deforms a micro cantilever due to the force interaction between the tip and single pulsating myocardial cells, a laser is utilized to focus a laser beam on the back end of the atomic force probe, the laser beam is reflected to a four-quadrant photoelectric detector, a light spot generates an offset on the four-quadrant photoelectric detector due to the deformation of the atomic force probe, the four-quadrant photoelectric detector converts an optical change signal into an electrical signal, the electrical signal is taken as a feedback signal in a force feedback system and is taken as an internal adjusting signal, and the piezoelectric ceramic nano displacement platform is driven to move appropriately, so that a constant acting force is kept between the sample and the tip, and a system for adjusting the acting force is formed as a force feedback system; the method comprises the steps of collecting and amplifying electrical signals obtained by measurement through a four-quadrant photoelectric detector voltage collecting module, converting the electrical signals through a D/A data converter, transmitting the electrical signals to a computer terminal control system, setting to measure surface height information of single pulsating myocardial cells on a Z axis in a time sequence at one point on the cell surface with constant force, giving a set value of measurement times at one point on the cell surface, judging whether the set value is reached, transmitting feedback signals which are compared with the set value to the D/A data converter, converting the feedback signals and transmitting the feedback signals to a piezoelectric ceramic nano displacement platform controller, regulating a sample platform above the piezoelectric ceramic nano displacement platform to move in the Z axis direction to form a closed loop, and enabling a system participating in control to be called a closed loop feedback system; the method is characterized in that: moving an atomic force probe, a sample stage, an atomic force microscopic imaging system, a force feedback system and a closed loop feedback system, so that the atomic force probe is accurately positioned on a single pulsating myocardial cell to be detected, measuring and determining the spatial position of a point on the surface of the cell at any moment by adopting the atomic force probe in a constant force single point time sequence multiple acquisition mode, scanning the single pulsating myocardial cell in the mode, and realizing the measurement of three-dimensional dynamic morphology, and specifically comprising the following steps:
(1) Controlling the nano displacement platform to move in the X, Y, Z axial direction, searching single pulsating myocardial cells, and moving the atomic force probe, the sample stage, the atomic force microscopic imaging system, the force feedback system and the closed loop feedback system of the atomic force microscopic imaging system, so that the atomic force probe is accurately positioned on the single pulsating myocardial cells to be detected;
(2) Under an optical microscope, observing the state of single-pulse myocardial cells, and moving an atomic force probe to place the tip of the atomic force probe right above the single-pulse myocardial cells to be detected;
(3) Setting measurement parameters, according to an atomic force microscopic imaging system, a force feedback system and a closed loop feedback system, the atomic force probe is contacted with a single pulsating myocardial cell with constant force, when the single pulsating myocardial cell contracts or expands, the atomic force probe moves along the Z axis direction along with the single pulsating myocardial cell, and in the pulsation period of the single pulsating myocardial cell, the atomic force probe is always contacted with the single pulsating myocardial cell in a constant force single point time sequence multiple acquisition mode, so that the contact between the atomic force probe and the surface of the single pulsating myocardial cell membrane is stable, the spatial position of a point on the cell surface at any moment is determined, the single pulsating myocardial cell is scanned in the acquisition mode, the position and the shape of the single pulsating myocardial cell at any moment in the pulsation period are accurately positioned, a measurement mode with stable contact is constructed, and the measurement of three-dimensional dynamic shape is realized, so that the three-dimensional dynamic shape of the single pulsating myocardial cell is obtained on the nanometer scale;
the constant force single point time sequence multiple acquisition mode is characterized in that the surface height information of single pulsating myocardial cells on a Z axis is measured by an atomic force probe according to the time sequence of a constant force at a cell surface point by setting measurement parameters, the spatial position of the single pulsating myocardial cells at any time at the cell surface point in a pulsating period is obtained, the atomic force probe is moved to the next position to continue to measure, the single pulsating myocardial cells are scanned by the mode, the position and the morphology of the single pulsating myocardial cells at any time in the pulsating period are accurately positioned, and the measurement of the three-dimensional dynamic morphology is realized, and the method specifically comprises the following steps:
(1) According to the measurement requirement, setting to measure the surface height information of single beating myocardial cells 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 times of a point on the cell surface reaches a set value or not through a closed loop feedback system, if so, analyzing to obtain the spatial position of a point on the cell surface of a single pulsating myocardial cell at any moment in a pulsating period, and moving an atomic force probe to the next position to continue measurement;
(3) The single pulsating myocardial cell is scanned in the mode, the spatial position of the single pulsating myocardial cell at any moment in the pulsating period is accurately positioned, the three-dimensional dynamic morphology of the single pulsating myocardial cell is obtained through analysis, and the measurement of the three-dimensional dynamic morphology is realized;
the atomic force probe is a silicon nitride probe, the micro-cantilever is triangular, the tip of the micro-cantilever is conical, the radius of the 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 mu m.
2. The method for measuring the three-dimensional dynamic morphology of single pulsating cardiomyocytes by using the atomic force probe according to claim 1, wherein: the measurement parameters in the step (3) are set as follows: setting an atomic force probe coarse needle feeding to gradually approach to single pulsating myocardial cells to be detected at a step length of 30-40nm, stopping the coarse needle feeding when the distance between a needle tip and the single pulsating myocardial cell membrane is 5-10nm, starting a constant force single point time sequential multiple acquisition mode of an atomic force microscopic imaging system, performing fine needle feeding by the atomic force probe at a step length of 0.01-0.05nm, and tracking and measuring the spatial position and morphology of the single pulsating myocardial cells at any moment in a pulsating period in real time in the constant force single point time sequential multiple acquisition mode.
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