CN111122913B - Super-alignment force clamp experiment method based on biomembrane mechanics probe system - Google Patents
Super-alignment force clamp experiment method based on biomembrane mechanics probe system Download PDFInfo
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Abstract
The invention discloses a super-standard force clamp experimental method based on a biomembrane mechanical probe system. The power pincers experiment relates to and has probe end and target end, and the probe end is including probe pellet, erythrocyte, its characterized in that: in the biomembrane mechanical probe force clamp experiment, a reference small ball is added at a probe end, the reference small ball is adhered to the end part of a first micro-pipette for sucking red blood cells through the strong interaction between protein molecules, the relative distance between the reference small ball positioned at the side boundary close to the red blood cells and the probe small ball positioned at the side boundary close to the red blood cells is tracked in real time, and the movement of a target end is controlled through a feedback algorithm to perform the force clamp experiment. According to the invention, by adding the reference beads and developing a 'double-boundary' force tracking mode based on a biomembrane mechanical probe system, the accuracy of the force acting on the molecular bond is obviously improved, accurate and stable acting force can be applied to the molecular bond with a slow dissociation rate, the recording time limit of the binding time of the molecular bond is obviously prolonged, and long-time recording is carried out.
Description
Technical Field
The invention relates to a quantitative detection method for detecting the dissociation rate of interaction between biological macromolecules, in particular to a super-alignment force clamp experimental method based on a biological membrane mechanics probe system.
Background
With the development of the monomolecular force spectrum technology, the regulation and control rules of various biological materials on monomolecular bonds have been analyzed by the techniques such as an atomic force microscope, optical tweezers, magnetic tweezers, a biomembrane mechanical probe and the like at the monomolecular level. The biological membrane mechanics probe (BFP) system has the advantages of small elasticity coefficient, low operation difficulty, easy operation on living cells and the like, and is particularly suitable for in-situ detection on the interaction of biological macromolecules on the surfaces of the living cells. As an important component of a dynamic force spectrum experiment, the 'force clamp' experiment quickly loads the acting force acting on the molecular bond to a set value, observes and collects the binding time of the single molecular bond under the action of the set force value, thereby directly obtaining the stress regulation rule of the molecular bond without model fitting, and obtaining the dissociation rate (k) of the molecular bond under the '0' force condition according to 'bell' model fittingoff). Therefore, the 'force clamp' experiment based on the biological membrane mechanical probe system is particularly suitable for in-situ detection of the dissociation rate of the molecular bond related to the membrane protein.
Although the bio-mechanical force clamp experiment has detected a variety of transient intermolecular interactions with faster off-rates, it is still not applicable to the study of intermolecular interactions with slower off-rates. This is because, in the absence of a force feedback control function during the detection process, various perturbations lead to a continuous shift in the force values acting on the molecular bonds over a longer binding time of the molecular bonds; in addition, in the recording process of the binding time of the overlong molecular bond, the probe end can also generate overall drift, even if the position of the small ball at the probe end is maintained at a set position, the force actually acting on the molecular bond can also generate larger deviation, and finally, the accuracy of the measurement result is poor, and even an error conclusion is obtained.
Therefore, the improvement of the stability and accuracy of the force acting on the molecular bond is the first condition for accurately detecting the dissociation rate of the stronger intermolecular interaction by applying the 'force clamp' technology based on a biomembrane mechanical probe system.
Disclosure of Invention
In order to solve the problems in the background art, the invention aims to provide an over-standard force clamp experimental method based on a biomembrane mechanical probe system.
According to the invention, a reference small ball is additionally added in a 'force clamp' experiment to correct the overall drift of a probe end, so that the accuracy of force value recording is improved, a PID feedback control algorithm is embedded in a 'molecular bond binding time recording' stage in the 'force clamp' experiment control, the force value recorded in the 'molecular bond binding time recording' stage is stably 'clamped' at a set position, and finally, the stable and accurate clamping of the force acting on the molecular bond is realized.
In order to achieve the purpose, the invention adopts the technical scheme that:
in the biomembrane mechanical probe force clamp experiment, a probe end and a target end are provided, the probe end comprises a probe small ball and red blood cells, a reference small ball is added at the probe end and is adhered to the end part of a first micro-sucker for sucking the red blood cells through the interaction between protein molecules, and the relative distance between the reference small ball positioned at the side boundary close to the red blood cells and the probe small ball positioned at the side boundary close to the red blood cells is tracked in real time to control the movement of the target end so as to carry out the force clamp experiment.
The method comprises the following specific steps:
1) an initialization stage:
the piezoelectric motion platform controls the target end to be far away from the probe end to be in an initial position and to be static, at the moment, the red blood cell is in an initial state, the distance between two boundaries of the reference small ball, which are close to one side boundary of the red blood cell, and the distance between two boundaries of the probe small ball, which are close to one side boundary of the red blood cell, are used as relative distances, the relative distances are tracked in real time through high-speed addition shooting, the average value of the relative distances recorded within 0.2 second is used as an initial relative distance, and then the;
2) and (3) an impact stage: monitoring the relative distance in real time, driving the cells/globules at the target end to move close to the red blood cells by the piezoelectric motion platform through the second micropipette at a preset speed, pushing the probe globules to compress the red blood cells in a centering manner until the relative distance is reduced by the preset compression distance, and entering the next stage;
3) and (3) a contact stage: monitoring the relative distance in real time, keeping the piezoelectric motion platform static, enabling the probe ball to be in stable contact with the cell/ball, and entering the next stage after preset contact time;
4) and (3) withdrawal stage: the piezoelectric motion platform drives the cells/small balls at the target end to withdraw and move away from the red blood cells at a preset speed through the second micropipette;
in the withdrawal process, monitoring the relative distance, if the relative distance is increased by the length corresponding to the preset force value compared with the initial relative distance, the force value is the value of the acting force between molecular bonds, namely the red blood cells are stretched by the set length, the experiment is an adhesion event, the piezoelectric motion platform immediately stops the current withdrawal motion, and the stage of combining time recording is entered;
if the relative distance is recovered to the initial relative distance and is not stretched, directly entering 6) a resetting stage;
5) and (3) combining a time recording stage: monitoring the relative distance in real time, and meanwhile, implementing feedback compensation by the piezoelectric motion platform according to the deviation between the relative distance corresponding to the preset force value and the actual relative distance until the relative distance is restored to the initial relative distance again, namely, the molecular bond is automatically broken, and entering a resetting stage;
the relative distance corresponding to the preset force value is the length obtained by adding the preset force value to the initial relative distance, but the tail end of the first micropipette deviates from the offset due to environmental disturbance, so that the actual relative distance deviates from the relative distance corresponding to the preset force value, and the relative distance is stabilized at the relative distance corresponding to the preset force value through real-time feedback compensation.
6) A reset phase: and monitoring the relative distance in real time, withdrawing the piezoelectric motion platform to the initial position, finishing the current cycle, storing the data of the relative distance, the time and the like of the current experiment, and preparing the next cycle.
In the biomembrane mechanical probe system, the action of the red blood cells is a force sensor, the force sensor is similar to a linear spring in a certain force range, and the change of the relative distance can be converted into a force value acting on the molecular bond in real time according to the elastic coefficient of the red blood cells.
The invention adds a reference small ball in the experiment of 'force clamp', utilizes the interaction between protein molecules to stick on the tip of a micro-pipette for sucking red blood cells, accurately tracks the force acting on the molecular bond in a 'double-boundary' tracking mode, adds the feedback control function of the force and can stably and accurately clamp the force acting on the molecular bond.
In the step 5), the relative distance monitored in real time is used as an actual relative distance, the actual relative distance is subjected to filtering processing by a filter of a PID controller and then subtracted from a reference relative distance corresponding to a preset force value to obtain a deviation, the deviation is input into the PID controller, and a deviation control quantity output by the PID controller is input into a motion module for controlling the piezoelectric motion platform to execute feedback compensation motion.
The surface of the first micropipette adsorbs Spy-tag protein molecules, the surface of the reference small ball is coated with the Spy-catcher protein molecules, and the reference small ball is fixedly adhered to the side surface of the end part of the first micropipette through the interaction between the Spy-tag and the Spy-catcher protein molecules.
The diameter of the reference small ball is larger than that of the probe small ball, and the reference small ball can be visually distinguished through the size of the small ball in the experiment process.
The reference small ball is a glass ball.
In the force clamp experiment, 3 times of the initial data sampling time interval is used as a data recording time interval to sample the force value data and record the force value data in a register, and when the molecular bond binding time exceeds 5 seconds, the data recording time interval is increased to 20 times of the sampling time interval; when the number of times of recording data exceeds 5000 times, the data recording time interval is further increased by 5 times.
The invention has the beneficial effects that:
according to the invention, the accuracy of the force acting on the molecular bond is obviously improved by adding the reference small ball and developing a 'double-boundary' force tracking mode based on a biomembrane mechanics probe system; the feedback control of the embedding force in the stage of recording the molecular bond combination time in the experiment control of the force clamp realizes the stable clamping of the force acting on the molecular bond in the molecular bond combination time in the experiment of the force clamp; and the recording time interval can be dynamically adjusted, and the recording time limit of the molecular bond binding time is obviously prolonged.
The invention mainly aims at the experiment of regulating and controlling the dissociation rate under the action of stronger intermolecular interaction in the field of life science, and can more accurately and effectively obtain the change condition of the combination time of the single molecular bond under the action of biological force. Compared with the original 'force clamp' experiment, the 'force clamp' experiment related by the invention has the following advantages:
1) the force acting on the molecular bond in the binding time is more accurately and stably clamped at a set force value;
2) the recording time limit of the molecular bond binding time is prolonged to more than 200 seconds.
Therefore, the invention can apply accurate and stable acting force to the molecular bond with slower dissociation rate, record the binding time of the molecular bond for a long time, and is particularly suitable for the in-situ detection of the dissociation rate stress regulation rule of stronger intermolecular interaction.
Drawings
FIG. 1 is a schematic diagram of a biomembrane based mechanical probe system according to the present invention.
Fig. 2 is a schematic diagram of feedback control according to the present invention.
Fig. 3 is an example of dynamically adjusting data recording time in accordance with the present invention.
Fig. 4 is an example of data recorded from a raw biomembrane mechanical probe experiment.
In the figure: 1. the method comprises the following steps of firstly, preparing a first micropipette, 2, red blood cells, 3, a probe ball, 4, cells/balls, 5, a second micropipette, 6, a piezoelectric motion platform, 7, a reference ball, 8, a probe ball boundary, 9, a reference ball boundary, 10, a relative distance, 11, a force-time data example of an ultra-accurate 'force clamp' technology, and 12, a recording time interval-recording time data example of the ultra-accurate 'force clamp' technology.
Detailed Description
The invention is further illustrated by the following figures and examples.
As shown in fig. 1, the embodied bio-membrane mechanical probe system comprises a probe end and a target end, wherein the probe end comprises a first micro-pipette 1, a red blood cell 2, a probe bead 3 and a reference bead 7, and the target end comprises a cell/bead 4 and a second micro-pipette 5; the end part of the first micropipette 1 sucks red blood cells 2, the side surface of the end part of the first micropipette 1 is stably connected with a reference small ball 7 through strong interaction among protein molecules, the end part of the second micropipette 5 sucks cells/small balls 4, the cells/small balls 4 express/are coated with a target protein molecule, if the cells 4 express a target protein molecule, and if the cells 4 are coated with a target protein molecule; the surface of the probe bead 3 is coated with another target protein molecule, and the probe bead 3 is connected to the front end of the red blood cell 2 through strong intermolecular interaction (such as interaction between streptavidin and biotin); the second micro-pipette 5 sucks the cell/pellet 4 and is connected to the piezoelectric motion platform 6, and the piezoelectric motion platform 6 drives the second micro-pipette 5 to move so as to drive the cell/pellet 4 to be far away from or close to the probe pellet 3 at the probe end to perform a force clamp experiment.
The first micropipette 1, the red blood cell 2, the probe small ball 3 and the reference small ball 7 form a probe end, and the probe end is fixed but is easily influenced by environmental disturbance and can slightly move; the target end is formed by the cells/small balls 4 and the second micropipette 5, and the piezoelectric motion platform 6 drives the control target end to move.
Spy-tag protein molecules are adsorbed on the surface of the first micropipette 1, the surface of the reference small ball 7 is connected with the Spy-catcher protein molecules, and the reference small ball 7 is fixedly adhered to the side surface of the end part of the first micropipette 1 through the interaction between the Spy-tag and the Spy-catcher protein molecules.
The diameter of reference sphere 7 is larger than the diameter of probe sphere 3. In a specific implementation, reference bead 7 is about 5 microns in diameter, significantly larger than about 2 microns in diameter of probe bead 3.
The reference ball 7 is a glass ball made of borosilicate.
In a specific embodiment, the first micropipette 1 at the probe end is soaked in a solution containing Spy-tag protein molecules in advance, so that the first micropipette 1 physically adsorbs a large amount of Spy-tag-bearing protein molecules.
The invention discovers that in the existing force clamp experiment, the probe end is kept fixed, but the first micro straw part of the probe end is easily influenced by environmental disturbance and can slightly move, and the actual deformation quantity of the red blood cell 3 cannot be accurately measured by simply tracking the position information of the boundary of the small probe ball 3.
In fig. 1, the positions of the right boundary 9 of the reference sphere 7 and the left boundary 8 of the probe sphere 3 are synchronously tracked in real time by shooting with a high-speed camera, and the deformation of the red blood cell 3 is represented by the change of the relative distance between the two boundaries. Because the reference small ball 7 is stably adhered to the side surface of the first micropipette 1 at the probe end and moves synchronously with the first micropipette 1, the position change of the reference small ball 7 can be regarded as the position change of the whole probe end of the first micropipette 1. Even in the case of the overall drift of the probe ends 1, 2, 3, 7 caused by environmental disturbances, the relative distance between the two boundaries can still accurately characterize the deformation quantity of the red blood cells 3.
The force acting on the molecular bond in the invention is stably and accurately clamped, so that the binding time of the molecular bond under the stress condition can be stably and accurately detected for a long time. Meanwhile, the technical scheme of automatically adjusting the data recording time interval according to the molecular bond combination time length is adopted, the data acquisition time can be greatly prolonged, and the technical problems that the capacity of a register in a computer memory in the experimental process of the biomembrane mechanical probe system is limited and long-time experimental data cannot be effectively stored are solved.
The specific embodiment and the implementation process of the invention are as follows:
1) an initialization stage:
the piezoelectric motion platform 6 controls the target ends 4 and 5 to be far away from the probe ends 1, 2, 3 and 7, and is in an initial position and static, at the moment, the red blood cell 2 is in an initial state, the distance between a reference small ball 7 located at a boundary 9 close to one side of the red blood cell 2 and a probe small ball 3 located at a boundary 8 close to one side of the red blood cell 2 is taken as a relative distance 10, the relative distance is tracked in real time through high-speed addition shooting, the average value of the relative distances recorded in 0.2 second is taken as an initial relative distance, and then the next stage is started;
2) and (3) an impact stage: monitoring the relative distance in real time, driving the cell/globule 4 at the target end to move towards the direction close to the red blood cell 2 by the piezoelectric motion platform 6 through the second micropipette 5 at a preset speed, pushing the probe globule 3 to compress the red blood cell 2 in a centering way until the relative distance is reduced by the preset compression distance, and entering the next stage;
3) and (3) a contact stage: monitoring the relative distance in real time, keeping the piezoelectric motion platform 6 static, stably contacting the probe bead 3 with the cell/bead 4, and entering the next stage after preset contact time;
4) and (3) withdrawal stage: the piezoelectric motion platform 6 drives the cells/globules 4 at the target end to withdraw and move away from the red blood cells 2 at a preset speed through the second micropipette 5;
in the withdrawal process, monitoring the relative distance, if the relative distance is increased by the length corresponding to the preset force value compared with the initial relative distance, the force value is the value of the acting force between molecular bonds, namely the red blood cells 2 are stretched by the set length, the experiment is an adhesion event, the piezoelectric motion platform 6 immediately stops the current withdrawal motion, and the next stage is entered, namely the combined time recording stage;
if the relative distance is only recovered to the initial relative distance and is not stretched, directly entering 6) a resetting stage;
5) and (3) combining a time recording stage: monitoring the relative distance in real time, and meanwhile, implementing feedback compensation on the piezoelectric motion platform 6 according to the deviation between the relative distance corresponding to the preset force value and the actual relative distance until the relative distance is restored to the initial relative distance again, namely, the molecular bond is automatically broken, and entering a resetting stage;
as shown in fig. 2, in step 5), the relative distance monitored in real time is used as the actual relative distance, the actual relative distance is filtered by a filter of a PID control module of the Labview platform, and then subtracted from a reference relative distance corresponding to a preset force value to obtain a deviation, the deviation is input to a PID controller, and a deviation control quantity output by the PID controller is input to a motion module controlling the piezoelectric motion platform 6 to execute feedback compensation motion.
The specific PID controller adopts pure proportional feedback control, and the proportional parameter is 1.
In the double-boundary tracking mode, the sampling frequency of the biomembrane mechanical probe system is higher than 600Hz, and the piezoelectric motion platform is controlled to execute feedback compensation motion according to the deviation value calculated by each sampling, so that the piezoelectric motion platform can not normally run, and the recorded data is lost due to the occupation of computer host resources. In a specific implementation, therefore, the compensation described above is performed once after each 10 sampling experiments.
Thus, the invention utilizes the high-speed camera of the biomembrane mechanical probe system to acquire the relative distance 10 between the probe small ball and the reference small ball in real time, and executes feedback compensation motion through the piezoelectric motion platform Physik Instrument P-753.1CD at the target end according to the actual relative distance and the preset reference relative distance, so as to stably maintain the force acting on the molecular bond at the set force value. The preset reference relative distance is the initial relative distance plus the deformation amount of the red blood cells corresponding to the preset force value.
6) A reset phase: and monitoring the relative distance in real time, withdrawing the piezoelectric motion platform 6 to the initial position, ending the current cycle, and preparing the next cycle by measuring the force value data and storing the data.
In the force clamp experiment, 3 times of the initial data sampling time interval is used as the data recording time interval to sample the force value data and record the force value data in the register, and when the molecular bond binding time exceeds 5 seconds, the data recording time interval is increased to 20 times of the sampling time interval; when the number of times of recording data exceeds 5000 times, the data recording time interval is further increased by 5 times. The invention can stably record the molecular bond binding time of more than 200 seconds.
Data from experiments recording ultra-long binding times (about 185s) the results are shown in figure 3, for example "force-time" data (10) and "recording time interval-recording time" data (11) under steady, accurate force clamp. The same experiment as above was carried out with the original biomembrane mechanical probe system without the addition of the reference bead (7) and without the force feedback control, and only 15 seconds of data results were stored after several runs, which are shown in fig. 4. The comparison shows that the measured result is very stable and accurate, and the data within 200s of binding time can be stored and obtained under the condition of the same register capacity, so that the deformation quantity of the red blood cells (2) can be accurately measured, and the problems of inaccurate data measurement and storage effectiveness caused by the integral drift of the probe end due to environmental disturbance are solved.
Therefore, the invention can realize dynamic adjustment of data recording time interval and prolong the recording time limit of molecular bond binding time by referring to the arrangement of the small balls.
The invention dynamically increases the time interval of data recording according to the length of the recorded combination time on the basis of ensuring the highest sampling frequency to be 0.6kHz, thereby avoiding the overflow of the computer memory caused by overlarge recorded data.
The foregoing detailed description is intended to illustrate and not limit the invention, which is intended to be within the spirit and scope of the appended claims, and any changes and modifications that fall within the true spirit and scope of the invention are intended to be covered by the following claims.
Claims (6)
1. The utility model provides a super accurate power pincers experimental method based on biomembrane mechanics probe system, power pincers experiment relates to and has probe end and target end, and the probe end is including probe bobble (3), red blood cell (2), its characterized in that: in the biomembrane mechanical probe force clamp experiment, a reference small ball (7) is additionally arranged at the probe end, the reference small ball (7) is adhered to the end part of a first micro-suction pipe (1) for sucking red blood cells (2) through the interaction between protein molecules, and the relative distance between the boundary of one side close to the red blood cells (2) of the reference small ball (7) and the boundary of one side close to the red blood cells (2) of the probe small ball (3) is shot by a high-speed camera and tracked in real time to control the movement of a target end so as to carry out the force clamp experiment;
the method comprises the following specific steps:
1) an initialization stage:
the piezoelectric motion platform (6) controls the target ends (4 and 5) to be far away from the probe ends (1, 2, 3 and 7) to be at an initial position and to be static, at the moment, the red blood cells (2) are in an initial state, the distance between a reference small ball boundary (9) of the reference small ball (7) on one side close to the red blood cells (2) and the two boundaries of a probe small ball boundary (8) of the probe small ball (3) on one side close to the red blood cells (2) is used as a relative distance (10), the relative distance is tracked in real time through high-speed addition shooting, the average value of the relative distances recorded in 0.2 second is used as an initial relative distance, and then the next stage is started;
2) and (3) an impact stage: monitoring the relative distance in real time, driving a cell/pellet (4) at a target end to move towards the red blood cell (2) by a piezoelectric motion platform (6) through a second micropipette (5) at a preset speed, pushing a probe pellet (3) to compress the red blood cell (2) in a centering manner until the relative distance is reduced by the preset compression distance, and entering the next stage;
3) and (3) a contact stage: monitoring the relative distance in real time, keeping the piezoelectric motion platform (6) static, stably contacting the probe small ball (3) with the cell/small ball (4), and entering the next stage after preset contact time;
4) and (3) withdrawal stage: the piezoelectric motion platform (6) drives the cells/small balls (4) at the target end to retreat and move away from the red blood cells (2) at a preset speed through the second micropipette (5);
in the withdrawal process, monitoring the relative distance, if the relative distance is increased by the length corresponding to the preset force value compared with the initial relative distance, taking the experiment as an adhesion event, immediately stopping the current withdrawal movement of the piezoelectric movement platform (6), and entering a combined time recording stage;
if the relative distance is recovered to the initial relative distance and is not stretched, directly entering 6) a resetting stage;
5) and (3) combining a time recording stage: monitoring the relative distance in real time, and meanwhile, implementing feedback compensation by the piezoelectric motion platform (6) according to the deviation between the relative distance corresponding to the preset force value and the actual relative distance until the relative distance is restored to the initial relative distance again, namely, the molecular bond is automatically broken, and entering a resetting stage;
6) a reset phase: and monitoring the relative distance in real time, withdrawing the piezoelectric motion platform (6) to the initial position, finishing the current cycle, storing the data of the relative distance, the time and the like of the current experiment, and preparing the next cycle.
2. The method for testing the super-standard force clamp based on the biomembrane mechanical probe system as claimed in claim 1, wherein: in the step 5), the relative distance monitored in real time is used as an actual relative distance, the actual relative distance is subjected to filtering processing by a filter of a PID controller and then subtracted from a reference relative distance corresponding to a preset force value to obtain a deviation, the deviation is input into the PID controller, and a deviation control quantity output by the PID controller is input into a motion module for controlling the piezoelectric motion platform (6) to execute feedback compensation motion.
3. The method for testing the super-standard force clamp based on the biomembrane mechanical probe system as claimed in claim 1, wherein: spy-tag protein molecules are adsorbed on the surface of the first micropipette (1), Spy-catcher protein molecules are coated on the surface of the reference small ball (7), and the reference small ball (7) is fixedly adhered to the side face of the end part of the first micropipette (1) through the interaction between the Spy-tag and the Spy-catcher protein molecules.
4. The method for testing the super-standard force clamp based on the biomembrane mechanical probe system as claimed in claim 1, wherein: the diameter of the reference small ball (7) is larger than that of the probe small ball (3), and the reference small ball can be visually distinguished through the size of the small ball in the experiment process.
5. The method for testing the super-standard force clamp based on the biomembrane mechanical probe system as claimed in claim 1, wherein: the reference small ball (7) is a glass ball.
6. The method for testing the super-standard force clamp based on the biomembrane mechanical probe system as claimed in claim 1, wherein: in the force clamp experiment, 3 times of the initial data sampling time interval is used as a data recording time interval to sample the force value data and record the force value data in a register, and when the molecular bond binding time exceeds 5 seconds, the data recording time interval is increased to 20 times of the sampling time interval; when the number of times of recording data exceeds 5000 times, the data recording time interval is further increased by 5 times.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911268349.2A CN111122913B (en) | 2019-12-11 | 2019-12-11 | Super-alignment force clamp experiment method based on biomembrane mechanics probe system |
| PCT/CN2020/115248 WO2021114789A1 (en) | 2019-12-11 | 2020-09-15 | Biomembrane force probe system-based super-alignment force clamp experimental method |
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| US5291775A (en) * | 1992-03-04 | 1994-03-08 | Topometrix | Scanning force microscope with integrated optics and cantilever mount |
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