CN113654971B - Photoinduction electrode scanning microscope and method for measuring electrical characteristics of biological cells - Google Patents
Photoinduction electrode scanning microscope and method for measuring electrical characteristics of biological cells Download PDFInfo
- Publication number
- CN113654971B CN113654971B CN202110825087.6A CN202110825087A CN113654971B CN 113654971 B CN113654971 B CN 113654971B CN 202110825087 A CN202110825087 A CN 202110825087A CN 113654971 B CN113654971 B CN 113654971B
- Authority
- CN
- China
- Prior art keywords
- light
- electrode
- light pattern
- photosensitive chip
- positive lens
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 238000000034 method Methods 0.000 title claims abstract description 25
- 210000004027 cell Anatomy 0.000 claims description 105
- 229910021417 amorphous silicon Inorganic materials 0.000 claims description 30
- 238000006073 displacement reaction Methods 0.000 claims description 30
- 230000002107 myocardial effect Effects 0.000 claims description 16
- AMGQUBHHOARCQH-UHFFFAOYSA-N indium;oxotin Chemical compound [In].[Sn]=O AMGQUBHHOARCQH-UHFFFAOYSA-N 0.000 claims description 14
- 230000003287 optical effect Effects 0.000 claims description 13
- 230000001464 adherent effect Effects 0.000 claims description 12
- 230000000737 periodic effect Effects 0.000 claims description 11
- 230000006698 induction Effects 0.000 claims description 9
- 210000004413 cardiac myocyte Anatomy 0.000 claims description 7
- 239000000969 carrier Substances 0.000 claims description 7
- 238000003384 imaging method Methods 0.000 claims description 5
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 4
- 241000700159 Rattus Species 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 4
- 239000011521 glass Substances 0.000 claims description 4
- 238000012545 processing Methods 0.000 claims description 4
- 239000000758 substrate Substances 0.000 claims description 4
- 238000012300 Sequence Analysis Methods 0.000 claims description 3
- 238000006243 chemical reaction Methods 0.000 claims description 3
- 238000004621 scanning probe microscopy Methods 0.000 claims description 2
- 238000005259 measurement Methods 0.000 abstract description 16
- 239000000463 material Substances 0.000 abstract description 4
- 230000008569 process Effects 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 101100327165 Arabidopsis thaliana CCD8 gene Proteins 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 238000004364 calculation method Methods 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000005286 illumination Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 210000000170 cell membrane Anatomy 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000010349 pulsation Effects 0.000 description 2
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 102000004310 Ion Channels Human genes 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003491 array Methods 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005530 etching Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 238000000059 patterning Methods 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000001338 self-assembly Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/1031—Investigating individual particles by measuring electrical or magnetic effects
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/84—Systems specially adapted for particular applications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N2015/1006—Investigating individual particles for cytology
Landscapes
- Chemical & Material Sciences (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Dispersion Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Electrochemistry (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention discloses a photoinduction electrode scanning microscope and a method for measuring the electrical characteristics of biological cells. The designed light pattern is irradiated on the chip plated with the photosensitive material, so that the conductivity of the irradiated area of the photosensitive layer is increased, and an electrode defined by the light pattern, namely a light-induced electrode, is generated on the chip. The light-induced electrode replaces the traditional fixed electrode, the position and the shape of the electrode can be changed in real time according to the needs, the non-invasive and flexible measurement of the electrical characteristics of the biological cells is realized, and the light-induced electrode scanning mode can be realized by moving the position of the light pattern or the photosensitive chip and is used for measuring and positioning the electrical characteristics of the biological cells on a two-dimensional plane or a three-dimensional space.
Description
Technical Field
The invention relates to the field of measurement of electrical characteristics of biological cells, in particular to a photoinduction electrode scanning microscope and a photoinduction electrode scanning method for measuring the electrical characteristics of the biological cells.
Background
The research on the electrical characteristics of the biological cells has important significance on the prevention and treatment of human diseases, and the measurement of the electrical characteristics of the biological cells can bring about great breakthrough in the fields of disease diagnosis, cell regeneration and rehabilitation, high-throughput drug screening and the like. Therefore, the development of the detection technology of the electrical characteristics of the biological cells provides a powerful tool for studying the physiological and pathological processes of organisms at a cellular level.
At present, microelectrode arrays are widely used for detecting the electrical characteristics of biological cells, and the detection technology is used for directly culturing the biological cells on the surface of a device, connecting with external signal acquisition equipment through pins on a chip and acquiring signals. The electrodes with various shapes can be designed and prepared by selecting various materials, including disc-shaped, inserted finger-shaped, volcanic-shaped, well-shaped and the like, and the preparation method mainly comprises a template method, an etching method, a self-assembly method and the like. The preparation of the traditional electrodes needs to design complicated physical electrodes, the design of the physical electrodes has the problems of complicated design, long manufacturing period, high cost and the like, and only the electrical characteristics of cells at fixed positions can be detected. The photo-induced electrode without contact and damage to biological cells can be used for cell manipulation, separation or particle capture and the like at present, and is not applied to the field of measurement of the electrical characteristics of the biological cells. The invention provides a photoinduction electrode scanning microscope for measuring the electrical characteristics of biological cells, which replaces the traditional fixed electrode with the photoinduction electrode, avoids the complex processing process, greatly reduces the cost, can change the position and the shape of the electrode in real time according to the requirement so as to realize the flexible measurement of the electrical characteristics of the biological cells, and can realize the photoinduction electrode scanning mode by carrying out X and Y direction movement on a chip through dynamic scanning of a light pattern or a displacement platform so as to position the cells.
Disclosure of Invention
The invention solves the problems: 1) The traditional electrode preparation has the problems of complex electrode manufacturing, difficult sample preparation and high cost; 2) The traditional electrode is fixed in shape and position, the measurement of the electrical characteristics of biological cells is not flexible enough, the resolution ratio is low, and the measurement dead angle is formed; 3) Because the conventional electrode is fixed in position, the position of the cell cannot be determined by measuring the electrical characteristics of the cell. The invention provides a photoinduction electrode scanning microscope and a method for measuring the electrical characteristics of biological cells, wherein the photoinduction electrode is used for replacing the traditional physical electrode, so that the complex electrode manufacturing process is avoided, and the electrode is continuous (has no measuring dead angle), high in resolution, high in efficiency and low in cost; furthermore, the shape and the position of the light induction electrode can be changed in real time according to the requirement so as to realize the patterning and the flexible activation of the measurement of the electrical characteristics of the biological cells; furthermore, a light-induced electrode scanning mode can be realized by utilizing dynamic scanning of a light pattern or moving of a displacement platform in X and Y directions on the chip, so that adherent cells are positioned on a two-dimensional plane; furthermore, the spatial position of the electric signals generated by the upper surfaces of the adherent cells and the suspension cells can be obtained through signal sequence analysis and calculation in the scanning process, and then the cell positioning in the three-dimensional space is realized. Furthermore, the light induction electrode is applied to the field of measuring the electrical characteristics of biological cells, and the limitation of a fixed electrode or an electrode array is broken through.
The invention provides a photoinduction electrode scanning microscope and a method for measuring the electrical characteristics of biological cells, and the purpose can be realized by the following technical measures:
a light induced electrode scanning microscope for measuring electrical properties of biological cells, comprising: the device comprises a displacement platform (1), a projection device (2), a first positive lens (3), a second positive lens (4), an objective lens (5), a photosensitive chip (6), an optical microscope (7), a CCD (8) and a computer (9).
Displacement platform (1) comprising: a micrometer displacement table (coarse adjustment, 25mm stroke and 0.1 μm resolution) and a nanometer displacement table (fine adjustment, 100 μm stroke and 0.2nm resolution) for three-dimensionally adjusting the photosensitive chip (6), wherein the X direction and the Y direction are used for horizontally adjusting the position of the photosensitive chip to realize a light-induced electrode scanning mode, and the Z direction is used for vertically adjusting the position of the photosensitive chip to adjust the imaging focal length to image cells on a display of a computer (9).
The projection device (2), the first positive lens (3), the second positive lens (4) and the objective lens (5) are used for projecting the designed light pattern onto the photosensitive chip (6), and the size of the light pattern is changed by adjusting the distance among the projection device (2), the first positive lens (3) and the second positive lens (4).
A photo-sensitive chip (6) on which photo-generated carriers are generated in the illuminated areas of the photo-sensitive layer to increase the conductivity, thereby creating a photo-induced electrode defined by a photo-pattern. And adhering the culture dish ring on the photosensitive chip (6) to form a photosensitive chip culture dish, extracting the myocardial cells, inoculating the myocardial cells into the culture dish, and performing an electrical characteristic measurement experiment.
An optical microscope (7), a CCD (8) for acquiring light patterns and cell images.
And the computer (9) is used for observing the light pattern and the cell image in real time and simultaneously used as an external acquisition device for acquiring and processing the cell electric signal. The computer (9) is provided with a signal amplifier and a data acquisition card (24bits, 200KS/s).
The photosensitive chip (6) is composed of an intrinsic hydrogenated amorphous silicon photosensitive layer (11), an n-type doped hydrogenated amorphous silicon layer (12), an indium tin oxide conductive layer (13) and a glass substrate (14) from top to bottom in sequence. Wherein the intrinsic hydrogenated amorphous silicon photosensitive layer (11) has a thickness of 100nm to 1000nm and is of n-typeThe thickness of the doped hydrogenated amorphous silicon layer (12) is 20nm-100nm, and the thickness of the indium tin oxide conducting layer is 20nm-200nm. The indium tin oxide conductive layer serves to collect carriers longitudinally and transmit them to an external circuit, while reducing optical reflection. The hydrogenated amorphous silicon is sensitive to the photo-electrode, the resistance becomes small under the illumination condition, the light-induced electrode is obtained by utilizing the characteristics of conduction at the illumination position and cut-off at the non-illumination position, and the hydrogenated amorphous silicon has high light-dark conductance ratio (more than or equal to 10)3) And high absorptivity, so hydrogenated amorphous silicon is selected as the photosensitive material. The photosensitive material consists of an intrinsic hydrogenated amorphous silicon photosensitive layer (11) and an n-type doped hydrogenated amorphous silicon layer (12). The intrinsic hydrogenated amorphous silicon photosensitive layer (11) is a generation region of photon-generated carriers, and only the intrinsic hydrogenated amorphous silicon photosensitive layer (11) has photoelectric conversion function; the n-type doped hydrogenated amorphous silicon layer (12) is used for reducing the contact resistance between the intrinsic hydrogenated amorphous silicon photosensitive layer (11) and the indium tin oxide conductive layer (13).
Further, the projection device (2) projects the designed light pattern, and the light pattern is irradiated on the photosensitive chip (6) through the convergence of the first positive lens (3) and the second positive lens (4) and the zooming of the objective lens (5) in sequence.
Further, the projection device (2) can also be realized by the following method: 1) scanning micro or nano fringes generated by two-beam laser interference, 2) controlling light spot scanning by a laser galvanometer, or 3) imaging scanning by combining a spatial light modulator with a photosensitive chip.
3 newborn rats were collected for 1 to 3 days, and cardiomyocytes were extracted and inoculated in a culture dish for the following electrical characteristic test.
A light-induced electrode scanning microscopy method for measuring the electrical characteristics of biological cells uses a microscope as described in any one of the above steps, and comprises the following steps:
myocardial cells of newborn rats are extracted for 1 to 3 days and cultured in a culture dish of a photosensitive chip (6). The projection device (2) projects the designed light pattern, and the light pattern is irradiated on the photosensitive chip (6) through the convergence of the first positive lens (3) and the second positive lens (4) and the zooming of the objective lens (5). The illuminated areas of the photo-sensitive chip (6) generate photo-generated carriers that increase the conductivity, thereby creating a photo-induced electrode on the chip defined by the light pattern. The size of the light pattern is adjusted by adjusting the distance between the projection device (2), the first positive lens (3) and the second positive lens (4). Simultaneously, the cells and the light pattern are observed in real time by using an optical microscope (7), a CCD (8) and a computer (9). Adjusting the projection position of the light pattern, or moving the displacement platform (1) to make the light pattern irradiate under the cell to be detected. One end of the lead is connected with an indium tin oxide conducting layer (13) of the photosensitive chip (6) to be used as a measuring electrode, the other end is connected with an external acquisition equipment computer (9), and a reference electrode is arranged above the cell. The observation computer (9) collects the readings of the software and detects the electrical characteristics of the cells in real time, thereby realizing the measurement of the electrical characteristics of the biological cells by utilizing the light-induced electrodes.
Two implementations of the scanning mode of the light-induced electrode scanning microscope. Method one, dynamic scanning of the light pattern: the light pattern is moved by a projection device (2), and the line pattern is moved from the left end to the right end of the chip to perform X-direction scanning and from the upper end to the lower end of the chip to perform Y-direction scanning. And secondly, the horizontal position of the photosensitive chip (6) is finely adjusted by using the displacement platform (1) to realize the continuous scanning of the light induction electrode in the X and Y directions. The indications of the software collected by the computer (9) are observed while the light induction electrode is scanned, and in the process of scanning in the X direction, the coordinate X is recorded when the strongest periodic voltage signal appears1Recording the coordinate Y when the strongest periodic voltage signal is present during the Y-direction scan1At this time (X)1，Y1) Namely, the cell position, the cell positioning on a two-dimensional plane by utilizing the light-induced electrode scanning mode is realized.
Further, the cell to be tested may also be other excitable cells, or cell membrane potential, and changes in cell membrane potential/ion channel.
Furthermore, the spatial position of the electric signals generated by the upper surface of the adherent cells and the suspension cells can be obtained through signal sequence analysis and calculation in the scanning process, and further the positioning of the myocardial cells on a three-dimensional space is realized.
The invention realizes the flexible measurement of the electrical characteristics of biological cells and the cell positioning by manufacturing the light-induced electrode scanning microscope and using the light-induced electrode to replace the traditional fixed electrode. Has the following advantages:
1) The electrode is simple to prepare, high in efficiency and low in cost, and a complex physical electrode manufacturing process is avoided.
2) The position and the shape of the electrode can be continuously adjusted in real time according to the requirement, so that a dynamic light induction electrode is generated, and the high-resolution flexible measurement of the electrical characteristics of the biological cells is realized.
3) The light-induced electrode scanning mode realizes the positioning of adherent cells on a two-dimensional plane by utilizing the dynamic scanning of a light pattern or the fine adjustment of a displacement platform to a photosensitive chip in the horizontal direction.
4) The spatial position of the electric signals generated by the upper surface of the adherent cells and the suspension cells can be obtained through the analysis and calculation of the signal sequence in the scanning process, and further, the cell positioning in the three-dimensional space is realized.
5) The light induction electrode is applied to the field of measuring the electrical characteristics of biological cells, and the limitation of a fixed electrode or an electrode array is broken through.
Drawings
FIG. 1 is a schematic structural diagram of a light-induced electrode scanning microscope according to the present invention;
wherein: 1 is a displacement platform, 2 is a projection device, 3 is a first positive lens, 4 is a second positive lens, 5 is an objective lens, 6 is a photosensitive chip, 7 is an optical microscope, 8 is a CCD, and 9 is a computer;
FIG. 2 is a schematic diagram of a structure of a photosensitive chip;
wherein: 11 is an intrinsic hydrogenated amorphous silicon photosensitive layer, 12 is an n-type doped hydrogenated amorphous silicon layer, 13 is an indium tin oxide conducting layer, and 14 is a glass substrate;
FIG. 3 is a schematic view of a photo-induced electrode scan;
FIG. 4 is an experimental graph of electrical characteristics of biological cells obtained by implementing a photo-induced electrode scanning mode by dynamic scanning of a light pattern;
fig. 5 is an experimental graph of the electrical characteristics of biological cells obtained by implementing a light-induced electrode scanning mode through the movement of a displacement platform.
Detailed Description
The invention is further described below with reference to the accompanying drawings.
As shown in fig. 1, the photoinduced electrode scanning microscope of the invention comprises a displacement platform 1, a projection device 2, a first positive lens 3, a second positive lens 4, an objective lens 5, a photosensitive chip 6, an optical microscope 7, a CCD8 and a computer 9. The photosensitive chip 6 is arranged on the displacement platform 1. The projection device 2 projects the designed light pattern, and the light pattern is irradiated on the photosensitive chip 6 through the convergence of the first positive lens 3 and the second positive lens 4 and the zooming of the objective lens 5 in sequence. The size of the light pattern is adjusted by adjusting the distance between the projection device 2, the first positive lens 3 and the second positive lens 4. The conductivity of the irradiated area of the photosensitive chip 6 is increased, a light-induced electrode defined by a light pattern is generated, the complex preparation process of the traditional electrode is avoided, and the shape and the position of the electrode are flexible and variable. The cell and the light pattern are observed by using an optical microscope 7, a CCD8 and a computer 9, the light pattern is adjusted according to the position of the cell, so that the light induction electrode is positioned below the cell to be detected, and the computer 9 is used as an external acquisition device to acquire the electrical characteristics of the biological cell. An optical microscope 7 and a CCD8 for acquiring light patterns and cell images. The computer 9 is provided with a signal amplifier and a data acquisition card (24bits, 200KS/s) and is used for observing light patterns and cell images in real time and simultaneously used as external acquisition equipment for acquiring and processing cell electric signals.
One end of a lead is connected with the indium tin oxide conducting layer 13 of the photosensitive chip 6 to be used as a measuring electrode, the other end of the lead is connected with the external acquisition equipment computer 9, and a silver wire is selected to be used as a reference electrode, is placed above the cells and is connected with the external acquisition equipment computer 9.
The invention provides two photo-induced electrode scanning modes: first, a direct scan mode is performed by projecting a dynamic light pattern using the projection device 2; secondly, the displacement platform 1 is used for finely adjusting the horizontal position of the photosensitive chip 6 to realize an indirect scanning mode of the photo-induced electrode. As shown in FIG. 2, the photo chip 6 is composed of an intrinsic hydrogenated amorphous silicon photo sensitive layer 11, an n-type doped hydrogenated amorphous silicon layer 12, an indium tin oxide conductive layer 13 and a glass substrate 14 in this order from top to bottom. Wherein, the thickness of the intrinsic hydrogenated amorphous silicon photosensitive layer 11 is 400nm, the thickness of the n-type doped hydrogenated amorphous silicon layer 12 is 50nm, and the thickness of the indium tin oxide conducting layer 13 is 120nm. Only the intrinsic hydrogenated amorphous silicon photosensitive layer 11 has a photoelectric conversion effect, and the irradiated region generates photo-generated carriers to increase conductivity, thereby generating a photo-induced electrode defined by a photo pattern on the chip. The n-type doped hydrogenated amorphous silicon layer 12 is used to reduce the contact resistance between the intrinsic hydrogenated amorphous silicon photosensitive layer 11 and the indium tin oxide conductive layer 13, resulting in better coupling between the two.
As shown in FIG. 3, the projection device 2 projects a vertical line pattern onto the photosensitive chip 6, the pattern is moved in the X direction or the displacement platform is moved in the X direction to move the vertical line pattern from the left end to the right end of the chip, and the reading of the software is acquired by observing the computer 9, when the strongest periodic voltage signal appears, the light line pattern is at the cell position, and the coordinate at this moment, namely X, is recorded1. The projector 2 projects the horizontal line pattern onto the photosensitive chip 6, the pattern is moved along the Y direction or the displacement platform is moved along the Y direction to move the horizontal line pattern from the upper end to the lower end of the chip, when the strongest periodic voltage signal appears in the software collected by the computer 9, the coordinate at the moment, namely the Y-axis, is recorded1. At this time, (X)1，Y1) Namely the position of the cell, the measurement and the positioning of the electrical characteristics of the cell on a two-dimensional plane are realized by utilizing a light-induced electrode scanning microscope.
As shown in fig. 4, the projection position of the light pattern is continuously changed by dynamic scanning of the light pattern, and a photo-induced electrode scanning mode is implemented, and a voltage signal is collected by the computer 9. When a light pattern is projected under the cell, the computer 9 collects a periodic voltage signal that appears as a graph to the software.
As shown in fig. 5, the photo-induced electrode scanning mode is realized by moving the displacement platform, that is, by moving the displacement platform to change the projection position of the vertical line pattern in the X direction and to change the projection position of the horizontal line pattern in the Y direction, and the computer 9 is used to collect the voltage signal. When a light pattern is projected under the cell, the computer 9 collects a periodic voltage signal that appears as a graph to the software.
The process of the present invention will be described in detail below.
Example 1:
3 newborn rats in 1-3 days are taken, ventricles are extracted, the myocardial tissues are digested by adding type II collagenase, and cells are separated. Filtering, centrifuging, discarding supernatant, adding DMEM high sugar medium containing 10% FBS into the precipitate, blowing to obtain cardiomyocyte suspension, and inoculating into culture dish. Placing the culture dish at 37 ℃ and 5% CO2The culture chamber of (3) is cultured for 1.5-2 hours for differential adherence, at which time the cardiomyocytes are present in the supernatant. Collecting supernatant, adding culture medium containing Brdu with final concentration of 0.1mmol/L for inhibiting proliferation of non-myocardial cells, inoculating myocardial cell suspension in the photosensitive chip 6 culture dish, culturing for 24 hr, changing culture medium, and culturing without Brdu. The cells are basically attached to the wall after 12 hours of culture, and a few attached single cells have spontaneous pulsation, the pulsation frequency is slow, and some cells are only attached several times per minute.
The individual components or devices are connected in the manner of fig. 1, and the light pattern is designed. The projection device 2 projects the designed light pattern, and the light pattern is irradiated onto the photosensitive chip 6 after being converged by the first positive lens 3 and the second positive lens 4 and zoomed by the objective lens 5. The size of the light pattern is adjusted by adjusting the positions of the first positive lens 3, the second positive lens 4, the objective lens 5 and the photo chip 6. The irradiated regions of the intrinsic hydrogenated amorphous silicon photosensitive layer 11 generate photo-generated carriers to increase the conductivity, thereby creating photo-induced electrodes defined by the photo-pattern on the chip. The light pattern and the cell position are observed by an optical microscope 7, a CCD8, and a computer 9, and the light pattern is projected at the cell position. The voltage signal of the cell is collected by the collection software of the computer 9, so that the measurement of the electrical characteristics of the biological cell of the light-induced electrode scanning microscope is realized.
Example 2:
the cardiomyocytes were seeded on the photosensitive chip 6 petri dish and the various elements or devices were connected in the manner shown in FIG. 1.
Scanning mode one of the photo-induced electrode: as shown in fig. 3, a series of vertical linear patterns are designed, and the X-direction scanning mode of the light-induced electrode is realized by controlling the vertical linear patterns to move from the left end to the right end of the photosensitive chip 6 in sequence. The light-induced electrode scans and simultaneously observes the computer 9 to acquire real-time signals of software, when the strongest periodic voltage signal appears, the computer 9 observes the light pattern and the adherent myocardial cells, the light pattern is positioned at the position of the cells, and the coordinate X at the moment is recorded1(X1=232.5 μm). As shown in fig. 3, a series of horizontal line patterns are designed, and the horizontal line patterns are controlled to move from the upper end to the lower end of the photosensitive chip 6 in sequence, so that the Y-direction scanning mode of the photoinduction electrode is realized. The computer 9 is observed to collect real-time signals of software while the light-induced electrode is scanned, when the strongest periodic voltage signal appears, the computer 9 is used to observe the light pattern and the cell, the light pattern is positioned at the position of the cell, and the coordinate of the light pattern at the moment, namely Y, is recorded1(Y1＝344.1μm)。(X1＝232.5μm，Y1=344.1 μm) is the location of the cell. The experimental graph of the electrical characteristics of the cells obtained by the scanning mode of the light-induced electrode realized by the dynamic scanning of the light pattern is shown in fig. 4, and the measurement and the positioning of the electrical characteristics of the adherent myocardial cells of the light-induced electrode scanning microscope on a two-dimensional plane are completed.
A second photo-induced electrode scanning mode: as shown in fig. 3, a vertical line pattern is designed, and the displacement platform 1 is adjusted in the X direction to move the vertical line pattern along the X direction, so that the X direction scanning mode of the light-induced electrode is realized. Inducing electrode scanning while observing the real-time signal of software collected by computer 9, observing adherent myocardial cell and light pattern via computer 9 when the strongest periodic voltage signal appears, the light pattern is at the position of cell, and recording the coordinate, namely X1(X1=232.5 μm). As shown in FIG. 3, a horizontal line pattern is designed, and the displacement platform 1 is adjusted in the Y direction to make a horizontal lineThe pattern is moved in this direction and when the strongest periodic voltage signal is present, the coordinate at that moment, Y, is recorded1(Y1＝344.1μm)。(X1＝232.5μm，Y1=344.1 μm) is the location of the cell. An experimental graph of electrical characteristics of cardiomyocytes obtained by realizing the light-induced electrode scanning mode through the movement of the displacement platform is shown in fig. 5, and the measurement and the positioning of the electrical characteristics of adherent cardiomyocytes on a two-dimensional plane by the light-induced electrode scanning microscope are completed.
The above-described embodiments are not intended to limit the present invention, and various modifications and equivalents may be made by those skilled in the art without departing from the spirit and scope of the present invention, and therefore the scope of the present invention is to be defined by the appended claims.
Claims (6)
1. A light-induced electrode scanning microscopy method for measuring electrical properties of biological cells, using a microscope of the structure comprising: the device comprises a displacement platform (1), a projection device (2), a first positive lens (3), a second positive lens (4), an objective lens (5), a photosensitive chip (6), an optical microscope (7), a CCD (8) and a computer (9); the photosensitive chip (6) is arranged on the displacement platform (1);
displacement platform (1) comprising: the micro-displacement table and the nano-displacement table are used for carrying out three-dimensional adjustment on the photosensitive chip (6), the X direction and the Y direction are used for horizontally adjusting the position of the photosensitive chip to realize a light induction electrode scanning mode, and the Z direction is used for vertically adjusting the position of the photosensitive chip to adjust the imaging focal length so as to image cells on a display of a computer (9);
the projection device (2), the first positive lens (3), the second positive lens (4) and the objective lens (5) are used for projecting a designed light pattern onto the photosensitive chip (6), and the size of the light pattern is changed by adjusting the distance among the projection device (2), the first positive lens (3) and the second positive lens (4);
a photosensitive chip (6) on which photo-generated carriers are generated in the irradiated areas of the photosensitive layer to increase the conductivity, thereby creating a photo-induced electrode defined by a light pattern;
an optical microscope (7) and a CCD (8) for acquiring light patterns and cell images;
the computer (9) is provided with a signal amplifier and a data acquisition card and is used for observing the optical pattern and the cell image in real time and simultaneously used as external acquisition equipment for acquiring and processing cell electric signals;
the method comprises the following steps: extracting myocardial cells of newborn rats for 1-3 days, and culturing in a culture dish of a photosensitive chip (6); the projection device (2) projects a designed light pattern, and the light pattern is irradiated on the photosensitive chip (6) after being converged by the first positive lens (3) and the second positive lens (4) and zoomed by the objective lens (5); observing adherent myocardial cells and a light pattern through an optical microscope (7), a CCD (8) and a computer (9), and moving the light pattern to the position below the myocardial cells to be detected; the computer (9) is also used as an external acquisition device to acquire the electrical characteristics of the myocardial cells in real time;
the pattern movement is carried out by utilizing the projection device (2) so as to directly adjust the projection position of the light pattern, the X and Y directions of the chip are scanned, or the position of the photosensitive chip (6) in the X and Y directions is finely adjusted by utilizing the displacement platform (1) so as to indirectly realize the movement of the light pattern, thereby realizing the light induction electrode scanning mode; and the computer (9) is observed in real time to acquire the readings of the software, and when the strongest periodic voltage signal appears, the coordinates in the X direction and the Y direction are respectively recorded, and the coordinates are the positions of the myocardial cells, so that the adherent myocardial cells on a two-dimensional plane are positioned.
2. The method of claim 1, wherein: the photosensitive chip (6) consists of an intrinsic hydrogenated amorphous silicon photosensitive layer (11), an n-type doped hydrogenated amorphous silicon layer (12), an indium tin oxide conducting layer (13) and a glass substrate (14) from top to bottom in sequence; wherein, the thickness of the intrinsic hydrogenated amorphous silicon photosensitive layer (11) is 100nm-1000nm, the thickness of the n-type doped hydrogenated amorphous silicon layer (12) is 20nm-100nm, and the thickness of the indium tin oxide conducting layer (13) is 20nm-200nm; the intrinsic hydrogenated amorphous silicon photosensitive layer (11) is a photogenerated carrier generating region and has a photoelectric conversion function, and the n-type doped hydrogenated amorphous silicon layer (12) can reduce the contact resistance between the intrinsic hydrogenated amorphous silicon photosensitive layer (11) and the indium tin oxide conducting layer (13).
3. The method of claim 1, wherein: the projection device (2) projects the designed light pattern, and the light pattern is irradiated on the photosensitive chip (6) through the convergence of the first positive lens (3) and the second positive lens (4) and the zooming of the objective lens (5) in sequence.
4. The method according to claim 1, wherein the projection device (2) is also implemented by: 1) scanning micro or nano stripe generated by laser interference of two light beams, 2) scanning light spots controlled by a laser galvanometer, or 3) imaging scanning by combining a spatial light modulator with a photosensitive chip.
5. The method according to claim 1, characterized in that one end of the wire is connected to the indium tin oxide conducting layer (13) of the light sensitive chip (6) as a measuring electrode and the other end is connected to an external acquisition device computer (9), and a reference electrode is placed above the cell.
6. The method of claim 1, wherein the spatial position of the electrical signals generated by the upper surface of the adherent cells and the suspended cells can be obtained by signal sequence analysis during scanning, so as to realize the positioning of the cardiomyocytes in three-dimensional space.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110825087.6A CN113654971B (en) | 2021-07-21 | 2021-07-21 | Photoinduction electrode scanning microscope and method for measuring electrical characteristics of biological cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110825087.6A CN113654971B (en) | 2021-07-21 | 2021-07-21 | Photoinduction electrode scanning microscope and method for measuring electrical characteristics of biological cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113654971A CN113654971A (en) | 2021-11-16 |
| CN113654971B true CN113654971B (en) | 2022-11-01 |
Family
ID=78489661
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110825087.6A Active CN113654971B (en) | 2021-07-21 | 2021-07-21 | Photoinduction electrode scanning microscope and method for measuring electrical characteristics of biological cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113654971B (en) |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1154475A (en) * | 1995-06-20 | 1997-07-16 | 松下电器产业株式会社 | Two-dimensional sensor having photoconductive layer for measuring cell activity |
| CN101135680A (en) * | 2007-07-13 | 2008-03-05 | 东南大学 | Light-induction dielectrophoresis auxiliary unicellular dielectric spectrum automatic test equipment and testing method |
| CN104419642A (en) * | 2013-08-22 | 2015-03-18 | 中国科学院沈阳自动化研究所 | Cell physiological information detection system and detection method |
| CN107304414A (en) * | 2016-04-19 | 2017-10-31 | 长庚大学 | Method and device for purifying and separating blood circulation tumor cells by non-contact and automatic identification |
| CN107357034A (en) * | 2017-08-02 | 2017-11-17 | 长春理工大学 | A kind of light-induction dielectrophoresis device of laser interference pattern light field as light source |
| CN107843541A (en) * | 2017-10-24 | 2018-03-27 | 上海大学 | A kind of real-time monitoring system and method for unicellular organism physical characteristic |
| CN108732094A (en) * | 2018-04-23 | 2018-11-02 | 江苏大学 | The three-dimensional image information acquisition methods and device of Cell-oriented Morphological Reconstruction |
| CN109810894A (en) * | 2019-03-01 | 2019-05-28 | 长春理工大学 | A kind of group's cell three-dimensional structure manipulation based on light-induction dielectrophoresis and building system and method |
| CN110257218A (en) * | 2019-03-19 | 2019-09-20 | 金鸿医材科技股份有限公司 | Electric field force is manipulated using image to screen the device and its operating method of biomone |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002078906A2 (en) * | 2001-03-29 | 2002-10-10 | Cellect Technologies Corp. | Methods devices and systems for sorting and separating particles |
-
2021
- 2021-07-21 CN CN202110825087.6A patent/CN113654971B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1154475A (en) * | 1995-06-20 | 1997-07-16 | 松下电器产业株式会社 | Two-dimensional sensor having photoconductive layer for measuring cell activity |
| CN101135680A (en) * | 2007-07-13 | 2008-03-05 | 东南大学 | Light-induction dielectrophoresis auxiliary unicellular dielectric spectrum automatic test equipment and testing method |
| CN104419642A (en) * | 2013-08-22 | 2015-03-18 | 中国科学院沈阳自动化研究所 | Cell physiological information detection system and detection method |
| CN107304414A (en) * | 2016-04-19 | 2017-10-31 | 长庚大学 | Method and device for purifying and separating blood circulation tumor cells by non-contact and automatic identification |
| CN107357034A (en) * | 2017-08-02 | 2017-11-17 | 长春理工大学 | A kind of light-induction dielectrophoresis device of laser interference pattern light field as light source |
| CN107843541A (en) * | 2017-10-24 | 2018-03-27 | 上海大学 | A kind of real-time monitoring system and method for unicellular organism physical characteristic |
| CN108732094A (en) * | 2018-04-23 | 2018-11-02 | 江苏大学 | The three-dimensional image information acquisition methods and device of Cell-oriented Morphological Reconstruction |
| CN109810894A (en) * | 2019-03-01 | 2019-05-28 | 长春理工大学 | A kind of group's cell three-dimensional structure manipulation based on light-induction dielectrophoresis and building system and method |
| CN110257218A (en) * | 2019-03-19 | 2019-09-20 | 金鸿医材科技股份有限公司 | Electric field force is manipulated using image to screen the device and its operating method of biomone |
Non-Patent Citations (2)
| Title |
|---|
| High-purity separation of cancer cells by optically induced dielectrophoresis;Hsiu-Hsiang Chen et al.;《Journal of Biomedical Optics》;20170430;第19卷(第4期);全文 * |
| 基于光诱导介电泳的微粒自动化操作实验研究;王淑娥等;《微纳电子技术》;20110215;第48卷(第02期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113654971A (en) | 2021-11-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Korchev et al. | Specialized scanning ion‐conductance microscope for imaging of living cells | |
| CN101173885B (en) | Near-field optical microscope system for micro-cell mesomeric state/transient state photoelectric detection and scanning image | |
| CN102353817B (en) | Probe of conducting atomic force microscope and measuring methods employing probe | |
| CN101430321B (en) | Non-contact biological detection scanning nano-glass probe microscope and its operation method | |
| CN103558367B (en) | System and method for measuring electrical characteristics of biological cells through nano-electrode array under physiological conditions | |
| CN105527462B (en) | A kind of method that AFM measures single Single Cardiac Cell living and pulsating force | |
| CN110108905B (en) | Method and device for detecting membrane potential and nerve cell membrane repairing behaviors of nerve cells | |
| CN107843541B (en) | A kind of real-time monitoring system and method for unicellular organism physical characteristic | |
| CN102071135A (en) | High resolution patch clamp based on scanning probe microscopy technology and operating method thereof | |
| CN111920404A (en) | Flexible microelectrode array and preparation method and application thereof | |
| WO2017028341A1 (en) | Method for generating single-cell three-dimensional image based on optical flow analysis | |
| CN102579043B (en) | Electric impedance imaging system with open electrode scanning mode | |
| CN113654971B (en) | Photoinduction electrode scanning microscope and method for measuring electrical characteristics of biological cells | |
| CN102353818A (en) | Device and method for evaluating neuron-like differentiation degree of PC12 cell | |
| CN102586092B (en) | Cell impedance imaging system and cell impedance imaging method for dynamically monitoring wound healing | |
| CN104419642B (en) | A kind of cell physiological information detecting system and detection method thereof | |
| CN107638176A (en) | A kind of application of hydrogel in the electrode for preparing detection electrocorticogram | |
| CN103439532A (en) | Microelectrode technology for metal surface microcell current distribution in-situ detection | |
| CN106442432A (en) | Super-localized photocurrent scanning imaging system based on aperture type conductive probe | |
| CN102455371A (en) | Device and method for evaluating malignancy degree of neuroblastoma | |
| CN104913731A (en) | Laser differential confocal microscope measurement and control system | |
| US20160356739A1 (en) | Electrochemical approach for cancer detection | |
| CN205364548U (en) | Optimal control system of three -dimensional bioprinting aquogel support | |
| CN111493865B (en) | Cortical electroencephalogram electrode and array capable of being used for multi-modal observation of brain | |
| CN210053477U (en) | Array transmission type microscopic image acquisition system |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |