Scanning imaging system and method based on optical feedback positioning and ionic conductance technology

Abstract

The invention belongs to the field of scanning ion conductance microscopes and scanning electrochemical cell microscopes, and discloses a scanning imaging system and method based on optical feedback positioning and ion conductance technology, the scanning imaging system comprises an SICM ion current circuit, an optical path reflector and an adapter bracket, the adapter bracket is arranged on a motion platform, and a laser generator is arranged on the upper part of the adapter bracket; the light path reflector is rotationally connected with the adapter bracket, a prism system and a CCD camera are sequentially arranged on a reflected light path of the light path reflector, and the prism system is fixed on the adapter bracket; the lower part of the adapter bracket is provided with a micromotor, the output end of the micromotor is connected with a Z-direction piezoelectric ceramic, the Z-direction piezoelectric ceramic is connected with a piezoelectric ceramic controller and is connected with the flexible hinge, the Z-direction piezoelectric ceramic is provided with a probe holder, the SICM probe is arranged on the probe holder, and the probe electrode is arranged in the SICM probe. According to the invention, the position of the probe can be rapidly and accurately controlled, and the problem of low scanning speed of scanning ionic conductivity detection equipment is solved.

Description

Technical Field

[0001] The present invention belongs to the field of scanning ion conductance microscopy and scanning electrochemical cell microscopy, and particularly relates to a scanning imaging system and method based on optical feedback positioning and ion conductance technology. Background Art

[0002] The level of microscopy technology determines the research boundary of human science and technology at the spatial scale, and is the basis and essential tool for the development of frontier fields such as life science and electrochemistry. Over the years, people have developed various microscopes based on sound, light, electricity, etc., which have greatly promoted the development of related fields, but at the same time, they all have certain limitations. Traditional optical microscopes (OMs) are well-developed and widely used, but their resolution is relatively low and it is difficult to meet the requirements for high-resolution use; super-resolution fluorescence microscopy breaks through the limitation of the optical diffraction limit, but the sample needs to be fluorescently treated and "in-situ" imaging of cells cannot be achieved; electron microscopes (EMs) have high resolution, but the sample needs to be processed and they need to be used in a vacuum environment, and live cells cannot be imaged. In recent years, scanning probe microscopes (SPMs) represented by atomic force microscopes (AFMs) have been widely used in the measurement field, but AFMs are based on atomic force for feedback control, which may cause scratches or deformation on the sample surface.

[0003] Scanning Ion Conductance Microscopy (SICM) and Scanning Electrochemical Cell Microscopy (SECCM), as new members of the SPM family, use a glass pipette with a tip opening of a few nanometers to a few hundred nanometers as a sensing probe for scanning detection. By detecting the magnitude of the current in the ion conductance circuit, precise positioning of the probe tip is achieved. By recording and analyzing the ion current or redox current, simultaneous detection of information such as the nano-scale three-dimensional morphology, electrochemistry, and mechanics of the sample under in-situ and non-contact conditions is realized. However, in existing research fields such as life science and materials science, the development has advanced from traditional static research to dynamic research, posing higher requirements for time resolution. Improving the time resolution of scanning ion conductance detection technology helps people better analyze dynamic behaviors such as cell proliferation, differentiation, and migration, observe the dynamic distribution changes of cell surface charges, explore the physiological behaviors of immune cells, monitor the distribution and dynamic changes of cell surface proteins, and observe the mechanism of action of drugs on cells in the field of life science. In the field of electrochemical measurement, it is conducive to carrying out research on the morphological changes and charge transfer processes on the interface of nanoelectrode materials, which has important scientific value for clarifying the structure-function relationships such as interface structure, composition, charge transfer, and electrocatalysis, and deeply exploring the reaction kinetics and internal mechanisms of material interfaces. However, although this type of technology and related instrument devices have made great progress in the past 20 years, the analysis sensitivity and spatial resolution of scanning ion conductance detection technology are still difficult to reach the single-molecule level. In addition, the scanning speed is as slow as the minute level, making it difficult to meet the requirements of single-molecule real-time three-dimensional localization imaging in living cells.

[0004] Currently, scanning ion conductance detection technology usually uses a piezoelectric ceramic with closed-loop control to control the probe position. Since this mode requires real-time measurement of the feedback value of the capacitance sensor, it will greatly reduce the response speed of the piezoelectric ceramic (only about one-tenth of the open-loop state), making it difficult to achieve fast scanning. At the same time, even if closed-loop control is used to offset the nonlinear effects such as hysteresis and creep of the piezoelectric ceramic, the noise in the signal transmission and acquisition process will also affect the final axial spatial resolution, making it still difficult for scanning ion conductance detection technology to meet the application requirements in dynamically and rapidly capturing key information. Summary of the Invention

[0005] To solve the problems existing in the prior art, the purpose of the present invention is to provide a scanning imaging system and method based on optical feedback positioning and ion conductance technology. The present invention can quickly and accurately control the probe position, solve the problem of slow scanning speed of scanning ion conductance detection equipment, and achieve the simultaneous improvement of imaging speed and accuracy.

[0006] The object of the present invention is achieved by the following technical solutions: A scanning imaging system based on optical feedback positioning and ion conductance technology, comprising an SICM ion current circuit, an optical path mirror and an adapter bracket. The adapter bracket is arranged on a moving platform capable of driving its movement. A laser generator is provided on the upper part of the adapter bracket. The optical path mirror is connected to the adapter bracket through a flexible hinge. The optical path mirror is used to horizontally reflect the emitted light of the laser generator. A prism system and a CCD camera are successively arranged on the reflected light path. The prism system is fixed on the adapter bracket. A micro motor is provided on the lower part of the adapter bracket. The output end of the micro motor is connected with a Z-direction piezoelectric ceramic. The Z-direction piezoelectric ceramic is connected with a piezoelectric ceramic controller and is connected with the flexible hinge. A probe holder is further provided on the Z-direction piezoelectric ceramic. The SICM probe of the SICM ion current circuit is arranged on the probe holder. The probe electrode of the SICM ion current circuit is arranged in the SICM probe.

[0007] Preferably, the laser generator is vertically arranged, and the laser generator can emit laser vertically downward. The optical path mirror adopts a triangular reflection lens.

[0008] Preferably, the flexible hinge includes a first connecting plate, a second connecting plate and a third connecting plate. One end of the first connecting plate is fixedly connected with the adapter bracket. The other end of the first connecting plate is elastically connected with one end of the second connecting plate through a first elastic connecting plate. The other end of the second connecting plate is a free end and is provided with the optical path mirror. The upper end of the third connecting plate is elastically connected with the body of the second connecting plate through a second elastic connecting plate. The lower end of the third connecting plate is fixedly connected with the Z-direction piezoelectric ceramic.

[0009] Preferably, the lower end of the first elastic connecting plate and the first connecting plate are of an integral structure. A U-shaped groove is provided on the first connecting plate at the connection with the first elastic connecting plate. The upper end of the first elastic connecting plate and the second connecting plate are of an integral structure. A U-shaped groove is provided on the second connecting plate at the connection with the first elastic connecting plate. The lower end of the second elastic connecting plate and the top of the third connecting plate are of an integral structure. The upper end of the second elastic connecting plate and the body of the second connecting plate are of an integral structure. U-shaped grooves are provided on both sides of the second connecting plate at the connection with the second elastic connecting plate.

[0010] Preferably, the CCD camera is arranged on a sliding bracket capable of adjusting the position and angle of the CCD camera. Both the sliding bracket and the moving platform are slidably arranged on the slide rail.

[0011] Preferably, the prism system adopts a roof prism module.

[0012] Preferably, the opening radius of the SICM probe is 10 - 500 nm.

[0013] Preferably, a sample cell for holding a sample to be measured is provided below the probe holder.

[0014] Preferably, the CCD camera is connected to a host computer. The host computer is used to calculate the displacement of the Z-axis piezoelectric ceramic based on the spot position information in the CCD camera. The host computer is also connected to a main controller. A signal generation and acquisition device is connected to the main controller. The signal generation and acquisition device includes an AD module and a DA module. The signal input end of the DA module is connected to the main controller. The positive electrode of the DA module is connected to the probe electrode of the SICM ion current circuit. The sample electrode of the SICM ion current circuit is connected to the input end of a current amplifier. The output end of the current amplifier is connected to the input end of the AD module. The output end of the AD module is connected to the negative electrode of the DA module. The motion platform, the piezoelectric ceramic controller, and the micro-motor are all connected to the main controller; The main controller can send an instruction to the DA module to make the DA module output a voltage in a preset form; The main controller can control the motion platform to move to control the horizontal movement of the Z-axis piezoelectric ceramic; The main controller can control the micro-motor to rotate to control the up and down movement of the Z-axis piezoelectric ceramic; The main controller can send an instruction to the piezoelectric ceramic controller to make the piezoelectric ceramic controller control the Z-axis piezoelectric ceramic to extend or shorten a preset length in the vertical direction.

[0015] The present invention also provides a scanning imaging method based on optical feedback positioning and ion conductance technology. The scanning imaging method is carried out by the scanning imaging system based on optical feedback positioning and ion conductance technology as described above in the present invention. The scanning imaging method includes: Preparation work: Place a sample cell below the probe holder, add an ionic solution to the sample cell and place the sample to be measured in the sample cell; adjust the light plate center of the light emitted by the laser generator to be located at the imaging zero position of the CCD camera; add an ionic solution to the SICM probe, and adjust the SICM probe to move to the initial position in the Z direction, and insert the sample electrode of the SICM ion current circuit into the sample cell; Positioning movement and information recording: The SICM probe moves downward in the Z direction. Using the spot displacement output by the spot displacement sensor of the CCD camera as the feedback of the SICM probe position information and the ionic current of the SICM ion current circuit as the detection feedback, the SICM probe is moved to a preset distance from the surface of the sample to be measured to achieve the positioning movement of the SICM probe. The preset distance is the opening radius of the SICM probe; record the SICM probe position information, ionic current information, and redox current information at this time, and then move the SICM probe to the initial position in the Z direction; Move the SICM probe to the next scanning point through the motion platform, and then repeat the process of the positioning movement and information recording for scanning until all scanning points are scanned; According to the SICM probe position information, ion current information, and redox current information of all the scanned points recorded, perform data processing to obtain the three-dimensional topography map and electrochemical information of the sample to be measured.

[0016] The present invention has the following beneficial effects: Through analysis, it is found that in the existing piezoelectric ceramics with closed-loop control, the feedback value of the capacitance sensor is measured in real time during operation, which greatly reduces the response speed of the piezoelectric ceramics, making it difficult to achieve fast scanning. Based on this, in the scanning imaging system based on optical feedback positioning and ion conductance technology of the present invention, a high-performance open-loop controlled piezoelectric ceramic in the Z direction is used as the SICM probe position controller, and the response speed is ten times that of the closed-loop state; an optical feedback positioning module is introduced in the feedback part, that is, the CCD camera can receive the spot displacement information caused by the displacement of the piezoelectric ceramic in the Z direction at high speed during use, so as to realize the high-speed synchronous reception and positioning of the SICM probe position; the planar CCD camera can not only detect the spot displacement in the Z direction, but also detect whether the spot generates displacement in the horizontal position, and realize the vertical calibration of the SICM probe through the spot displacement information in the two-dimensional plane; at the same time, the open-loop controlled piezoelectric ceramic in the Z direction avoids the hysteresis, creep and other non-linear effects of the piezoelectric ceramic in the closed-loop state, as well as the noise in the signal transmission and acquisition process, and improves the axial spatial resolution. Description of the Drawings

[0017] Figure 1 It is a schematic structural diagram of the adapter bracket and related connecting components in the scanning imaging system based on optical feedback positioning and ion conductance technology in the embodiment of the present invention; Figure 2 It is a schematic overall structural diagram of the scanning imaging system based on optical feedback positioning and ion conductance technology in the embodiment of the present invention; Figure 3(a) is a three-dimensional structural diagram of the flexible hinge in the embodiment of the present invention; Figure 3(b) is an indication diagram of the flexible hinge in the embodiment of the present invention; Figure 4 It is a flowchart of the scanning imaging method based on optical feedback positioning and ion conductance technology in the embodiment of the present invention.

[0018] In the figure: 1 is a laser generator, 2 is a transfer bracket, 3 is a prism system, 4 is a CCD camera, 5 is a sliding bracket, 6 is a motion platform, 7 is a Z-axis piezoelectric ceramic, 8 is an SICM probe, 9 is a probe holder, 10 is a triangular reflecting mirror, 11 is a flexible hinge, 11-1 is a first connecting plate, 11-2 is a second connecting plate, 11-3 is a third connecting plate, 11-4 is a first elastic connecting plate, 11-5 is a second elastic connecting plate, 11-6 is a groove, 12 is a cantilever beam, 13 is a sample electrode, 14 is a probe electrode, 15 is a sample box, 16 is a sample to be measured, and 17 is a slide rail. Detailed implementation manner

[0019] Combined with the accompanying drawings and the embodiments of the present invention below, the present invention will be clearly and completely described. The described embodiments are only a part of the embodiments of the present invention, not all of the embodiments.

[0020] Through analysis, the slow response speed of the piezoelectric controller in the existing scanning ion conductance microscope and scanning electrochemical cell microscope with closed-loop feedback causes the problem of slow scanning speed. Therefore, the present invention proposes a scanning imaging system and method based on optical feedback positioning and ion conductance technology. This system replaces the piezoelectric ceramic closed-loop feedback control with optical feedback control, changes the positioning method that only uses the piezoelectric ceramic capacitance sensor as feedback in such devices in the past, and fundamentally solves the problem of slow response speed of scanning ion conductance detection devices, greatly improving the scanning speed of such devices.

[0021] See Figure 1 and Figure 2 , the scanning imaging system based on optical feedback positioning and ion conductance technology in this embodiment includes an SICM ion current circuit, an optical path mirror, and a transfer bracket 2. The transfer bracket 2 is arranged on a motion platform 6 that can drive its movement. A laser generator 1 is provided on the upper part of the transfer bracket 2. The optical path mirror is connected to the transfer bracket 2 through a flexible hinge 11. The optical path mirror is used to horizontally reflect the emitted light of the laser generator 1. A prism system 3 and a CCD camera 4 are successively arranged on the reflected light path. The prism system 3 is fixed on the transfer bracket 2; a micro motor is provided on the lower part of the transfer bracket 2. The output end of the micro motor is connected to a Z-axis piezoelectric ceramic 7. The Z-axis piezoelectric ceramic 7 is connected to a piezoelectric ceramic controller and is connected to the flexible hinge 11. A probe holder 9 is also provided on the Z-axis piezoelectric ceramic 7. The SICM probe 8 of the SICM ion current circuit is arranged on the probe holder 9, and the probe electrode 14 of the SICM ion current circuit is arranged in the SICM probe 8.

[0022] See Figure 4 , using the scanning imaging method of the scanning imaging system based on optical feedback positioning and ion conductance technology in the above embodiment of the present invention, includes: Preparation: Place a sample box 15 below the probe holder 9, add an ionic solution to the sample box 15 and place the sample to be measured 16 in the sample box 15; adjust the center of the light plate of the light emitted by the laser generator 1 to be at the imaging zero position of the CCD camera 4; add an ionic solution to the SICM probe 8, and adjust the SICM probe 8 to move to the initial position in the Z direction, and insert the sample electrode 13 of the SICM ionic current circuit into the sample box 15; Positioning movement and information recording: The SICM probe 8 moves downward in the Z direction, using the spot displacement output by the spot displacement sensor of the CCD camera 4 as the position information feedback of the SICM probe 8, and using the ionic current of the SICM ionic current circuit as the detection feedback, so that the SICM probe 8 moves to a preset distance from the surface of the sample to be measured 16, realizing the positioning movement of the SICM probe 8. The preset distance is the opening radius of the SICM probe 8. During the process of moving the SICM probe 8 to a preset distance from the surface of the sample to be measured 16, the micro-motor can be controlled to drive the Z-direction piezoelectric ceramic 7 to quickly move in the Z direction first. During the movement, the displacement of the Z-direction piezoelectric ceramic 7 can be obtained through the optical information detected by the CCD camera 4. When the SICM probe 8 approaches the surface of the sample to be measured 16, then the piezoelectric ceramic controller is used to control the Z-direction piezoelectric ceramic 7 to perform Z-direction (i.e., vertical direction) displacement, so that the SICM probe 8 gradually approaches the surface of the sample to be measured 16. When the distance between the SICM probe 8 and the surface of the sample to be measured 16 reaches the opening radius of the SICM probe 8, at this time, an ionic current will be generated between the probe electrode 14 and the sample electrode 13 of the SICM ionic current circuit; record the position information, ionic current information and redox current information of the SICM probe 8 at this time, and then move the SICM probe 8 to the initial position in the Z direction; Move the SICM probe 8 to the next scanning point through the motion platform 6, and then repeat the process of the positioning movement and information recording for scanning until all scanning points are scanned; According to the recorded position information, ionic current information and redox current information of the SICM probe 8 at all scanning points, perform data processing to obtain the three-dimensional topography map and electrochemical information of the sample to be measured 16.

[0023] As a preferred embodiment of the present invention, for the convenience of calculation and calibration, in this embodiment, the laser generator 1 is vertically arranged, so that the laser generator 1 can emit a vertically downward laser, and the optical path reflector uses a triangular reflector 10.

[0024] As a preferred embodiment of the present invention, referring to Figures 3 (a) and 3 (b), in this embodiment, the flexible hinge 11 includes a first connecting plate 11-1, a second connecting plate 11-2 and a third connecting plate 11-3, one end of the first connecting plate 11-1 is fixedly connected to the adapter bracket 2, the other end of the first connecting plate 11-1 is elastically connected to one end of the second connecting plate 11-2 through a first elastic connecting plate 11-4, the other end of the second connecting plate 11-2 is a free end and is provided with the optical path reflector, the upper end of the third connecting plate 11-3 is elastically connected to the plate body of the second connecting plate 11-2 through a second elastic connecting plate 11-5, and the lower end of the third connecting plate 11-3 is fixedly connected to the Z-direction piezoelectric ceramic 7. In this embodiment, when the length of the Z-direction piezoelectric ceramic 7 changes, the Z-direction piezoelectric ceramic 7 can drive the third connecting plate 11-3 to move up and down. At this time, the third connecting plate 11-3 will apply an upward thrust or a downward pull to the second connecting plate 11-2. When the third connecting plate 11-3 pushes and pulls the second connecting plate 11-2, the elastic deformation of the first elastic connecting plate 11-4 can make the first connecting plate 11-1 and the second connecting plate 11-2 deflect at a certain angle at the first elastic connecting plate 11-4. At this time, the free end (i.e., the right end) of the second connecting plate 11-2 will be displaced up and down, and the light spot of the reflected light of the optical path reflector on the CCD camera 4 will be displaced. The displacement of the Z-direction piezoelectric ceramic 7 can be obtained through the displacement of the light spot and the fitting relationship. Moreover, when the third connecting plate 11-3 pushes and pulls the second connecting plate 11-2, the elastic deformation of the second elastic connecting plate 11-5 can make the third connecting plate 11-3 and the second connecting plate 11-2 deflect at a certain angle at the second elastic connecting plate 11-5. At this time, the third connecting plate 11-3 and the second connecting plate 11-2 can be adaptively deformed, thereby ensuring the deformation of the entire flexible hinge 11.

[0025] Further, as a further improvement of the above example of the present invention, referring to FIGS. 3(a) and 3(b), in this embodiment, the lower end of the first elastic connecting plate 11-4 and the first connecting plate 11-1 are of an integral structure, and a U-shaped groove 11-6 is provided on the first connecting plate 11-1 at the connection with the first elastic connecting plate 11-4; the upper end of the first elastic connecting plate 11-4 and the second connecting plate 11-2 are of an integral structure, and a U-shaped groove 11-6 is provided on the second connecting plate 11-2 at the connection with the first elastic connecting plate 11-4; the lower end of the second elastic connecting plate 11-5 and the top of the third connecting plate 11-3 are of an integral structure, the upper end of the second elastic connecting plate 11-5 and the body of the second connecting plate 11-2 are of an integral structure, and U-shaped grooves 11-6 are provided on both sides of the second connecting plate 11-2 at the connection with the second elastic connecting plate 11-5. Through the design of the above grooves 11-6, on the one hand, it can ensure that the deformation of the first elastic connecting plate 11-4 and the second elastic connecting plate 11-5 is easier and more flexible, thus ensuring the sensitivity of the position change of the optical path mirror. At the same time, it can also avoid the stress concentration phenomenon at the roots of the first elastic connecting plate 11-4 and the second elastic connecting plate 11-5.

[0026] Further, as a further improvement of the above example of the present invention, referring to Figure 1 , in this embodiment, a cantilever beam 12 can also be provided above the second connecting plate 11-2. One end of the cantilever beam 12 is connected to the second connecting plate 11-2, and the other end of the cantilever beam 12 is installed with the optical path mirror. Fixing the fixed end of the cantilever beam 12 at different positions of the second connecting plate 11-2 can realize flexible adjustment of the left and right positions of the optical path mirror, making the adjustment range of the present invention wider.

[0027] As a preferred embodiment of the present invention, the CCD camera 4 is arranged on a sliding bracket 5 capable of adjusting the position and angle of the CCD camera 4. Both the sliding bracket 5 and the moving platform 6 are slidably arranged on the slide rail 17, which is convenient for adjusting the relative position and angle between the CCD camera 4 and the prism system 3. In addition, the prism system 3 adopts a roof prism module. The opening radius of the SICM probe 8 is 10 - 500 nm. These settings help to improve the detection accuracy of the present invention.

[0028] As a preferred embodiment of the present invention, a sample box 15 can also be provided in this embodiment. The sample box 15 is arranged below the probe holder 9 and is used for holding the solution and the sample to be measured 16; during use, both the sample electrode 13 and the probe electrode 14 of the SICM ion current circuit are immersed in the solution of the sample box 15.

[0029] In addition, the SICM ion current circuit further includes a host computer, which is connected to the CCD camera 4. The host computer can calculate the displacement of the Z-axis piezoelectric ceramic 7 according to the spot position information in the CCD camera 4. The host computer is also connected to a main controller, and a signal generation and acquisition device is connected to the main controller. The signal generation and acquisition device includes an AD module and a DA module. The signal input end of the DA module is connected to the main controller. The positive electrode of the DA module is connected to the probe electrode 14 of the SICM ion current circuit. The sample electrode 13 of the SICM ion current circuit is connected to the input end of a current amplifier. The output end of the current amplifier is connected to the input end of the AD module. The output end of the AD module is connected to the negative electrode of the DA module. The motion platform 6, the piezoelectric ceramic controller, and the micro-motor are all connected to the main controller. The main controller can send an instruction to the DA module to make the DA module output a voltage in a preset form. The current amplifier can amplify the redox current information and the ion current signal between the probe electrode 14 and the sample electrode 13 and send them to the AD module. The AD module can convert the current signal (i.e., the redox current information and the ion current signal) into a digital signal and send it to the DA module. The DA module sends the current signal to the main controller, and the main controller transmits the current signal to the host computer. The main controller can control the movement of the motion platform 6 to control the movement of the Z-axis piezoelectric ceramic 7 in the horizontal direction. The main controller can control the rotation of the micro-motor to control the up and down movement of the Z-axis piezoelectric ceramic 7. The main controller can send an instruction to the piezoelectric ceramic controller to make the piezoelectric ceramic controller control the Z-axis piezoelectric ceramic 7 to extend or shorten a preset length in the vertical direction.

[0030] The present invention also provides a scanning imaging method based on optical feedback positioning and ion conductance technology. The scanning imaging method is carried out by the scanning imaging system based on optical feedback positioning and ion conductance technology as described above in the present invention. The scanning imaging method Embodiment 1 As Figure 2 shown in FIGS. 3(a) and 3(b), and with reference to Figure 1, the scanning imaging system based on optical feedback positioning and ion conductance technology in this embodiment mainly includes a PC, a main controller, a signal generation and acquisition device, a three-dimensional motion module, a laser generator 1, an optical path deflection module, a CCD camera 4, a SICM probe 8, a SICM ion current circuit, and a current amplifier. The Z-axis piezoelectric ceramic 7 of the three-dimensional motion module is fixedly connected to the three-axis sliding motion platform 6 through an adapter bracket 2. The optical path deflection module is installed on the Z-axis piezoelectric ceramic 7. The optical path deflection module includes a flexible hinge 11 connected to the adapter bracket 2 and the Z-axis piezoelectric ceramic 7, a cantilever beam 12 installed at the free end of the second connecting plate 11-2 of the flexible hinge 11, a triangular reflecting mirror 10 installed on the cantilever beam 12, and a probe holder 9 installed on the lower surface of the piezoelectric ceramic 7. The probe holder 9 is fixedly connected to the flexible hinge 11; the SICM probe 8 is installed on the probe holder 9, and the movement is controlled by the Z-axis piezoelectric ceramic 7; the laser generator 1 is installed above the left of the adapter bracket 2, and is calibrated by fine-tuning with nuts to keep the initial position of the laser generator 1 at a 45° angle with the optical path reflecting mirror; the prism system 3 is installed behind the adapter bracket 2, aligned with the center of the opening of the adapter bracket 2, and fixed by a prism clamp fastened with a bolt through a groove on the adapter bracket. The CCD camera 4 is installed on the sliding bracket 5, placed behind the adapter bracket 2, and the sensor direction of the CCD camera 4 is aligned with the center of the prism system 3 and the opening of the adapter bracket 2. The sample electrode 13 of the ion current circuit is connected to the current amplifier, the current amplifier is connected to the AD module of the signal generation and acquisition device, and the DA module of the signal generation and acquisition device is connected to the probe electrode 14 of the ion current circuit; the CCD camera 4 is connected to the main controller, and the main controller and the three-dimensional motion module are connected to the upper computer. The prism system 3 uses a roof prism module.

[0031] Among them, the PC is mainly used for human-computer interaction and mainly has the following functions: 1) Position display and setting of the micro-motor and piezoelectric ceramic: Display the real-time positions of the micro-motor and piezoelectric ceramic in the X, Y, and Z directions, and input and set the target positions of the micro-motor and piezoelectric ceramic in the X, Y, and Z directions.

[0032] 2) Search for the scanning starting position: Before scanning, it is necessary to first search for the approximate position of the sample, and based on prior knowledge, add the maximum amount of the sample's undulation height to the searched position and leave a certain margin to determine the starting position of the probe in the Z direction during scanning.

[0033] 3) Setting of scanning parameters: Set the scanning parameters before scanning imaging, such as the number of scanning points, the scanning point spacing, the starting and ending positions of the probe, the probe stop threshold, the scanning speed, the probe dwell time, the magnitude of the normal excitation voltage, the waveform of the variable excitation voltage signal, and the sampling frequency. By customizing the scanning parameters, different scanning requirements can be met.

[0034] 4) Preview of scanning data: During the scanning process, the human-machine interface will display the ion current magnitude and curve, the piezoelectric ceramic position change and curve, and the sample three-dimensional topography preview in real time, which can help us judge and detect abnormal situations during the scanning process.

[0035] 5) Saving of scanning data: After the scanning is completed, save the scanning data as text data through the human-machine interface for deeper analysis using Matlab data analysis software.

[0036] The main controller is connected to the PC through the RS232 interface, and receives various action instructions input by the human-machine interface. The instructions include the movement of the piezoelectric ceramic, scanning imaging under specified parameters, and transmission of scanning data. After receiving the action instructions, the main controller controls the movement of the Z-direction piezoelectric ceramic 7 through the piezoelectric ceramic controller, using the ion current signal collected by the signal generator collector or the laser spot signal received by the CCD camera 4 as feedback. When the ion current signal or the spot position received by the CCD camera 4 reaches their respective set thresholds, the Z-direction piezoelectric ceramic 7 stops moving. The movement of the micro-motor is controlled by directly connecting the PC, the main controller, and the micro-motor controller through the RS232 interface.

[0037] The main controller communicates with the signal generator collector through an optical fiber, transmitting the ion current and redox current collected by the signal generator collector for real-time feedback control of the micro-motor or piezoelectric ceramic, and saving the record and then transmitting it to the PC for subsequent in-depth analysis. At the same time, using optical fiber transmission can completely isolate the signal generator collector from the outside world, avoiding interference from the mains system to the signal generator collector. The CCD camera 4 is directly connected to the upper computer, and processes the received spot image through image processing algorithms to obtain the coordinate information of the spot center point.

[0038] The signal generation and acquisition device includes an AD module and a DA module. The AD module is used to collect voltage signals or current signals converted by a current amplifier and transmit them to the main controller. The DA module can output voltages of ±10V and is used to output different waveform voltage signals as needed, such as constant voltage, rectangular wave, triangular wave, sine wave, etc. The most commonly used is to use a triangular wave to test the cyclic voltammetry curve to obtain the electrochemical activity information of the sample (i.e., the sample to be tested 16). In scanning imaging, the ion current and redox current are usually only in the pA level or even smaller. Therefore, a current amplifier is used to amplify and convert the ion current and redox current, and then the converted current is transmitted to the AD module for acquisition. In addition, the signal generation and acquisition device is powered by a storage battery, effectively ensuring its complete isolation from the mains system.

[0039] The three-dimensional motion module (i.e., the motion platform 6) mainly includes micro-motors and piezoelectric ceramics in the X, Y, and Z directions and the corresponding controllers. Among them, the stroke of the X, Y, and Z-axis micro-motors is 15 mm each, which is used for the large-range movement of the probe. The stroke of the X and Y-axis piezoelectric ceramics is 100 μm, and the stroke of the Z-axis piezoelectric ceramic 7 is 30 μm. The Z-axis piezoelectric ceramic 7 is used for the high-precision movement of the probe, and the positioning accuracy of the Z-axis piezoelectric ceramic 7 can reach 0.1 nm.

[0040] The scanning imaging method based on optical feedback positioning and ion conductance technology in the embodiments of the present invention mainly includes the following steps: Step 1, fill the ion solution into the first channel of the SICM probe 8, insert the metal electrode (i.e., the probe electrode 14), fill the neodymium iron boron powder into the second channel, connect the electrodes at the sample end (i.e., the sample electrode 13), connect the bias voltage, TMR sensor, and current amplifier loop, and check whether the output current signals of the piezoelectric ceramics, micro-motors, ion current, and TMR sensor are normal; Step 2, search and determine the initial scanning position. Set the search speed, stop judgment method, and threshold value on the human-computer interaction interface, and then the Z-axis piezoelectric ceramic 7 and the micro-motor start to cooperate with each other for the downward exploration movement. When the Z-axis piezoelectric ceramic 7 has completed a stroke (30 μm), if it has not reached near the sample, the PC drives the micro-motor controller through the RS232 interface to control the Z-axis micro-motor to move downward by 25 μm, and at the same time, the Z-axis piezoelectric ceramic 7 retracts to the highest point to start the search and detection of the next stroke. This process is repeated until the SICM probe 8 moves near the sample surface (the distance is about the size of the probe opening radius, generally 10 nanometers to 500 nanometers). Then, based on prior knowledge, estimate the maximum undulation amount of the sample, add this undulation amount to the current position of the Z-axis piezoelectric ceramic 7, and leave a margin of 1 - 5 μm to avoid the SICM probe 8 colliding with the sample, resulting in the fragmentation of the SICM probe 8 and the failure of the scan.

[0041] The feedback control during the lowering process of the SICM probe 8 is divided into two parts, namely optical feedback control and ion current feedback control. The principle of optical control is as follows: When the Z-axis piezoelectric ceramic 7 moves to drive the SICM probe 8 to lower, it will drive the third connecting plate 11-3 of the flexible hinge 11 to displace synchronously. The third connecting plate 11-3 pulls the second connecting plate 11-2 downward. After the second connecting plate 11-2 is stressed, it will pull the first elastic connecting plate 11-4 to bend. At this time, the free end of the second connecting plate 11-2 will move downward, thereby causing the triangular mirror 10 and the reflection optical path to deflect, changing the position of the light spot irradiated on the photodetector. By calculating the center position of the light spot through Gaussian fitting of the light spot image, the position of the nano-capillary during the scanning process can be obtained in real time. The principle of ion current feedback control is as follows: The ion current only changes when the SICM probe 8 is less than the opening radius of the SICM probe 8 from the sample. Therefore, the ion current change curve can be used to accurately position the probe, so that the SICM probe 8 moves to a position where the distance from the sample surface is the opening radius of the SICM probe 8 (usually 10 nanometers to 500 nanometers).

[0042] Step 3: Set the scanning parameters on the human-machine interface, such as the number of scanning points, the scanning point spacing, the starting and ending positions of the probe, the probe stop threshold, the scanning speed, the probe residence time, the voltage magnitude of the normal excitation voltage, the waveform of the variable excitation voltage signal, and the sampling frequency. The number of scanning points and the scanning point spacing jointly determine the size of the scanning area, and the scanning point spacing directly affects the fineness of the scanning result. Increase the scanning speed as much as possible within the allowable range (10 - 500 μm / ms), but too fast scanning speed may lead to a decline in imaging quality, or even the probe colliding with the sample and breaking. At the same time, the probe can be controlled to stay for a period of time when it moves near the sample to perform actions such as substance delivery, cyclic voltammetry curve detection, charge measurement, and sample stiffness detection.

[0043] Step 4: Start scanning after the parameters are set. First, use the optical signal and ion current as feedback. The optical signal provides the position feedback signal, and the ion current serves as the threshold signal for reaching the specified position. The SICM probe 8 descends at a high speed (10 - 500 μm / ms) in the Z direction until it moves to a position where the distance from the sample surface is the opening radius of the probe (usually 10 nanometers to 500 nanometers). Record the position information, ion current information, and redox current information of the SICM probe 8 at this time. The SICM probe 8 quickly retracts to the initial Z position and moves to the next scanning point along the X-axis or Y-axis, repeating the above steps until the detection imaging of all scanning points is completed.

[0044] Step 5: Read the scanning result data and process data on the human-machine interface, save them as text, and process the data in the Matlab data analysis software to obtain the three-dimensional topography map and electrochemical information of the sample.

[0045] Among them, the initial position of the SICM probe 8 in the Z direction is determined by combining the ion current change curve with the prior knowledge of the sample undulation height. According to the probe position and the corresponding spot displacement, the relationship curve between the probe position and the corresponding spot displacement is fitted, and the spot displacement amount is converted into probe position information according to this relationship curve. The spot image is calculated by the Gaussian fitting method to obtain the center point coordinates and calculate the displacement amount.

[0046] In summary, the present invention introduces an optical feedback positioning module in the existing scanning ion conductance detection instrument, which is used to detect the optical path deflection caused by the angular deformation of the flexible hinge due to the probe displacement, so as to position the probe with the laser spot displacement amount as the feedback, replacing the closed-loop feedback control with the open-loop control of the piezoelectric ceramic, changing the positioning method of such devices that only use the piezoelectric ceramic capacitance sensor as the feedback in the past, fundamentally solving the problem of slow response speed of the scanning ion conductance detection device, and greatly improving the scanning speed of such devices. The main controller takes the FPGA as the core, can monitor the change of the ion current, respond quickly to the ion current change exceeding the threshold, and lift the probe. The optical feedback directly connects the CCD camera to the upper computer, fits the laser spot image to obtain the center coordinate point, and obtains the corresponding curve between the piezoelectric ceramic displacement and the spot displacement. The scanning imaging method of the present invention based on optical feedback positioning and ion conductance technology uses the laser spot displacement and the ion current as the composite feedback to control the movement of the probe. First, taking the laser spot displacement received by the CCD camera as the position feedback, the probe quickly descends in the Z direction and maintains the ion current feedback monitoring. When the probe moves to a distance from the sample surface equal to the probe opening radius, the ion current changes, and the probe position information, ion current information, and redox current information at this time are recorded to complete the scanning of this point. Then the probe quickly retreats to the initial position in the Z direction and moves to the next scanning point along the X axis or Y axis, repeating the above steps to perform the scanning of the next point until the detection imaging of all scanning points is completed, and the three-dimensional topography map and electrochemical information of the sample are obtained by processing the above data. This method can retain the advantages of in-situ, non-contact, high-precision, and multi-dimensional imaging of the scanning ion conductance detection device while solving the current bottleneck problems of such devices, greatly improving its scanning imaging speed, and having broad application prospects in the fields of life science, medicine, electrochemistry, and materials science. The present invention combines and complements the optical feedback positioning and ion conductance detection technologies, and proposes a scanning imaging system and method based on optical feedback positioning and ion conductance technology. This system replaces the piezoelectric ceramic closed-loop control with optical feedback positioning, approaches the sample with the ion current as the feedback, realizes nano-level detection, and fundamentally solves the problem of slow response speed of the piezoelectric ceramic in the scanning ion conductance detection technology, greatly improving the detection speed of the scanning ion conductance detection device.

[0047] Obviously, the described embodiments are only partial embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0048] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the above embodiments, those of ordinary skill in the art should understand that modifications or equivalent replacements can still be made to the specific implementation manners of the present invention. Any modification or equivalent replacement without departing from the spirit and scope of the present invention shall be covered by the protection scope of the present invention.

Claims

1. A scanning imaging system based on optical feedback positioning and ion conductivity technology, characterized in that: The invention comprises a SICM ion current circuit, an optical path reflector and an adapter bracket (2). The adapter bracket (2) is arranged on a motion platform (6) capable of driving the adapter bracket to move. A laser generator (1) is arranged on the upper part of the adapter bracket (2). The optical path reflector is connected to the adapter bracket (2) via a flexible hinge (11). The optical path reflector is used to horizontally reflect the output light of the laser generator (1). A prism system (3) and a CCD camera (4) are arranged in sequence on the reflected light path. The prism system (3) is fixed on the adapter bracket (2). A micro motor is arranged on the lower part of the adapter bracket (2). The output end of the micro motor is connected to a Z-direction piezoelectric ceramic (7). The Z-direction piezoelectric ceramic (7) is connected to a piezoelectric ceramic controller and is connected to the flexible hinge (11). A probe holder (9) is also arranged on the Z-direction piezoelectric ceramic (7). A SICM probe (8) of the SICM ion current circuit is arranged on the probe holder (9). A probe electrode (14) of the SICM ion current circuit is arranged in the SICM probe (8).

2. The scanning imaging system based on optical feedback positioning and ion conductivity technology according to claim 1, characterized in that: The laser generator (1) is arranged vertically, and is capable of radiating laser light vertically downwards; the optical path reflector is a triangular reflector lens (10).

3. The scanning imaging system based on optical feedback positioning and ion conductivity technology according to claim 1, characterized in that: The flexible hinge (11) comprises a first connecting plate (11-1), a second connecting plate (11-2) and a third connecting plate (11-3); one end of the first connecting plate (11-1) is fixedly connected to the adapter bracket (2); the other end of the first connecting plate (11-1) is elastically connected to one end of the second connecting plate (11-2) via a first elastic connecting plate (11-4); the other end of the second connecting plate (11-2) is a free end and is provided with the optical path reflector; the upper end of the third connecting plate (11-3) is elastically connected to the body of the second connecting plate (11-2) via a second elastic connecting plate (11-5); and the lower end of the third connecting plate (11-3) is fixedly connected to the Z-direction piezoelectric ceramic (7).

4. The scanning imaging system based on optical feedback positioning and ion conductivity technology according to claim 3, characterized in that: The lower end of the first elastic connecting plate (11-4) is an integral structure with the first connecting plate (11-1), and a U-shaped groove (11-6) is provided on the first connecting plate (11-1) at the connection with the first elastic connecting plate (11-4); the upper end of the first elastic connecting plate (11-4) is an integral structure with the second connecting plate (11-2), and a U-shaped groove (11-6) is provided on the second connecting plate (11-2) at the connection with the first elastic connecting plate (11-4); the lower end of the second elastic connecting plate (11-5) is an integral structure with the top of the third connecting plate (11-3), the upper end of the second elastic connecting plate (11-5) is an integral structure with the body of the second connecting plate (11-2), and U-shaped grooves (11-6) are provided on both sides of the connection between the second connecting plate (11-2) and the second elastic connecting plate (11-5).

5. The scanning imaging system based on optical feedback positioning and ion conductivity technology according to claim 1, characterized in that: The CCD camera (4) is arranged on a sliding bracket (5) capable of adjusting the position and angle of the CCD camera (4), and the sliding bracket (5) and the motion platform (6) are both slidably arranged on a slide rail (17).

6. The scanning imaging system based on optical feedback positioning and ion conductivity technology according to claim 1, characterized in that: The prism system (3) adopts a roof prism module.

7. The scanning imaging system based on optical feedback positioning and ion conductivity technology according to claim 1, characterized in that: The opening radius of the SICM probe (8) is 10~500nm.

8. The scanning imaging system based on optical feedback positioning and ion conductivity technology according to claim 1, characterized in that: A sample box (15) for containing a sample (16) to be tested is provided below the probe holder (9).

9. The scanning imaging system based on optical feedback positioning and ion conductivity technology according to claim 1, characterized in that: The CCD camera (4) is connected to a host computer, and the host computer is used to calculate the displacement of the piezoelectric ceramic (7) in the Z direction according to the light spot position information in the CCD camera (4); the host computer is also connected to a main controller, and the main controller is connected to a signal generator and collector, and the signal generator and collector includes an AD module and a DA module, the signal input end of the DA module is connected to the main controller, the positive electrode of the DA module is connected to the probe electrode (14) of the SICM ion current circuit, the sample electrode (13) of the SICM ion current circuit is connected to the input end of a current amplifier, the output end of the current amplifier is connected to the input end of the AD module, and the output end of the AD module is connected to the negative electrode of the DA module, and the motion platform (6), the piezoelectric ceramic controller and the micromotor are all connected to the main controller; The main controller can send instructions to the DA module to make the DA module output a voltage in a preset form; The main controller is capable of controlling the movement of the motion platform (6) to control the Z-direction piezoelectric ceramic (7) to move in a horizontal direction; The main controller can control the rotation of the micromotor to control the up and down movement of the Z-axis piezoelectric ceramic (7); The main controller can send instructions to the piezoelectric ceramic controller, so that the piezoelectric ceramic controller controls the Z-direction piezoelectric ceramic (7) to extend or shorten a preset length in the vertical direction.

10. A scanning imaging method based on optical feedback positioning and ion conductivity technology, characterized in that: The scanning imaging method is performed by a scanning imaging system based on optical feedback positioning and ion conductivity technology according to any one of claims 1 to 9, and the scanning imaging method comprises: Preparation: Place a sample box (15) under the probe holder (9), add an ion solution into the sample box (15) and place a sample to be tested (16) in the sample box (15); adjust the center of the light plate of the laser generator (1) to be located at the imaging zero point position of the CCD camera (4); add an ion solution into the SICM probe (8), adjust the SICM probe (8) to move to the initial position in the Z direction, and insert the sample electrode (13) of the SICM ion current circuit into the sample box (15); Positioning movement and information recording: the SICM probe (8) moves downward along the Z direction, the spot displacement output by the spot displacement sensor of the CCD camera (4) is used as the position information feedback of the SICM probe (8), and the ion current of the SICM ion current circuit is used as the detection feedback, so that the SICM probe (8) moves to a preset distance from the surface of the sample to be tested (16), thereby realizing the positioning movement of the SICM probe (8), wherein the preset distance is the opening radius of the SICM probe (8); the position information, ion current information and redox current information of the SICM probe (8) are recorded at this time, and then the SICM probe (8) is moved to the initial position in the Z direction; The SICM probe (8) is moved to the next scanning point by the motion platform (6), and then the positioning movement and information recording process is repeated to perform scanning until all scanning points are scanned; Data processing is performed based on the position information, ion current information and redox current information of the SICM probe (8) at all the recorded scanning points to obtain a three-dimensional morphology image and electrochemical information of the sample to be tested (16).

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