Multifunctional scanning photoelectrochemical cell microscope and single-cell photoelectrochemical synchronous measurement method

Abstract

A multifunctional scanning photoelectrochemical cell microscope and a method for simultaneous photoelectrochemical measurement of individual nanoparticles are presented. By constructing a multifunctional scanning photoelectrochemical cell microscope, the SECCM system, optical imaging system, and spectroscopic measurement system are organically integrated. This enables simultaneous dynamic measurement and imaging of electrochemical and spectral characteristics of nanoparticle analytes participating in the reaction process. Furthermore, a method for simultaneous photoelectrochemical measurement of individual nanoparticles is provided, avoiding the complex process of secondary measurement to obtain information about individual samples. This invention significantly improves the timeliness, accuracy, and multidimensional fusion of data acquisition, providing a powerful tool platform and system solution for the analysis of individual physical structures, surface molecule recognition, and reaction mechanism research at the micro- and nano-scale.

Description

Technical Field

[0001] This invention relates to the field of electrochemical testing and analysis technology, and in particular to a multifunctional scanning photoelectrochemical cell microscope based on spectral imaging and a method for simultaneous photoelectric measurement of a single individual. Background Technology

[0002] Scanning Electrochemical Cell Microscopy (SECCM) is an electrochemical characterization technique that originated in 2010, first proposed by the Unwin research group at the University of Warwick, UK. The core of SECCM lies in achieving high spatial resolution for electrochemical detection by constructing tiny electrochemical cells, thus enabling the study of electrochemical processes at the microscopic scale. A typical SECCM setup employs a dual-electrode system, where the object under study serves as the working electrode, and a nanoprobe acts as a quasi-reference electrode, filled with solution and intercalated with Ag / AgCl wires to maintain a stable potential. The electrochemical information provided by SECCM primarily reflects electrochemical processes such as charge transfer and ion transport on the sample surface; this information often cannot be directly correlated with the sample's size and structure.

[0003] In practical applications, to infer the size and structure of a sample, it is usually necessary to combine SECCM characterization with other characterization techniques, such as scanning electron microscopy (SEM) and transmission electron microscopy (TEM), to obtain more comprehensive information. For example, the paper "Interfacial Structure and Energy Determine the Heterogeneity in the Electrochemical Metal Dissolution Activity at Grain Boundary," published in the journal *Chemistry of Materials* (2023, Vol. 35, pp. 4243–4249), systematically studied how the structure and energy of grain boundaries affect the activity of metal dissolution reactions by combining scanning electrochemical cell microscopy (SECCM), focused ion beam (FIB), and electron microscopy. This study pointed out that "SECCM electrochemical imaging must be performed first, followed by secondary verification using electron microscopy or other structural characterization methods" to infer the structural characteristics of the sample. Therefore, this secondary detection method makes it difficult to simultaneously obtain electrochemical and structural information, leading to a significant increase in experimental complexity, time cost, and error. Furthermore, errors can arise due to resolution differences between different techniques and variations during the secondary detection process. Therefore, it is very necessary to develop a device that can simultaneously and dynamically measure and image electrochemical information and spectral characteristics during the reaction process, but there is currently no such integrated device or method. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the present invention aims to provide a multifunctional scanning photoelectrochemical cell microscope and a single-particle photoelectrochemical synchronous measurement method, which can realize the synchronous dynamic measurement and imaging of electrochemical information and spectral characteristic information of nanoparticle single-particle analyte samples during the reaction process; it also provides a single-particle photoelectrochemical synchronous measurement method, which avoids the complex process of secondary measurement to obtain single-particle sample information, and has the advantages of timeliness, accuracy and multidimensional fusion.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] The multifunctional scanning photoelectrochemical cell microscope includes a SECCM system, an optical imaging system, and a spectroscopic measurement system connected to the optical imaging system; the optical imaging system transmits the collected light synchronization signal to the spectroscopic measurement system.

[0007] The optical imaging system is used for the positioning and imaging of a single sample, including a reverse dark-field microscope installed below the body of the reverse dark-field microscope. A dichroic mirror is provided below the reverse dark-field microscope. The dichroic mirror reflects the light signal from the reverse dark-field microscope in the horizontal direction and guides it to the color CCD camera to form a reflective dark-field microscopy imaging optical path.

[0008] The spectral measurement system is used to measure the fluorescence spectrum, Raman spectrum, SERS, and Rayleigh scattering spectrum of a single sample. It includes a white light or laser source incident vertically, a mirror, a spectrometer, and a spectral CCD camera, which together form the spectral analysis path. The white light or laser source is focused onto the sample area by a reverse dark-field microscope. The spectral signal generated after the sample interacts with the light is collected in reverse by the reverse dark-field microscope and transmitted to a dichroic mirror. The dichroic mirror, acting as a partial reflector, reflects a portion of the light signal to the color CCD camera, enabling real-time visualization and dark-field imaging of the sample. The remaining light signal continues in a straight line and is reflected by the mirror to the spectral analysis path, entering the spectrometer for wavelength decomposition. The multi-band spectral signal after dispersion is received and recorded by the spectral CCD camera, realizing the acquisition and digital processing of the spectral signal.

[0009] The SECCM system, used to measure the electrochemical information of samples, consists of a three-dimensional positioning system and a signal acquisition and processing module. The three-dimensional positioning system includes an electrochemical probe, which is fixed to a Z-axis piezoelectric ceramic displacement stage via a cantilever. The Z-axis piezoelectric ceramic displacement stage is connected to the output shaft of a Z-axis stepper motor to achieve vertical adjustment control. Horizontal XY-axis adjustment control is achieved collaboratively by the XY-axis piezoelectric ceramic and the stepper motor; that is, the power outputs of both the XY-axis piezoelectric ceramic and the stepper motor are connected to the stage. The signal acquisition and processing module includes a current amplifier. After the electrochemical probe contacts the sample surface, the generated electrical signal is first amplified by the current amplifier and then transmitted to an analog-to-digital converter to convert the analog signal into a digital signal. The converted signal is received and processed by a computer to record, analyze, and image the electrochemical signal. Simultaneously, the computer sends control commands to the XY-axis stepper motor, XY-axis piezoelectric ceramic, Z-axis stepper motor, and Z-axis piezoelectric ceramic displacement stage to complete the path planning and scanning execution of the electrochemical probe in three-dimensional space, achieving spatial electrochemical measurement.

[0010] The electrochemical probe is composed of an ultrathin gold film attached to the inner wall of the tip of a glass tube.

[0011] The single-cell photoelectric synchronous measurement method based on the above-mentioned multifunctional scanning photoelectrochemical cell microscope includes the following steps:

[0012] Step 1: Spray noble metal nanoparticles onto an ITO glass plate to form an electrode surface. The sprayed noble metal nanoparticles are individual particles. Place the ITO glass plate as a sample on the stage. Pour the electrolyte solution into the prepared electrochemical probe. Insert silver / silver chloride as a quasi-reference electrode into the electrochemical probe. The tip of the electrochemical probe contacts the electrode surface of the ITO glass plate through a liquid bridge to form a three-dimensional micro-electrochemical cell.

[0013] Step 2: Using the computer-controlled SECCM system's three-dimensional positioning system, the electrochemical probe is brought close to the electrode surface of the ITO glass plate. Under the set voltage, a weak current is formed between the electrochemical probe and the electrode surface. The feedback current information is amplified by a current amplifier and converted into a digital signal that the computer can process by an analog-to-digital converter. This process obtains the position information of the individual sample and the instantaneous electrochemical information of the electrode surface. The instantaneous electrochemical information includes cyclic voltammetry curves, polarization curves, impedance spectra, and linear sweep voltammetry curves.

[0014] Step 3: White light or laser light is incident vertically below the stage. The scattered light from the sample surface is collected by an inverted dark-field microscope. A dichroic mirror reflects part of the light signal from the dark-field microscope in the horizontal direction and guides it to a color CCD camera. The color CCD camera acquires the positioning and imaging information of a single sample.

[0015] Step 4: Another portion of the light signal from the reverse dark-field microscope is further reflected by the mirror and enters the spectrometer for analysis and conversion. It is received and recorded by the spectral CCD camera to obtain the instantaneous fluorescence spectrum, Raman spectrum, instantaneous SERS and instantaneous Rayleigh scattering spectrum of the individual sample.

[0016] The specific method for obtaining the location information of a single sample and the transient electrochemical information of the electrode surface is as follows:

[0017] First, the electrochemical probe is lowered a predetermined distance Δ along the Z-axis by controlling the Z-axis piezoelectric ceramic displacement stage. z and during the descent, at Δ t Record current values ​​in real time at time intervals I The speed of the electrochemical probe is controlled to be v vertical_down When the droplet at the tip of the electrochemical probe contacts the electrode surface of the ITO glass plate, a significant change in current occurs. The feedback current signal automatically determines whether the electrochemical probe has contacted the sample surface and determines its height information in the Z-axis direction. Subsequently, the movement distance Δ of the XY-axis piezoelectric ceramic and the stepper motor is set. x and Δ y With the electrochemical probe fixed, the stage is moved, and based on the feedback current signal of the electrochemical probe at each position, the spatial position information of the sample in the XY axis direction of the plane is further obtained, so as to realize the synchronous measurement of the sample position and its instantaneous electrochemical response at a specific position.

[0018] The method for obtaining the instantaneous fluorescence spectrum of a single sample is as follows: The sample is locally excited using a white light source with an excitation wavelength in the visible or ultraviolet band, causing fluorescent active molecules in the sample to transition to an excited state and release fluorescence during their return to the ground state. The fluorescence signal is collected in reverse by a reverse dark-field microscope and then transmitted to a dichroic mirror. The dichroic mirror, acting as a partial reflector, reflects a portion of the fluorescence signal to a color CCD camera, enabling real-time visualization and dark-field imaging of the sample. The remaining fluorescence signal continues to travel in a straight line and is reflected by the reflector to the spectral analysis path, entering the spectrometer for wavelength decomposition. The multi-band fluorescence spectrum signal after dispersion is received and recorded by a spectral CCD camera, realizing the acquisition and digital processing of the instantaneous fluorescence spectrum of a single sample.

[0019] The method for obtaining the Raman spectrum of a single sample specifically involves: introducing a laser source as the Raman excitation light to irradiate the sample surface, causing the sample molecules to undergo Raman scattering with the incident light. This Raman scattering signal is collected in reverse by a reverse dark-field microscope and then transmitted to a dichroic mirror. The dichroic mirror, acting as a partial reflector, reflects a portion of the scattered signal to a color CCD camera, enabling real-time visualization and dark-field imaging of the sample. The remaining scattered signal continues to travel in a straight line and is reflected by the reflector to the spectral analysis path, entering the spectrometer for wavelength decomposition. The dispersed Raman spectral signal is received by a spectral CCD camera, which records its Raman shift and intensity, thus realizing the acquisition and digital processing of the instantaneous Raman spectrum of a single sample.

[0020] The method for obtaining transient SERS of a single sample is as follows: The electrochemical probe is composed of an ultrathin gold film attached to the inner wall of the tip of a glass tube, which can generate a local surface plasmon resonance effect. Under laser irradiation, a high-intensity local electromagnetic field is formed, which enhances the molecular signal near the hot spot area of ​​the electrochemical probe. After the molecular signal is enhanced, it is collected in reverse by a reverse dark-field microscope and then transmitted to a dichroic mirror. The dichroic mirror acts as a partial reflector, reflecting a portion of the scattered enhanced signal to a color CCD camera to achieve real-time visualization, positioning, and dark-field imaging. The remaining enhanced signal continues to travel in a straight line and is reflected by the reflector to the spectral analysis path, where it enters the spectrometer for wavelength decomposition. The SERS spectral signal after dispersion is received and recorded by a spectral CCD camera, realizing the acquisition and digital processing of the transient SERS signal.

[0021] The method for obtaining the instantaneous Rayleigh scattering spectrum of a single sample is as follows: A laser or white light is irradiated onto the sample electrode surface coated with noble metal nanoparticles, causing photons to undergo elastic scattering after interacting with particles smaller than the wavelength. The scattered light signal is collected in reverse by a reverse dark-field microscope and then transmitted to a dichroic mirror. The dichroic mirror, acting as a partial reflector, reflects a portion of the scattered light signal to a color CCD camera, enabling real-time visualization and dark-field imaging of the sample. The remaining scattered light signal continues to travel in a straight line and is reflected by the reflector to the spectral analysis path, entering the spectrometer for wavelength decomposition. The dispersed Rayleigh scattering spectral signal is received and recorded by a spectral CCD camera, realizing the acquisition and digital processing of the instantaneous Rayleigh scattering spectrum.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] (1) This invention constructs a multifunctional scanning photoelectrochemical cell microscope, which organically integrates the SECCM system, optical imaging system and spectral measurement system, and can realize photoelectric synchronous dynamic measurement and real-time imaging of electrochemical information and spectral feature information at the individual scale such as a single nanoparticle.

[0024] (2) This invention can not only accurately obtain the instantaneous electrochemical response of a single organism during the reaction process (such as CV curve, LSV curve, impedance spectrum, etc.), but also simultaneously record its corresponding fluorescence spectrum, Raman spectrum, SERS and Rayleigh scattering spectrum and other spectroscopic features, thereby revealing the structural, composition and functional changes of a single research object in multiple dimensions, spatially, synchronously, and in multiple dimensions.

[0025] In summary, this invention significantly improves the timeliness, accuracy, and multidimensional fusion of data acquisition, providing a powerful tool platform and system solution for the analysis of individual physical structures, surface molecule recognition, and reaction mechanism research at the micro-nano scale. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the structure of a multifunctional scanning photoelectrochemical microscope.

[0027] In the figure, 1. Stage; 2. Electrochemical probe; 3. Cantilever; 41. Z-axis piezoelectric ceramic displacement stage; 42. Z-axis stepper motor; 5. Current amplifier; 6. Analog-to-digital converter; 7. Computer; 81. XY-axis stepper motor; 82. XY-axis piezoelectric ceramic; 9. Reverse dark-field microscope body; 10. Reverse dark-field microscope; 11. Dichroic mirror; 12. Color CCD camera; 13. Mirror; 14. Spectrometer; 15. Spectroscopic CCD camera. Detailed Implementation

[0028] The present invention will now be further described with reference to the accompanying drawings.

[0029] like Figure 1 As shown, the multifunctional scanning photoelectrochemical cell microscope includes a SECCM system located above stage 1, an optical imaging system located below stage 1, and a spectral measurement system connected to the optical imaging system; the optical imaging system transmits the collected light synchronization signal to the spectral measurement system.

[0030] The SECCM system, used to measure the electrochemical information of samples, consists of a three-dimensional positioning system and a signal acquisition and processing module. The three-dimensional positioning system includes an electrochemical probe 2, which is fixed to a Z-axis piezoelectric ceramic displacement stage 41 via a cantilever 3. The Z-axis piezoelectric ceramic displacement stage 41 is connected to the output shaft of a Z-axis stepper motor 42 to achieve vertical adjustment control. Horizontal XY-axis adjustment control is achieved collaboratively by an XY-axis piezoelectric ceramic 82 and a stepper motor 81; that is, the power outputs of both the XY-axis piezoelectric ceramic 82 and the stepper motor 81 are connected to the stage 1, forming a complete three-dimensional positioning system. The signal acquisition and processing module includes a current... After the electrochemical probe 2 contacts the sample surface, the generated electrical signal is first amplified by the current amplifier 5, and then transmitted to the analog-to-digital converter 6 to convert the analog signal into a digital signal. The converted signal is received and processed by the computer 7 to realize the recording, analysis and imaging of the electrochemical signal. At the same time, the computer 7 also sends control commands to the XY-axis stepper motor 81, XY-axis piezoelectric ceramic 82, Z-axis stepper motor 42 and Z-axis piezoelectric ceramic displacement stage 41 to complete the path planning and scanning execution of the electrochemical probe 2 in three-dimensional space, thereby realizing accurate spatial electrochemical measurement. Among them, the XY-axis stepper motor 81 and Z-axis stepper motor 42 realize large displacement adjustment, and the XY-axis piezoelectric ceramic 82 and Z-axis piezoelectric ceramic displacement stage 41 realize fine adjustment.

[0031] The optical imaging system is used for the positioning and imaging of individual samples. It includes a reverse dark-field microscope 10 mounted below the body 9 of the reverse dark-field microscope, used to focus the imaging light path and achieve sample observation. A dichroic mirror 11 is located below the reverse dark-field microscope 10. The dichroic mirror 11 horizontally reflects the image light signal from the reverse dark-field microscope 10 and guides it to a color CCD camera 12. The color CCD camera 12 receives the reflected light signal and performs real-time imaging, enabling real-time visualization, positioning, and dark-field imaging of the sample. All components in the optical imaging system are fixedly connected via standard optical interfaces, forming a stable reflective dark-field microscopy imaging light path.

[0032] The spectral measurement system is used to measure the fluorescence spectrum, Raman spectrum, SERS, and Rayleigh scattering spectrum of a single sample. It includes a white light or laser source incident vertically below the stage 1, a reflector 13, a spectrometer 14, and a spectral CCD camera 15, which together constitute the spectral analysis path. The white light or laser source is focused onto the sample area by a reverse dark-field microscope 10. After the sample interacts with the light, the resulting spectral signal is collected in reverse by the reverse dark-field microscope 10 and transmitted to a dichroic mirror 11. The dichroic mirror 11 acts as a partial reflector, reflecting a portion of the light signal to the color CCD camera 12, realizing real-time visualization and dark-field imaging of the sample. The remaining light signal continues to travel in a straight line and is reflected by the reflector 13 to the spectral analysis path, entering the spectrometer 14 for wavelength decomposition. The multi-band spectral signal after dispersion is received and recorded by the spectral CCD camera 15, realizing the acquisition and digital processing of spectral signals. The reflector 13 is connected to the spectrometer 14 via an optical fiber or a standard optical interface. The output of the spectrometer 14 is connected to the spectral CCD camera 15, forming a continuous and stable spectral imaging measurement path.

[0033] The electrochemical probe 2 is composed of an ultrathin gold film attached to the inner wall of the glass tube tip, which can generate a local surface plasmon resonance effect.

[0034] The specific preparation steps of the electrochemical probe 2 are as follows:

[0035] Step 1: Preparation of nanopipettes by laser drawing method: Using glass capillary tubes as molds, nanopipettes are prepared using a P-2000 laser needle drawing instrument;

[0036] Step 2: Preparation of ultrathin gold film-attached nanopipettes by chemical modification: C2H5OH and HAuCl4 solution were mixed in a volume ratio of 2:3 and filled into the glass nanopipettes described in Step 1. The mixture was irradiated under a 254 nm 8 W UV light source for 7 hours, rinsed with C2H5OH and deionized water, dried, and then annealed at 100 °C for 1 hour.

[0037] The method for simultaneous photoelectric measurement of a single organism based on the above-mentioned multifunctional scanning photoelectrochemical cell microscope includes the following steps:

[0038] Step 1: Spray noble metal nanoparticles onto an ITO glass plate to form an electrode surface. The sprayed noble metal nanoparticles are individual particles. Place the ITO glass plate as a sample on stage 1. Pour the electrolyte solution into the prepared electrochemical probe 2. Insert silver / silver chloride as a quasi-reference electrode into the electrochemical probe 2. The tip of the electrochemical probe 2 contacts the electrode surface of the ITO glass plate through a liquid bridge to form a three-dimensional micro-electrochemical cell.

[0039] Step 2: The three-dimensional positioning system of the SECCM system, controlled by computer 7, brings the electrochemical probe 2 close to the electrode surface of the ITO glass plate. Under the set voltage, a weak current is formed between the electrochemical probe 2 and the electrode surface. The feedback current information is amplified by the current amplifier 5 and converted into a digital signal that can be processed by the computer 7 by the analog-to-digital converter 6. This is to obtain the position information of the individual sample and the instantaneous electrochemical information of the electrode surface. The instantaneous electrochemical information includes the cyclic voltammetry curve (CV curve), polarization curve, impedance spectrum, and linear scan voltammetry curve (LSV curve).

[0040] Step 3: White light or laser light is incident vertically below the stage 1. The scattered light from the sample surface is collected by the inverted reverse dark field microscope 10. The dichroic mirror 11 reflects part of the light signal from the reverse dark field microscope 10 in the horizontal direction and guides it to the color CCD camera 12. The color CCD camera 12 acquires the positioning and imaging information of the individual sample.

[0041] Step 4: Another portion of the light signal from the reverse dark-field microscope 10 is further reflected by the mirror 13 and enters the spectrometer 14 for analysis and conversion. It is received and recorded by the spectral CCD camera 15 to obtain the transient fluorescence spectrum, Raman spectrum, transient SERS and transient Rayleigh scattering spectrum of the individual sample.

[0042] The specific method for obtaining the location information of a single sample and the transient electrochemical information of the electrode surface is as follows:

[0043] First, the electrochemical probe 2 is lowered along the Z-axis by a set distance Δ by controlling the Z-axis piezoelectric ceramic displacement stage 41. z and during the descent, at Δ t Record current values ​​in real time at time intervals I The speed of electrochemical probe 2 is controlled as follows: v vertical_down When the droplet at the tip of electrochemical probe 2 contacts the conductive electrode surface of the ITO glass plate, a significant change in current occurs (specifically, the threshold for the current abrupt change is determined based on the background current before the droplet at the tip of electrochemical probe 2 contacts the sample surface). I threshold When the droplet has not yet contacted the sample surface, the electrochemical circuit is not closed, and the current satisfies... I < I thresholdWhen the droplet contacts the sample surface and forms an electrochemical pathway, the current suddenly increases to I I threshold The feedback current signal automatically determines whether the electrochemical probe 2 has contacted the sample surface and determines its height information in the Z-axis direction; subsequently, the moving distance Δ of the XY-axis piezoelectric ceramic 82 and the stepper motor 81 is set. x and Δ y With the electrochemical probe 2 fixed, the stage 1 is moved, and based on the feedback current signals from the electrochemical probe 2 at various positions, the spatial position information of the sample in the XY axis direction is further acquired, realizing the synchronous measurement of the sample position and its instantaneous electrochemical response at a specific position. Whenever the probe moves to a new measurement position and contacts the sample surface again, the system automatically performs current acquisition and can simultaneously trigger laser excitation, realizing the synchronous acquisition of electrochemical and spectral signals.

[0044] The method for obtaining the instantaneous fluorescence spectrum of a single sample specifically involves: locally exciting the sample using a white light source with an excitation wavelength in the visible or ultraviolet band, causing fluorescent active molecules in the sample to transition to an excited state and release fluorescence during their return to the ground state. The fluorescence signal is collected in reverse by a reverse dark-field microscope 10 and then transmitted to a dichroic mirror 11. The dichroic mirror 11 acts as a partial reflector, reflecting a portion of the fluorescence signal to a color CCD camera 12, enabling real-time visualization and dark-field imaging of the sample. The remaining fluorescence signal continues to travel in a straight line and is reflected by a mirror 13 to the spectral analysis path, entering the spectrometer 14 for wavelength decomposition. The multi-band fluorescence spectrum signal after dispersion is received and recorded by a spectral CCD camera 15, realizing the acquisition and digital processing of the instantaneous fluorescence spectrum of a single sample.

[0045] The method for obtaining the Raman spectrum of a single sample is as follows: a laser light source of a specific wavelength is introduced as the Raman excitation light to irradiate the sample surface, causing the sample molecules to undergo Raman scattering with the incident light. The Raman scattering signal is collected in reverse by a reverse dark-field microscope 10 and then transmitted to a dichroic mirror 11. The dichroic mirror 11 acts as a partial reflector, reflecting a portion of the scattered signal to a color CCD camera 12, thereby achieving real-time visualization and dark-field imaging of the sample. The remaining scattered signal continues to travel in a straight line and is reflected by a reflector 13 to the spectral analysis path, entering the spectrometer 14 for wavelength decomposition. The Raman spectral signal after dispersion is received by a spectral CCD camera 15, which records its Raman shift and intensity, thereby realizing the acquisition and digital processing of the instantaneous Raman spectrum of a single sample.

[0046] The method for obtaining transient SERS of a single sample is as follows: the electrochemical probe 2 is composed of an ultrathin gold film attached to the inner wall of the tip of a glass tube, which can generate a local surface plasmon resonance effect. Under laser irradiation, it forms a high-intensity local electromagnetic field, which enhances the molecular signal near the hot spot area of ​​the electrochemical probe 2. After the molecular signal is enhanced, it is collected in reverse by the inverted dark-field microscope 10 and continues to be transmitted to the dichroic mirror 11. The dichroic mirror 11 acts as a partial reflector, reflecting a portion of the scattered enhanced signal to the color CCD camera 12, realizing real-time visualization and dark-field imaging of the sample. The remaining enhanced signal continues to travel in a straight line and is reflected by the reflector 13 to the spectral analysis path, entering the spectrometer 14 for wavelength decomposition. The SERS spectral signal after dispersion is received and recorded by the spectral CCD camera 15, thereby obtaining the transient SERS signal, which is suitable for detection and reaction process monitoring at the single-molecule level.

[0047] The method for obtaining the instantaneous Rayleigh scattering spectrum of a single sample is as follows: A laser or white light is irradiated onto the sample electrode surface coated with noble metal nanoparticles, causing photons to undergo elastic scattering after interacting with particles smaller than the wavelength. The scattered light signal is collected in reverse by a reverse dark-field microscope 10 and transmitted to a dichroic mirror 11. The dichroic mirror 11 acts as a partial reflector, reflecting a portion of the scattered light signal to a color CCD camera 12, enabling real-time visualization and dark-field imaging of the sample. The remaining scattered light signal continues along a straight line and is reflected by a mirror 13 to the spectral analysis path, entering a spectrometer 14 for wavelength decomposition. The dispersed Rayleigh scattering spectrum signal is received and recorded by a spectral CCD camera 15. The instantaneous Rayleigh scattering spectrum obtained in this way can reflect the particle size, morphology, and interaction characteristics of the nanoparticles with the surrounding medium, and can assist in electrochemical measurements to determine the sample type and surface structure.

[0048] This invention, while measuring single-point kinetics using a SECCM system, employs a reverse dark-field microscopy imaging and spectral measurement system to simultaneously collect and identify multiple spectral signals (fluorescence, Raman, SERS, Rayleigh scattering) from a single sample (corresponding to the matrix-like point-by-point movement of the electrochemical probe). The measurement optical path propagates from bottom to top, transmitting and splitting along the following path: a laser or white light source is emitted from below and enters the reverse dark-field microscope body 9 vertically; the light is focused by the reverse dark-field microscope 10 onto the sample area located on the stage 1; at the sample, light-matter interaction occurs, including... Multiple signals, including fluorescence emission, Raman scattering, and Rayleigh scattering, are emitted. All upward-scattered light is collected in reverse by the reverse dark-field microscope 10 and then transmitted upward to the dichroic mirror 11. The dichroic mirror 11 is a partial reflector used to reflect a portion of the light signal to the first color CCD camera 12, enabling real-time visualization and dark-field imaging of the sample. The remaining light signal continues to travel in a straight line and is reflected by the reflector 13 to the spectral analysis path. The spectrometer 14 receives the composite light signal introduced by the reflector and performs wavelength decomposition. The multi-band spectral signal after spectral separation is received by the spectral CCD 15 for image recording and digital acquisition.

[0049] The entire measurement optical path adopts a two-stage beam splitting path design to achieve parallel operation of "real-time imaging" and "spectral acquisition" of the signal, taking into account both spatial and spectral information acquisition, and providing key support for photoelectric synchronous measurement. Among them, the dichroic mirror 11 is mainly responsible for reflecting the visible light image to the color CCD camera 12 to achieve real-time positioning and tracking; while the reflector 13 is dedicated to transmitting the sorted spectral signal to the spectrometer 14 and the spectral CCD camera 15 for component analysis and signal interpretation.

[0050] This invention organically integrates a SECCM with a reverse dark-field microscopy imaging system and a spectral measurement system to construct a photoelectric collaborative measurement platform with high spatial resolution and high temporal response capability. This system can simultaneously acquire electrochemical signals and multimodal spectral signals (including fluorescence spectroscopy, Raman spectroscopy, SERS, Rayleigh scattering spectroscopy, etc.) the instant the electrochemical probe comes into contact with a single sample, enabling multidimensional, holographic measurement and real-time imaging of the dynamic response process of single samples such as single nanoparticles, single molecules, or micro-region active sites.

[0051] This invention utilizes the synergistic acquisition and fusion analysis of electrochemical information (such as CV curves, polarization curves, and charge transfer behavior) and spectral characteristics (such as Raman peak positions, fluorescence intensity, and scattering distribution) to precisely reveal the intrinsic properties of samples in terms of structure, composition, and reaction behavior at the micro- and nano-scale. This technology overcomes the time lag and spatial mismatch limitations of traditional asynchronous measurement methods, significantly enhancing the comprehensive understanding of the physical, chemical, and biological properties of individual research objects. It provides a universally applicable and cutting-edge research tool and methodology for fields such as single-molecule reaction mechanism analysis, nanomaterial heterogeneity research, and biomolecule recognition.

Claims

1. A multi-functional scanning photoelectrochemical cell microscope, characterized by, The SECCM system, the optical imaging system, and the spectral measurement system connected with the optical imaging system; the optical imaging system transmits the collected optical synchronization signal to the spectral measurement system; The optical imaging system is used for positioning and imaging of single samples, and includes an inverted dark field microscope (10) installed below the body (9) of the inverted dark field microscope, and a dichroic mirror (11) arranged below the inverted dark field microscope (10), which reflects the light signal from the inverted dark field microscope (10) in the horizontal direction and guides the light signal to a color CCD camera (12), forming a reflective dark field microscopic imaging light path; The spectral measurement system is used for measuring the fluorescence spectrum, Raman spectrum, SERS and Rayleigh scattering spectrum of single samples, and includes a white light or laser light source, a mirror (13), a spectrometer (14) and a spectral CCD camera (15) arranged in the vertical direction, which together constitute a spectral analysis path; wherein the white light or laser light source is focused by the inverted dark field microscope (10) and irradiated to the sample area; after the sample interacts with the light, the spectral signal is collected by the inverted dark field microscope (10) in the reverse direction and continues to be transmitted to the dichroic mirror (11), which reflects part of the light signal to the color CCD camera (12) as a partial mirror, realizing real-time visual positioning and dark field imaging of the sample; the remaining light signal continues to travel in a straight line and is reflected by the mirror (13) to the spectral analysis path, enters the spectrometer (14) for wavelength decomposition, and the multi-band spectral signal after the light is split is received and recorded by the spectral CCD camera (15), realizing the collection and digital processing of the spectral signal; The SECCM system is used for measuring the electrochemical information of the sample, and is composed of a three-dimensional positioning system and a signal acquisition and processing module; the three-dimensional positioning system includes an electrochemical probe (2), which is fixed on a Z-axis piezoelectric ceramic displacement table (41) through a cantilever (3), and the Z-axis piezoelectric ceramic displacement table (41) is connected with the output shaft of a Z-axis stepping motor (42) to realize adjustment and control in the vertical direction; the adjustment and control in the horizontal XY-axis direction is realized by the cooperation of an XY-axis piezoelectric ceramic (82) and a stepping motor (81), that is, the power output of the XY-axis piezoelectric ceramic (82) and the stepping motor (81) are connected with the object table (1); the signal acquisition and processing module includes a current amplifier (5), after the electrochemical probe (2) contacts the sample surface, the generated electrical signal is first amplified by the current amplifier (5), then transmitted to an analog-to-digital converter (6) to complete the conversion of the analog signal to the digital signal, and the converted signal is received and processed by a computer (7), realizing the recording, analysis and imaging of the electrochemical signal; at the same time, the computer (7) also sends control instructions to the XY-axis stepping motor (81), the XY-axis piezoelectric ceramic (82), the Z-axis stepping motor (42) and the Z-axis piezoelectric ceramic displacement table (41), completes the path planning and scanning execution of the electrochemical probe (2) in the three-dimensional space, and realizes the spatial electrochemical measurement.

2. The multi-functional scanning photoelectrochemical cell microscope according to claim 1, wherein, The electrochemical probe (2) is composed of an ultrathin gold film attached to the inner wall of the tip of a glass tube.

3. The method of single-body photoelectric synchronous measurement by multifunctional scanning photoelectrochemical cell microscope according to claim 2, characterized in that, The method comprises the following steps: Step one, noble metal nanoparticles are sprayed on the ITO glass plate to form an electrode surface, the sprayed noble metal nanoparticles are single bodies, the ITO glass plate is placed on the stage (1) as a sample, an electrolyte solution is filled into the prepared electrochemical probe (2), silver / silver chloride is used as a quasi-reference electrode and inserted into the electrochemical probe (2), the tip of the electrochemical probe (2) is in contact with the electrode surface of the ITO glass plate through a liquid bridge, and a three-dimensional micro electrochemical cell is formed; Step two, the three-dimensional positioning system of the SECCM system is controlled by the computer (7) to make the electrochemical probe (2) close to the electrode surface of the ITO glass plate, a weak current is formed between the electrochemical probe (2) and the electrode surface under a set voltage, the feedback current information is amplified by the current amplifier (5), the continuous current signal collected by the electrochemical probe (2) is converted into a digital signal that can be processed by the computer (7) by the analog-to-digital converter (6), so that the position information of the single body sample and the instantaneous electrochemical information of the electrode surface are obtained, and the instantaneous electrochemical information includes a cyclic voltammetry curve, a polarization curve, an impedance spectrum and a linear sweep voltammetry curve; Step three, white light or laser light is incident along the vertical direction below the stage (1), the scattered light of the sample surface is collected by the inverted reverse dark field microscope (10), the dichroic mirror (11) reflects part of the light signal from the reverse dark field microscope (10) in the horizontal direction and guides the light signal to the color CCD camera (12), and the color CCD camera (12) acquires the positioning and imaging information of the single body sample; Step four, another part of the light signal from the reverse dark field microscope (10) is further reflected by the mirror (13) into the spectrometer (14) for analysis and conversion, is received and recorded by the spectral CCD camera (15), and the instantaneous fluorescence spectrum, the Raman spectrum, the instantaneous SERS and the instantaneous Rayleigh scattering spectrum of the single body sample are obtained.

4. The single-body optoelectronic synchronous measurement method according to claim 3, characterized in that, The method for obtaining the position information of the single body sample and the instantaneous electrochemical information of the electrode surface is as follows: First, by controlling the Z-axis piezoelectric ceramic displacement table (41) to make the electrochemical probe (2) along the Z-axis direction to drop a set distance Δ z , and in the process of falling in Δ t time interval real-time recording current value I , the speed of the electrochemical probe (2) is v vertical_down ; when the electrochemical probe (2) tip droplet contact ITO glass plate electrode surface, the current generated will appear obvious change, feedback current signal automatically determine whether the electrochemical probe (2) has contacted the sample surface, and determine its height information in the Z-axis direction; Subsequently, the moving distance ΔX and ΔY of the XY-axis piezoelectric ceramic (82) and the stepping motor (81) are set x and Δ y The sample stage (1) is moved with the electrochemical probe (2) fixed, and the spatial position information of the sample in the XY-axis direction is further acquired according to the feedback current signals of the electrochemical probe (2) at each position, so that the synchronous measurement of the sample position and the instantaneous electrochemical response of the sample at a specific position is realized.

5. The single-body optoelectronic synchronous measurement method according to claim 3, characterized in that, The method for obtaining the instantaneous fluorescence spectrum of the single body sample is as follows: a white light source with an excitation wavelength in the visible light or ultraviolet band is used to locally excite the sample, so that the fluorescent active molecules in the sample transition to an excited state and release fluorescence in the process of returning to the ground state, the fluorescence signal is reversely collected by the reverse dark field microscope (10) and continues to be transmitted to the dichroic mirror (11); the dichroic mirror (11) is used as a partial mirror, a part of the fluorescence signal is reflected to the color CCD camera (12), real-time visual positioning and dark field imaging of the sample are realized, and the remaining fluorescence signal continues to travel along a straight line and is reflected by the mirror (13) to the spectral analysis path and enters the spectrometer (14) for wavelength decomposition, the multi-band fluorescence spectrum signal after spectral decomposition is received and recorded by the spectral CCD camera (15), and the collection and digital processing of the instantaneous fluorescence spectrum of the single body sample are realized.

6. The single-body optoelectronic synchronous measurement method according to claim 3, characterized in that, The method for acquiring the Raman spectrum of a single sample comprises the following steps: introducing a laser light source as a Raman excitation light to irradiate a sample surface, so that molecules of the sample are subjected to Raman scattering with the incident light; the Raman scattering signal is reversely collected by a reverse dark field microscope (10) and continuously transmitted to a dichroic mirror (11); the dichroic mirror (11) serves as a partial mirror, and a part of the scattering signal is reflected to a color CCD camera (12) to realize real-time visual positioning and dark field imaging of the sample; the remaining scattering signal continues to move in a straight line and is reflected by a mirror (13) to a spectral analysis path, and then enters a spectrometer (14) to be subjected to wavelength decomposition; and the Raman spectrum signal after being subjected to the wavelength decomposition is received by a spectral CCD camera (15) and recorded for Raman shift and intensity, so that the instantaneous Raman spectrum of the single sample is acquired and subjected to digital processing.

7. The single-body optoelectronic synchronous measurement method according to claim 3, characterized in that, The method for acquiring the instantaneous SERS of a single sample comprises the following steps: an electrochemical probe (2) is formed by attaching an ultrathin gold film to the inner wall of a glass tube tip, and can generate a localized surface plasmon resonance effect to form a high-intensity localized electromagnetic field under laser irradiation, so that the molecular signal near the hot spot region of the electrochemical probe (2) is enhanced; the enhanced molecular signal is reversely collected by a reverse dark field microscope (10) and continuously transmitted to a dichroic mirror (11); the dichroic mirror (11) serves as a partial mirror, and a part of the scattering enhanced signal is reflected to a color CCD camera (12) to realize real-time visual positioning and dark field imaging; the remaining enhanced signal continues to move in a straight line and is reflected by a mirror (13) to a spectral analysis path, and then enters a spectrometer (14) to be subjected to wavelength decomposition; and the SERS spectrum signal after being subjected to the wavelength decomposition is received by a spectral CCD camera (15) and recorded, so that the instantaneous SERS signal is acquired and subjected to digital processing.

8. The single-body optoelectronic synchronous measurement method according to claim 3, characterized in that, The method for acquiring the instantaneous Rayleigh scattering spectrum of a single sample comprises the following steps: laser or white light is irradiated to the surface of a sample electrode coated with noble metal nanoparticles, so that photons are elastically scattered after interacting with particles smaller than the wavelength; the scattered light signal after the elastic scattering is reversely collected by a reverse dark field microscope (10) and continuously transmitted to a dichroic mirror (11); the dichroic mirror (11) serves as a partial mirror, and a part of the scattered light signal is reflected to a color CCD camera (12) to realize real-time visual positioning and dark field imaging of the sample; the remaining scattered light signal continues to move in a straight line and is reflected by a mirror (13) to a spectral analysis path, and then enters a spectrometer (14) to be subjected to wavelength decomposition; and the Rayleigh scattering spectrum signal after being subjected to the wavelength decomposition is received by a spectral CCD camera (15) and recorded, so that the instantaneous Rayleigh scattering spectrum is acquired and subjected to digital processing.

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