In-situ active oxygen quantum detection equipment based on fluorescent nano-diamond nitrogen vacancy color center

Abstract

The invention relates to the technical field of quantum precision measurement, and discloses fluorescent nano-diamond nitrogen vacancy color center-based in-situ active oxygen quantum detection equipment, which comprises a to-be-detected sample coupling module, an integrated laser module, a scanning confocal light path, an integrated circuit module and a data processing module. The to-be-tested sample coupling module is used for bearing samples in different forms; an acousto-optic modulator is arranged in the integrated laser module, and the integrated laser module is used for generating first-order diffraction light in a continuous wave mode and a pulse mode; the scanning confocal light path performs sample positioning and in-situ scanning by using a three-dimensional piezoelectric nano displacement table, and collects NV color center fluorescence; the integrated circuit module and the data processing module cooperatively execute an all-optical detection pulse sequence, double-index fitting is performed on a fluorescence-free evolution attenuation curve, and the spin depolarization time inversion active oxygen concentration of an internal color center is extracted. According to the invention, the influence of sample drift is effectively overcome, and high-spatial-resolution active oxygen in-situ quantitative detection is realized.

Description

Technical Field

[0001] This invention relates to the field of quantum precision measurement technology, specifically to an in-situ reactive oxygen quantum detection device based on fluorescent nanodiamond nitrogen vacancy color centers. Background Technology

[0002] Reactive oxygen species (ROS), as key signaling molecules and metabolites in living organisms, are closely related to various physiological and pathological processes due to abnormal changes in their concentrations. Nitrogen vacancy (NV) centers in fluorescent nanodiamonds, as quantum sensors, possess excellent photostability and biocompatibility, and are gradually becoming an important means of ROS detection. However, existing detection technologies based on fluorescent nanodiamonds still face several technical bottlenecks in practical applications.

[0003] First, when performing in-situ detection in liquid environments or inside living cells, nanoscale sensor particles are subject to random displacement due to Brownian motion. Furthermore, the system is susceptible to thermal drift caused by changes in ambient temperature during long-term observation. This spatial instability makes it difficult to lock the detection focus onto the same particle for extended periods, easily leading to signal loss or measurement interruptions, and hindering long-term stable monitoring.

[0004] Secondly, traditional NV color center sensing schemes typically rely on external microwave fields to manipulate spin states. High-power microwave radiation can easily generate thermal effects in biological samples, altering their physiological state or introducing additional measurement interference. Furthermore, nanodiamond particles contain both internal and surface color centers, and current technologies often lack effective optical and algorithmic methods to separate these signals. This results in the detection result being an average of numerous color center signals, reducing the sensitivity to changes in external reactive oxygen species concentrations.

[0005] Finally, in the detection of complex biological samples, background fluorescence and stray light outside the focal plane significantly reduce the signal-to-noise ratio, affecting the accuracy of photon counting. Furthermore, existing detection devices are typically designed for a single type of sample, lacking universal adaptability to different sample morphologies. This makes it difficult to maintain high spatial resolution while ensuring operational flexibility, thus limiting the applicability of the detection equipment. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides an in-situ reactive oxygen species (ROS) quantum detection device based on fluorescent nanodiamond nitrogen-vacancy color centers. This device solves the problem that existing ROS detection technologies struggle to simultaneously achieve nanoscale spatial resolution, in-situ real-time monitoring, and long-term stable tracking in complex intracellular microenvironments or solution environments.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution: an in-situ reactive oxygen quantum detection device based on nitrogen-vacancy color centers in fluorescent nanodiamonds. This device utilizes the characteristic that nitrogen-vacancy color centers in fluorescent nanodiamonds are sensitive to magnetic noise, and measures the spin depolarization time of the color centers through an integrated opto-electro-mechanical system, thereby inverting the concentration of reactive oxygen in the environment.

[0008] The testing equipment mainly includes a sample coupling module, a scanning confocal optical path, an integrated laser module, an integrated circuit module, and a data processing module; The sample coupling module is used to carry the sample containing fluorescent nanodiamonds. It can be connected to the three-dimensional piezoelectric nano-displacement stage in the scanning confocal optical path through a mechanical interface. The integrated laser module generates first-order diffraction light in response to the driving signal. The first-order diffraction light includes a continuous wave mode for fluorescence imaging of the sample under test and a pulse mode for optical polarization and readout of the spin state of the color center in the fluorescent nanodiamond. The scanning confocal optical path is used to establish the optical transmission path between the first-order diffraction light and the sample to be tested. The first-order diffraction light is focused onto the sample to be tested. The sample is then subjected to coarse positioning and in-situ scanning with nanometer-level precision by a manual three-dimensional micrometer displacement stage and a three-dimensional piezoelectric nanometer displacement stage, and the fluorescence signal emitted by the stimulated emission of the color center is collected. The data processing module is connected to the integrated circuit module to collaboratively execute quantum detection timing, obtain the spin depolarization time of fluorescent nanodiamonds based on the acquired fluorescence signals, and inversely determine the reactive oxygen concentration in the environment of the sample under test based on the change in spin depolarization time.

[0009] Preferably, the data processing module incorporates all-optical detection and measurement logic, which controls the integrated circuit module. The drive signal output from the RF generation unit controls the integrated laser module to generate an optical sequence including polarization pulses and a period of free evolution without illumination. The digital delayed pulse generator controls the single-photon detector to generate a readout window of a specific width, i.e., a readout pulse. First, the color center spin is polarized to the ground state using polarization pulses. Then, during the period of free evolution without illumination, the color center spin depolarization is affected by environmental magnetic noise. After the period of free evolution without illumination ends, the next polarization pulse arrives synchronously with the readout pulse, which reads the fluorescence signal intensity corresponding to the aforementioned free evolution period. Finally, the data processing module controls the hardware to repeatedly execute the sequence by traversing different free evolution durations to obtain the fluorescence decay curve.

[0010] Preferably, based on the difference in relaxation characteristics between surface and internal color centers in fluorescent nanodiamonds, the data processing module performs a double-exponential fitting analysis on the fluorescence decay curve to extract the slow component of spin depolarization time corresponding to the spin of the internal color centers in the fluorescent nanodiamonds. The data processing module then calls a preset relaxation rate and paramagnetic substance concentration calibration curve and calculates the quasi-real-time concentration of reactive oxygen species based on this slow component of spin depolarization time.

[0011] Preferably, to achieve optical pulses in the nanosecond to microsecond range, the integrated circuit module employs discrete RF generation and modulation links. The microcontroller configures an RF source chip and an RF attenuation chip to generate an initial RF signal with a specific frequency and power. A digital delayed pulse generator outputs a TTL-level signal corresponding to the all-optical detection and measurement logic to control the on / off state of the RF switch chip. The controlled RF signal is amplified by an RF amplifier to form a drive signal, which drives the acousto-optic modulator in the integrated laser module to convert continuous laser light into first-order diffracted light using the acousto-optic effect, and generates an optical sequence according to the measurement logic.

[0012] Preferably, the scanning confocal optical path employs a spatial filtering structure. First-order diffracted light enters the optical path after passing through an aspherical lens. An integrated LED light source provides wide-field white light illumination for auxiliary positioning of the sample field of view. The objective lens focuses the laser onto the sample surface and collects the sample's fluorescence signal and reflected white light. The collected beam passes through a dichroic mirror and a filter, then is split into two paths by a beam splitter: one path enters the industrial camera for imaging; the other path passes through a pinhole filter structure to remove stray light and is coupled into a multimode fiber for transmission to the single-photon detector. The LED light source must be turned off during photon counting. The single-photon detector converts the photon signal into electrical pulses, which are counted by a digital delay pulse generator within a synchronized readout time window.

[0013] Preferably, to address the sample position drift issue during long-term measurements, the data processing module runs an automatic tracking program. This program sets an initial fluorescence count baseline and percentage threshold, and monitors the current fluorescence count in real time. When the fluorescence count change exceeds the threshold, the system triggers a position correction mechanism: it sends instructions to the integrated circuit module to drive the three-dimensional piezoelectric nanostage to perform step scans along the X, Y, and Z axes of the target region, performs Gaussian fitting on the scan data to determine the new three-dimensional coordinates, and then updates the stage position to ensure the stability of long-term detection.

[0014] Preferably, the substrate surface of the sample coupling module is machined with rectangular grooves for adapting to glass slides and circular grooves for adapting to culture dishes, and is fixed by a mechanical clamping assembly, which can accommodate samples of different shapes. A light-transmitting hole is provided at the center of the substrate to ensure unobstructed light path between the objective lens and the bottom of the sample.

[0015] Compared with the prior art, the present invention has the following beneficial technical effects: 1. This invention uses a data processing module in conjunction with a three-dimensional piezoelectric nano-displacement stage to execute an automatic tracking program based on fluorescence feedback, monitors photon count in real time and corrects the spatial coordinates of the sample, overcoming the detection failure problem caused by Brownian motion of nanoparticles and thermal drift of the system in liquid environment, and realizing long-term and stable in-situ monitoring of single-particle fluorescent nanodiamonds.

[0016] 2. By using a fully optical detection pulse sequence and a double exponential fitting analysis performed by a data processing module, this invention can effectively extract the slow component of spin depolarization time corresponding to the spin of the color center inside fluorescent nanodiamonds. Spin state readout can be achieved without an external microwave field, thereby enabling low-interference and high-sensitivity quantitative detection of reactive oxygen species concentration in complex biological environments.

[0017] 3. This invention, through the pinhole filter structure and single-photon detection logic in the scanning confocal optical path, combined with the multi-specification mechanical interface design of the sample coupling module, suppresses background stray light and improves the signal-to-noise ratio, while being compatible with the loading requirements of different morphological samples such as standard solutions and live cells, providing a universal detection platform with nanometer-level spatial resolution. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the sample coupling module according to an embodiment of the present invention; Figure 2 This is a schematic diagram of different states of a sample to be tested according to an embodiment of the present invention; Figure 3 This is a schematic diagram of the scanning confocal optical path according to an embodiment of the present invention; Figure 4 This is a schematic diagram of the optical path principle of a scanning confocal optical path according to an embodiment of the present invention; Figure 5 This is a partial structural schematic diagram of a scanning confocal optical path according to an embodiment of the present invention; Figure 6 This is a schematic diagram of the structure of an integrated laser module according to an embodiment of the present invention; Figure 7 This is a schematic diagram of the optical path principle of an integrated laser module according to an embodiment of the present invention; Figure 8 This is a connection block diagram illustrating the signal interaction relationship between the integrated circuit module and its functional sub-modules according to an embodiment of the present invention. Figure 9 This is a schematic diagram of the overall quantum detection results based on fluorescent nanodiamonds according to an embodiment of the present invention.

[0019] Among them, 100, scanning confocal optical path; 101, three-dimensional piezoelectric nano-displacement stage; 102, manual three-dimensional micro-displacement stage; 103, objective lens; 104, mirror one; 105, dichroic mirror; 106, beam splitter one; 107, LED light source; 108, aspherical lens one; 109, filter; 110, lens; 111, industrial camera; 112, beam splitter two; 113, pinhole filter structure; 200, integrated laser module; 201, laser diode; 202, rotating half-wave plate; 203, polarized beam splitter cube; 204, acousto-optic modulator; 205, mirror two; 206, aperture; 207, aspherical lens two; 300, sample coupling module; 400, integrated circuit module; 500, data processing module; 600, mechanical clamping assembly; 700, rectangular groove; 800, circular groove. Detailed Implementation

[0020] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings and examples.

[0021] like Figure 1-8 As shown, this embodiment of the invention provides an in-situ reactive oxygen species quantum detection device based on fluorescent nanodiamond nitrogen-vacancy color centers. The detection device mainly includes a sample coupling module 300, a scanning confocal optical path 100, an integrated laser module 200, an integrated circuit module 400, and a data processing module 500. These five modules are interconnected via optical fibers, cables, and mechanical interfaces to form a closed-loop control and detection system.

[0022] The sample coupling module 300 is mainly composed of a substrate with positioning grooves adapted to different sample shapes, including a rectangular groove 700 for placing a glass slide and a circular groove 800 for placing a culture dish. The substrate is also equipped with a mechanical clamping assembly 600 for fixing the glass slide or culture dish. A light-transmitting hole is located at the center of the substrate to ensure the laser beam passes through and irradiates the sample. The sample coupling module 300 is fixed to the three-dimensional piezoelectric nano-displacement stage 101 of the scanning confocal optical path 100 by mechanical fasteners, allowing for spatial movement with nanometer-level precision along with the three-dimensional piezoelectric nano-displacement stage 101.

[0023] The scanning confocal optical path 100 adopts a modular optical cage structure. The excitation light input end is connected to the integrated laser module 200 via a single-mode fiber to receive the first-order diffracted light transmitted from it; the fluorescence output end is connected to the single-photon detector in the integrated circuit module 400 via a multimode fiber. The scanning confocal optical path 100 internally houses a dichroic mirror 105, a beam splitter group (beam splitter one 106 and beam splitter two 112), and several lenses 110. The scanning confocal optical path 100 also integrates an LED light source 107 and an industrial camera 111, forming a white light reflection imaging channel. Coarse sample positioning is achieved by a manual three-dimensional micron-level displacement stage 102, while fine scanning is achieved by a three-dimensional piezoelectric nanometer-level displacement stage 101 driven by the integrated circuit module 400 to perform three-dimensional spatial scanning.

[0024] An integrated laser module 200 is mounted on an independent optical breadboard and includes a laser diode 201, a beam shaping element, an acousto-optic modulator 204, and a second reflector 205. In this embodiment, the beam shaping element specifically includes a rotating half-wave plate 202 and a polarization beam splitter 203, which work together to adjust the power and polarization state of the incident laser. Additionally, the beam shaping element may include an aperture 206 to filter out other diffracted light. The laser diode 201 generates continuous laser light under constant current drive. After being adjusted by the rotating half-wave plate 202 and the polarization beam splitter 203, the laser light enters the acousto-optic modulator 204. The acousto-optic modulator 204 receives the radio frequency drive signal output from the integrated circuit module 400 and modulates the continuous laser light into first-order diffracted light using the acousto-optic effect. The first-order diffracted light is transmitted through the three-sided reflector 205 (with an aperture 206 interposed therebetween) and then coupled by the aspherical lens 207 into a single-mode optical fiber connected to the scanning confocal optical path 100.

[0025] The integrated circuit module 400 includes a microcontroller, an RF generation unit, a digital delayed pulse generator, a single-photon detector, and a voltage-controlled constant current source chip. The power supply section includes a power management circuit that converts the external input power into multiple DC voltages required by the device to power each component. The microcontroller acts as the main control unit and interacts with the data processing module 500 via a serial communication protocol. The microcontroller controls the voltage-controlled constant current source chip to output a constant current, driving the laser diode 201 in the integrated laser module 200. The RF generation unit generates, controls, and amplifies the RF signals required to drive the acousto-optic modulator 204.

[0026] In this embodiment, the radio frequency (RF) generation unit specifically includes an RF source chip, an RF attenuation chip, an RF switch chip, and an RF amplifier connected in sequence. During operation, the microcontroller configures the internal registers of the RF source chip and the RF attenuation chip to set a specific frequency and power RF signal. After being controlled by the RF switch chip, the RF signal is amplified by the RF amplifier and ultimately drives the acousto-optic modulator 204. The digital delayed pulse generator is connected to the RF switch chip and the single-photon detector. On one hand, it outputs a TTL level signal to control the on / off state of the RF switch chip, thereby achieving mode switching of the first-order diffracted light; on the other hand, it receives the pulse signal converted by the single-photon detector for photon counting.

[0027] The data processing module 500 runs on a computer and coordinates the work of various hardware components through software programs. The data processing module 500 performs the following functions: acquiring and displaying image data from the industrial camera 111; assisting operators in adjusting the manual three-dimensional micron-level displacement stage 102 to complete the initial positioning of the sample; acquiring the counting data from the single-photon detector and combining it with the real-time coordinates of the three-dimensional piezoelectric nanometer displacement stage 101 to construct a confocal scanning fluorescence image; and determining the precise coordinates of the fluorescent bright spots using a Gaussian fitting algorithm. During the detection process, the data processing module 500 connects to the integrated circuit module 400 to collaboratively execute the quantum detection timing sequence, acquire the fluorescence decay curve, perform bi-exponential fitting analysis on the data, and extract the spin depolarization time component characterizing the internal NV color center state. Subsequently, the data processing module 500 calls the preset relaxation rate and paramagnetic substance concentration calibration curve or corresponding relationship to invert the real-time concentration of reactive oxygen species, completing the in-situ reactive oxygen species detection.

[0028] This embodiment uses NV centers within fluorescent nanodiamonds as quantum sensors to detect the concentration of reactive oxygen species in the environment using an all-optical method. NV centers are point defects formed in the diamond lattice where carbon atoms are replaced by nitrogen atoms, with vacancies in adjacent positions. Under laser excitation at a specific wavelength (e.g., 532 nm), NV centers emit red fluorescence, and different spin states (…) and The corresponding fluorescence intensities show significant differences. By utilizing this characteristic and combining it with a pulse detection sequence, the initialization and readout of the spin state can be achieved.

[0029] This embodiment detects reactive oxygen species based on the measurement of the spin depolarization time of the NV color center. Spin depolarization time refers to the characteristic time for a spin system to recover from a non-equilibrium state to a thermal equilibrium state. The longitudinal relaxation rate of the NV color center... (i.e., spin depolarization time) The reciprocal of the intrinsic relaxation rate With environmental relaxation rate It is composed of linear superposition. Among them, It is mainly determined by the phonon Raman scattering process between the spin of the NV color center and the vibration of the diamond lattice, and this value remains basically stable under isothermal conditions; It mainly depends on the local magnetic field noise spectral density and root mean square magnetic field strength sensed by the NV color center.

[0030] Reactive oxygen species (ROS), as paramagnetic substances, generate random fluctuations in magnetic fields due to the unpaired electrons within their molecules, creating localized magnetic noise. When fluorescent nanodiamonds are placed in solutions or cellular environments containing ROS, the magnetic noise generated by ROS affects the spin of the NV color center, accelerating its spin relaxation process and increasing the environmental relaxation rate. The concentration of paramagnetic material in the environment increases significantly, thus shortening the spin depolarization time. Higher concentrations of paramagnetic material in the environment generate stronger magnetic noise, resulting in a shorter measured spin depolarization time. Therefore, measuring the spin depolarization time... By observing the changes in concentration and combining them with the pre-calibrated curves showing the relationship between paramagnetic substance concentration and relaxation rate, the near-real-time concentration of reactive oxygen species can be obtained.

[0031] The main sources of magnetic noise are the paramagnetic centers formed by unpaired electron dangling bonds on the surface of nanodiamonds and paramagnetic substances in the surrounding environment. Since the magnetic field detection scale of the NV center spins is on the nanometer scale, magnetic noise affects both the surface and internal NV centers of nanodiamonds. This results in the fluorescence decay curve of the nanodiamond ensemble exhibiting a double-exponential decay characteristic, containing a shorter time component corresponding to the surface NV centers and a longer time component corresponding to the internal NV centers. During data processing, these two components are distinguished through double-exponential fitting, and the longer spin depolarization time component corresponding to the internal NV centers is extracted, enabling the quantification of the reactive oxygen species concentration in the environment.

[0032] The specific measurements are achieved through a fully optical detection pulse sequence. The measurement cycle consists of three stages: First, a polarization pulse is applied to polarize the NV color center spin to its initial bright state; then, the spin system enters a period of free evolution without illumination; after the free evolution period ends, the next polarization pulse arrives synchronously with the readout pulse, which reads the fluorescence signal intensity corresponding to the aforementioned free evolution period. Finally, by changing the free evolution time and repeating the sequence, a decay curve of fluorescence intensity with evolution time is obtained, and the spin depolarization time value used for detection is then extracted through fitting. This process does not require microwave assistance and is suitable for in-situ detection in complex microenvironments such as inside cells.

[0033] See attached document Figure 6 and attached Figure 7The integrated laser module 200 is mounted on a 30×30cm independent optical breadboard. The integrated laser module 200 mainly consists of a laser diode 201, a rotating half-wave plate 202, a polarized beam splitter 203, an acousto-optic modulator 204, three reflectors 205, an aperture 206, an aspherical lens 207, and a single-mode optical fiber. In this embodiment, the rotating half-wave plate 202 is an LBTEK MHWP20-532BM model, the polarized beam splitter 203 is an LBTEK MPBS24-532 model, the acousto-optic modulator 204 is an SGT100-532-0 model, the aspherical lens 207 is an LBTEK MAC90657-A model, and the single-mode optical fiber is a JCOPTIX FCS1-PC-450-600 model.

[0034] Driven by a constant current, laser diode 201 generates a continuous laser beam with a wavelength of 532 nm and a maximum power of 80 mW. The beam output from laser diode 201 first enters a beam shaping element composed of a rotating half-wave plate 202 and a polarized beam-splitting cube 203. The regulated laser beam then enters an acousto-optic modulator 204, which has a driving frequency of 100 MHz. The RF source chip and RF attenuation chip in integrated circuit module 400 generate RF signals of specific frequency and power, which, after passing through an RF switch chip and an RF amplifier, drive the acousto-optic modulator 204. The acousto-optic modulator 204 uses the acousto-optic effect to modulate the continuous laser beam into first-order diffracted light. The first-order diffracted light undergoes optical path folding and guidance via three reflectors 205. An aperture 206 is placed between the reflectors 205 to block zero-order light and stray light, allowing only the first-order diffracted light to pass through. The first-order diffracted light, after spatial filtering, is finally focused by aspherical lens 207 and coupled into a single-mode fiber, and then transmitted through the single-mode fiber to the scanning confocal optical path 100.

[0035] See attached document Figure 3 To be continued Figure 5 In this embodiment, the scanning confocal optical path 100 adopts an optical cage structure and is mounted on a 60×40cm optical breadboard to ensure the stability and portability of the detection equipment. The scanning confocal optical path 100 mainly includes two optical flanges, one aspherical lens 108, one LED light source 107, four lenses 110, two beam splitters (beam splitter 106 and beam splitter 112), one dichroic mirror 105, one reflector 104, one objective lens 103, one filter 109, one industrial camera 111, two manual three-dimensional micron-level displacement stages 102, and one three-dimensional piezoelectric nanon-level displacement stage 101.

[0036] In this embodiment, the optical flange is model FAA-SM1-APC2 from JCOPTIX; the aspherical lens 108 is model OLSM0915021-T2M from JCOPTIX; the dichroic mirror 105 is model OFD1LP-650 from JCOPTIX; the objective lens 103 is a microscope objective with a magnification of 50×, a focal length of 4mm, a working distance of 7mm, and a numerical aperture of 0.55; the filter 109 is model OFE1LP-650 from JCOPTIX; the industrial camera 111 is model MER2-160-249U3C-HS from Daheng Imaging; the multimode fiber is model FCM2-PC-200L from JCOPTIX; and the three-dimensional piezoelectric nano-displacement stage 101 is model P-562.6CD from PI. Of the four lenses 110, one is a lens with a focal length of 100mm, and the other three are lenses with a focal length of 30mm.

[0037] The specific connection and working principle of the scanning confocal optical path 100 are as follows: The integrated laser module 200 couples the first-order diffracted light into the scanning confocal optical path 100 through a single-mode optical fiber. The laser beam passes sequentially through an aspherical lens 108, a beam splitter 106, and a dichroic mirror 105, and is then reflected by a reflecting mirror 104, finally being focused onto the sample by the objective lens 103. Simultaneously, the white light emitted by the LED light source 107 is reflected by the beam splitter 106 and merged into the optical path, also passing through the dichroic mirror 105, the reflecting mirror 104, and the objective lens 103 to illuminate the sample surface, achieving incident illumination.

[0038] The fluorescence and white light reflected from the surface of the nanodiamond are collected in the opposite direction by objective lens 103. The beam is reflected by mirror 104, passes through dichroic mirror 105 and filter 109, and is split into two paths at beam splitter 112. One beam is focused by lens 110 with a focal length of 100mm onto the photosensitive surface of industrial camera 111 to acquire white light reflection and fluorescence spot images. The other beam is focused by lens 110 with a focal length of 30mm, passes through pinhole filter structure 113, and is then collimated and focused by two other lenses 110 with a focal length of 30mm before being coupled into multimode fiber and transmitted to single-photon detector. LED light source 107 must be turned off when performing photon counting.

[0039] In addition, two manual three-dimensional micron-level displacement stages 102 and a three-dimensional piezoelectric nanon-level displacement stage 101 are mounted on the optical breadboard of the scanning confocal optical path 100 as positioning units. In actual operation, the sample coupling module 300 is fixed on the three-dimensional piezoelectric nanon-level displacement stage 101; by adjusting the manual three-dimensional micron-level displacement stage 102, combined with the image acquired by the industrial camera 111, the sample focal plane is coarsely adjusted and the detection area is selected; then, the three-dimensional piezoelectric nanon-level displacement stage 101 is used to achieve nanometer-level precision scanning and positioning of the sample, and in conjunction with the fluorescence collection function of the single-photon detector, confocal scanning fluorescence imaging and subsequent quantum detection experiments are realized.

[0040] Reference Appendix Figure 1 and attached Figure 2 The sample coupling module 300 is mainly composed of a substrate. The substrate can be connected to the three-dimensional piezoelectric nano-displacement stage 101 by screws, and its surface is provided with a clamping structure for fixing the sample. The substrate has a rectangular groove 700 and a circular groove 800: the rectangular groove 700 is used to place a glass slide, and the circular groove 800 is used to place a culture dish. A light-transmitting hole is provided at the center of the two grooves to allow first-order diffracted light to illuminate the sample through the objective lens 103.

[0041] See attached document Figure 8 The integrated circuit module 400 serves as the control and signal processing hub of the detection equipment. It mainly consists of a three-dimensional piezoelectric nanostage 101 driver, a digital delay pulse generator, a single-photon detector, a microcontroller, an RF source chip, an RF attenuation chip, an RF switch chip, an RF amplifier, and a voltage-controlled constant current source chip.

[0042] In this embodiment, the driver for the three-dimensional piezoelectric nanostage 101 is a P-562.6CD model from PI Corporation; the digital delay pulse generator is an ASG8100 model from QuantumCTek; and the single-photon detector is an SPCM-AQRH-14-FC model. The microcontroller and its peripheral RF and constant current drive circuits are integrated on a printed circuit board, wherein the microcontroller is an STM32F103ZET6 model from STMicroelectronics; the RF source chip is an ADF4351 model; the RF attenuation chip is an HMC624A model; the RF switch chip is a ZASWA-2-50DR+ model from Mini-Circuits; the RF amplifier is a KDT700MPA-035 model; and the voltage-controlled constant current source chip is a KW-VCCS1000 model.

[0043] In terms of power management, this embodiment converts AC mains power into 24V DC power via a power adapter, and then generates 12V and 5V DC voltages through an onboard switching step-down chip to power various electronic components. For independent devices such as the 101 driver for the 3D piezoelectric nanostage, a dedicated power adapter provided by the manufacturer can be used for independent power supply.

[0044] In terms of control logic and signal links, the driver of the three-dimensional piezoelectric nanostage 101 is directly connected to the data processing module 500 (i.e., the computer) via a serial communication interface. It receives and parses the instructions issued by the data processing module 500, driving the three-dimensional piezoelectric nanostage 101 to achieve nanoscale movement. The microcontroller establishes a connection with the data processing module 500 via a serial communication protocol. On one hand, the microcontroller connects to the control terminal of the voltage-controlled constant current source chip, controlling the chip to output a constant current, driving the laser diode 201 in the integrated laser module 200 to output continuous laser light. On the other hand, the microcontroller configures the internal registers of the RF source chip and the RF attenuation chip to control them to generate RF signals of specific frequency and power.

[0045] The radio frequency (RF) signal then enters the RF switch chip. A digital delay pulse generator is connected to the data processing module 500 and downloads timing control commands; one of its output TTL-level signals is connected to the control terminal of the RF switch chip. During the TTL high-level period, the RF switch chip is turned on, the RF signal passes through and is amplified by the RF amplifier, driving the acousto-optic modulator 204 in the integrated laser module 200; conversely, the RF signal will not reach the acousto-optic modulator 204; thus, the optical sequence required for the experiment can be generated.

[0046] A single-photon detector receives fluorescence signals from the scanning confocal optical path 100 via an optical fiber and converts the detected fluorescence signals into electrical pulse signals, which are then input into a digital delay pulse generator. The digital delay pulse generator counts the pulse signals within a specific time window and transmits the counting results to the data processing module 500, which is used to construct a confocal scanning image or obtain the spin depolarization time.

[0047] See attached document Figure 1 and attached Figure 2 The fluorescent nanodiamonds used in the experiment were purchased from Adamas Nano, with a stock solution concentration of 1 mg / mL and a size of 100 nm. Before the experiment, 500 μL of the fluorescent nanodiamond stock solution was taken out, diluted with deionized water to 0.02 mg / mL, and the diluted solution was sonicated for 15 minutes and stored for later use.

[0048] For equipment verification or sensitivity calibration experiments, gadolinium chloride solution was used as the standard paramagnetic sample. Gadolinium chloride powder (G119221-25g) was dissolved in ultrapure water to prepare a 1M stock solution, which was then serially diluted to obtain a series of solutions of 0.1μM, 10μM, 1mM and 100mM. Each solution was sonicated for 15 minutes before use.

[0049] For biological cell experiments, a fluorescent nanodiamond solution is added to the culture medium and co-incubated with the cells. The fluorescent nanodiamonds can enter the cells via endocytosis. After a specific incubation period, the cells are fixed and stained, and then mounted on a glass slide for subsequent experiments. For live cell assays, culture dishes can be used directly.

[0050] After sample preparation, the glass slide carrying the sample is embedded into the rectangular groove 700 of the sample coupling module 300, or the culture dish is placed into the circular groove 800 and fixed by a clamping structure. Then, the entire sample coupling module 300 is mounted on the three-dimensional piezoelectric nanodisplacement stage 101.

[0051] During the initial positioning phase, the software controls the LED light source 107 to remain constantly on, while the industrial camera 111 continuously acquires images and displays them in real time. The manual three-dimensional micron-level displacement stage 102 is adjusted until the sample surface is clearly displayed in the imaging field of view of the industrial camera 111. At this point, cell boundaries or the distribution area of ​​nanodiamonds can be confirmed through the images, completing the rough locking of the sample's focal plane.

[0052] After initial sample positioning, the detection equipment, through the data processing module 500, collaboratively controls the three-dimensional piezoelectric nanostage 101, the integrated laser module 200, and the single-photon detector to perform nanoscale fine scanning, target identification, and dynamic tracking. This control logic, developed using Python, achieves hardware-level instruction interaction and collaborative operation by calling the serial communication protocol provided by the manufacturer of the three-dimensional piezoelectric nanostage 101 and the software development kit provided by the manufacturer of the single-photon detector.

[0053] The data processing module 500 first sets the upper and lower limits of movement and the scanning step size for each motion axis of the three-dimensional piezoelectric nanostage 101, and sets the counting integration time of the single-photon detector. Then, the device drives the three-dimensional piezoelectric nanostage 101 to move through a dual-layer loop control logic, while simultaneously using a digital delayed pulse generator to record the fluorescence counts corresponding to each position coordinate. Finally, the data processing module 500 maps the position coordinates to the fluorescence counts, constructing and displaying a two-dimensional confocal scanning fluorescence image.

[0054] The scanning process of the detection equipment follows a logic of first axial and then planar. First, the three-dimensional piezoelectric nano-displacement stage 101 is controlled to perform XZ-plane scanning imaging. The Z-axis coordinates of the sample are determined by analyzing the distribution of bright spots in the XZ-plane fluorescence image. For example... Figure 9 As shown in a, Figure 9Figure a is a schematic diagram of XZ-plane confocal scanning fluorescence imaging according to an embodiment of the present invention. The horizontal axis is X (μm) (range 0-200μm), the vertical axis is Z (μm) (range 0-200μm), and the color bar on the right represents Counts (Mcps) (representing fluorescence count intensity, in millions of times). Bright spots in the figure indicate areas with strong fluorescence signals, which can be used to accurately locate interface positions.

[0055] Subsequently, the detection equipment drives the displacement stage to move the sample to the selected Z-axis, performing XY plane scanning imaging. For example... Figure 9 As shown in b. Figure 9 Figure b is a schematic diagram of XY-plane confocal scanning fluorescence imaging according to an embodiment of the present invention. The horizontal axis is X (μm) (range 0-200μm), and the vertical axis is Y (μm) (range 0-200μm). The color scale is also Counts (Mcps) (representing fluorescence count intensity, in millions of times). After acquiring the XY-plane fluorescence image, the detection device analyzes the distribution of bright spots in the image and automatically calibrates or manually selects bright spots as targets to be measured through image processing algorithms. Figure 9 The enlarged illustration in the upper right corner further showcases the details of the local particles.

[0056] For the selected target, the detection device enters a fine-tuning phase to obtain its center coordinates. This process employs a single-axis independent scanning strategy: the detection device fixes two coordinate axes of the target (e.g., the Y and Z axes), and controls a third coordinate axis (e.g., the X axis) to perform step-by-step scanning within a small range while recording fluorescence data; subsequently, Gaussian fitting is performed on the acquired fluorescence data, and the peak position of the fitted curve is marked as the target's position in the X-axis direction. The device repeats the above scanning and fitting process sequentially in three orthogonal directions to finally determine the target's center coordinates. Figure 9 As shown in c. Figure 9 Figure c is a flowchart illustrating the process of performing a three-dimensional fine scan of a selected target and quasi-real-time position tracking via fluorescence feedback in one embodiment of the present invention. The horizontal axis represents Coordinates (μm) (representing the scanning range), and the vertical axis represents Counts (Mcps) (representing fluorescence count intensity in millions of times). The figure shows three curves, corresponding from top to bottom to the fluorescence intensity distribution along the Z-axis (PL&Z), Y-axis (PL&Y), and X-axis (PL&X). The target can be accurately located by peak fitting.

[0057] After determining the coordinates, the detection equipment executes a target discrimination procedure. The three-dimensional piezoelectric nanostage 101 is moved to the target position, and a digital delayed pulse generator collects fluorescence counts over a preset time period (e.g., 10 ms), displaying the count values ​​in near real-time scrolling mode on the interface of the data processing module 500. Figure 9As shown in Figure c, this figure illustrates the process of near real-time tracking and repositioning of a selected target using fluorescence signals. The horizontal axis represents Time (s) (representing the acquisition time), and the vertical axis represents Counts (Mcps) (representing the fluorescence count intensity, in millions of times). If the displayed fluorescence count remains stable and the value is within the expected count range for a single nanodiamond, the detection device determines that the bright spot is a valid nanodiamond probe; if the fluorescence count decays rapidly over time or the value exceeds the expected range, the bright spot is determined to be an interference signal or a non-target particle.

[0058] To address the slow drift of nanodiamonds in solution environments caused by Brownian motion or mechanical displacement, the detection device runs an automatic tracking program during the detection process. This program predefines an initial fluorescence count and a percentage threshold. During the experiment, the detection device monitors the current fluorescence count in near real-time. Once the current fluorescence count is detected to be lower than the product of the initial fluorescence count and the percentage threshold, it is determined that the target has drifted. At this point, the detection device automatically triggers the aforementioned positioning process to reposition the target and correspondingly move the three-dimensional piezoelectric nanodisplacement stage 101, achieving long-term stable tracking of the nanodiamonds and ensuring that the nanodiamonds remain at the detection center of the confocal optical path during the spin depolarization time measurement.

[0059] During the spin depolarization time measurement experiment, the detection device intermittently executes the aforementioned automatic tracking procedure. If the device determines that the target has shifted position during the experiment, it will temporarily interrupt the current spin depolarization time measurement task and trigger a target repositioning process. This repositioning process invokes the aforementioned fine scanning strategy, moves the three-dimensional piezoelectric nanostage 101 to a new position, and then the detection device resumes the interrupted measurement task. Through this threshold-feedback-based dynamic tracking, the detection device can effectively overcome the signal attenuation problem in long-term experiments, achieving stable long-term detection of single-particle nanodiamonds.

[0060] See attached document Figure 9 d, Figure 9 d is a schematic diagram of the fluorescence intensity decay curve and fitting results with free evolution time in one embodiment of the present invention. The horizontal axis represents Time (ms) (representing free evolution time, ranging from 0-1.5ms), and the vertical axis represents Contrast (%) (representing fluorescence contrast, ranging from 0-14%). The inset shows the spin depolarization time measurement sequence. Spin depolarization time ( The measurement relies on the timing coordination control of the integrated laser module 200 and the single-photon detector by the digital delayed pulse generator.

[0061] In the specific measurement, the detection equipment was set to a laser power of 2.26mW. The measurement sequence mainly includes the following steps: The first stage is spin initialization. A digital delayed pulse generator outputs a TTL signal to control the hardware to generate a 20μs pulse-mode first-order diffraction beam, polarizing the NV color centers in the nanodiamond to the spin ground state. ).

[0062] This is followed by a phase of free evolution without illumination. During this period, the NV center spins undergo longitudinal relaxation under the influence of ambient magnetic noise and lattice phonons. The free evolution time increases sequentially according to a preset sequence (e.g., from hundreds of nanoseconds to milliseconds).

[0063] Finally, there is the readout phase. After the free evolution ends, the next 20μs polarization pulse arrives. The detection device, through a digital delayed pulse generator, synchronously triggers the counting gating of the single-photon detector at the moment the polarization pulse begins, with the gating width set to [value missing]. The number of photons collected during this period corresponds to the fluorescence signal for the aforementioned free evolution time. This polarization pulse also completes the next spin initialization.

[0064] To obtain high signal-to-noise ratio data, the above measurement sequence was repeated. This makes the total fluorescence counting time close to .

[0065] After acquiring fluorescence counts corresponding to different free evolution times through measurement sequences, the data processing module 500 converts the accumulated photon counts into fluorescence contrast, which reflects the population difference of the NV center spin quantum state at the readout time. Since nanodiamond particles simultaneously possess NV centers located deep within the crystal lattice and near the crystal surface, the data processing module 500 employs a double exponential decay model to perform nonlinear least-squares fitting on the obtained fluorescence decay curves to quantify the influence of environmental magnetic noise on the nanodiamond spin state. According to this model, the relationship between fluorescence contrast and free evolution time is described as the superposition of a rapid decay term, a slow decay term, and a steady-state baseline value. The rapid decay term is a decay process characterized by a rapid spin depolarization time and has a corresponding amplitude weight; this component mainly corresponds to the NV centers on the nanodiamond surface. The slow decay term is a decay process characterized by a slow spin depolarization time and has a corresponding amplitude weight; this component mainly corresponds to the NV centers inside the nanodiamond. The steady-state baseline value represents the constant term when evolution tends towards equilibrium.

[0066] Using the aforementioned double-exponential fitting model, the detection device can extract the spin depolarization time component corresponding to the NV color center inside the nanodiamond, and use this component to quantitatively analyze the concentration of reactive oxygen species or paramagnetic ions in the sample to be tested (such as intracellular or gadolinium chloride solution).

[0067] like Figure 9 As shown in e, Figure 9 e is a schematic diagram illustrating the experimental results of dynamically tracking and measuring the spin depolarization time of multiple nanodiamond particles within the same sample region in one embodiment of the present invention. The horizontal axis represents Time (min) (representing the experimental time, ranging from 3 to 15 min), and the vertical axis represents... (μs) (representing spin depolarization time, ranging from 120 to 300 μs). The three curves in the figure correspond to three different nanodiamonds, ND-1, ND-2, and ND-3, respectively, clearly showing the dynamic changes in their spin depolarization time over time, thus verifying the stability of the detection equipment.

[0068] To establish a quantitative correlation between the spin depolarization time of the internal NV center and the concentration of the analyte, the data processing module 500 performs calculations based on the superposition principle of spin relaxation rates. (NV center longitudinal relaxation rate) Defined as the reciprocal of the spin depolarization time, it consists of two parts: the intrinsic relaxation rate and the environmental relaxation rate. The intrinsic relaxation rate is primarily determined by the phonon Raman scattering process between the NV center spin and lattice vibrations; the environmental relaxation rate depends mainly on the magnetic field noise spectral density and root-mean-square magnetic field strength sensed by the NV center. Magnetic noise originates from paramagnetic centers formed by unpaired electron dangling bonds on the nanodiamond surface and paramagnetic substances in the environment. When the concentration of paramagnetic substances in the environment changes, the environmental relaxation rate of the NV center changes, leading to a change in the measured spin depolarization time.

[0069] Due to individual differences among nanodiamond particles, the data processing module 500 performs statistical analysis. The detection device independently tracks and measures the spin depolarization time of multiple nanodiamond particles within the same sample area, collecting the spin depolarization time data of these particles and calculating their statistical mean and variance, which serve as the final measurement result under these environmental conditions. Quantitative detection of the sample is achieved by constructing a calibration curve showing the change in average depolarization time as a function of the analyte concentration.

[0070] See attached document Figure 9 f, Figure 9 f is a calibration curve showing the relationship between the spin depolarization time of nanodiamond and the concentration of paramagnetic ions (such as gadolinium ions) in the environment, according to one embodiment of the present invention. The horizontal axis in the figure represents Gd. 3+ (M) (represents the molar concentration of gadolinium ions, ranging from 0 to 10) -1 M), with the ordinate as (μs) (representing spin depolarization time, ranging from 0 to 200 μs). Gadolinium chloride (GdCl3) was used as the standard paramagnetic material in the experiment. Following the aforementioned experimental preparation steps, gadolinium chloride solutions with concentration gradients of 0.1 μM, 10 μM, 1 mM, and 100 mM were prepared. Fluorescent diamond nanoparticles were immobilized on the surface of a glass slide, and solutions of different concentrations were added dropwise. Confocal scanning was used to lock onto a single diamond particle, and spin relaxation measurements were performed.

[0071] Experimental data show that as the gadolinium ion concentration in the solution increases (from 0.1 μM to 100 mM), the magnetic noise generated by the environment increases, the environmental relaxation rate increases, and consequently, the measured spin depolarization time decreases. The trend shows a decrease. By statistically analyzing the spin depolarization time values ​​under different gadolinium ion concentrations, the relationship between spin depolarization time and gadolinium ion concentration can be established, providing a calibration basis for subsequent quantitative analysis of reactive oxygen species concentration in unknown samples.

[0072] In another specific application example, a quantum sensing experiment for in-situ reactive oxygen species within cells was conducted. Cells that had taken up 100nm diamond nanoparticles were used in the experiment. White light reflectance imaging was used to determine the cell location, followed by switching to confocal scanning mode to locate the fluorescent nanodiamond nanoparticles within the cells. During the measurement process, image processing algorithms were used to dynamically track the nanodiamond nanoparticles in real time to overcome positional deviations caused by cell movement.

[0073] The experiment first measured the initial spin depolarization time of intracellular nanodiamonds under conditions without external stimulation. Subsequently, hydrogen peroxide was added to the cell culture environment, and ultraviolet (UV) irradiation was applied to simulate oxidative stress. Upon initiation of UV irradiation, the concentration of reactive oxygen species (ROS) in the environment increased sharply, and the spin depolarization time of the nanodiamonds significantly shortened. After the UV light was removed, the spin depolarization time gradually returned to its original level as ROS were quenched and metabolized. These results validate the device's ability to monitor the generation and destruction of ROS in cells in real time.

[0074] Furthermore, to enable the detection of specific cellular microenvironments, fluorescent nanodiamonds with surface-modified mitochondrial-specific antibodies were employed in the experiment. These nanodiamonds can specifically bind to the mitochondrial region, and by detecting changes in spin relaxation signals near the mitochondria, localized measurement of endogenous reactive oxygen species levels during mitochondrial metabolism was achieved. This detection device can also be further expanded into a general-purpose quantum sensing platform, executing spin manipulation sequences such as Rabi oscillations and Hahn echoes for measuring multidimensional physical quantities such as magnetic fields and temperature.

Claims

1. An in-situ reactive oxygen species quantum detection device based on fluorescent nanodiamond nitrogen-vacancy color centers, characterized in that, include: The sample coupling module (300) is used to carry the sample containing fluorescent nanodiamonds; An integrated laser module (200) has a built-in acousto-optic modulator (204). The integrated laser module (200) is used to generate first-order diffraction light in response to a driving signal. The first-order diffraction light includes a continuous wave mode for fluorescence imaging of the sample under test and a pulse mode for optical polarization and readout of the spin state of the nitrogen vacancy color center of the fluorescent nanodiamond. A scanning confocal optical path (100) is used to focus the first-order diffraction light onto the sample to be tested. A three-dimensional piezoelectric nano-displacement stage (101) is used to perform the positioning and in-situ scanning of the sample to be tested and to collect the fluorescence signal emitted by the nitrogen vacancy color center. The integrated circuit module (400) and the data processing module (500) work together to execute a full optical detection pulse sequence. The integrated circuit module (400) receives the fluorescence signal and performs photon counting. The data processing module (500) extracts the spin depolarization time of the fluorescent nanodiamond based on the photon counting result and inverts the reactive oxygen concentration in the environment of the sample to be tested.

2. The in-situ reactive oxygen species quantum detection device based on fluorescent nanodiamond nitrogen-vacancy color centers according to claim 1, characterized in that, The data processing module (500) works in conjunction with the integrated circuit module (400) to execute the all-optical detection pulse sequence, specifically as follows: The data processing module (500) controls the integrated circuit module (400) and the integrated laser module (200) to generate the all-optical detection pulse sequence; The all-optical detection pulse sequence includes polarization pulses, a free evolution time without illumination, and readout pulses; The polarization pulse is used to optically polarize the spin state, and the readout pulse is used to read out the spin state through the fluorescence signal. The data processing module (500) obtains the fluorescence decay curve by changing the free evolution time and controlling the integrated circuit module (400) to repeatedly generate the all-optical detection pulse sequence.

3. The in-situ reactive oxygen species quantum detection device based on fluorescent nanodiamond nitrogen-vacancy color centers according to claim 2, characterized in that, The data processing module (500) performs a double exponential fitting analysis on the fluorescence decay curve, extracts the slow component of spin depolarization time corresponding to the spin state of the color center inside the fluorescent nanodiamond, and inverts the reactive oxygen concentration according to the preset relaxation rate and paramagnetic substance concentration calibration curve.

4. The in-situ reactive oxygen species quantum detection device based on fluorescent nanodiamond nitrogen-vacancy color centers according to claim 1, characterized in that, The integrated circuit module (400) uses a microcontroller to control a voltage-controlled constant current source chip to drive the laser diode (201) inside the integrated laser module (200) to generate continuous laser light; The microcontroller is connected to the radio frequency generation unit to configure the parameters of the radio frequency signal. After the radio frequency signal is controlled to be turned on and off by the TTL level signal output by the digital delay pulse generator, the continuous laser is converted into first-order diffraction light in continuous wave mode or pulse mode by the acousto-optic modulator (204). The digital delayed pulse generator is also connected to a single-photon detector, receives the electrical pulse signal converted by the single-photon detector based on the fluorescence signal, and performs the photon counting.

5. The in-situ reactive oxygen species quantum detection device based on fluorescent nanodiamond nitrogen-vacancy color centers according to claim 4, characterized in that, The radio frequency generation unit uses a radio frequency source chip to generate an initial radio frequency signal. The initial radio frequency signal is adjusted in power by a radio frequency attenuation chip, then passes through a radio frequency switch chip controlled by the TTL level signal, and finally is amplified by a radio frequency amplifier to output the drive signal. The microcontroller is connected to the RF source chip and the RF attenuation chip for configuring internal registers.

6. The in-situ reactive oxygen species quantum detection device based on fluorescent nanodiamond nitrogen-vacancy color centers according to claim 4, characterized in that, The single-photon detector receives the fluorescence signal from the scanning confocal optical path (100) and converts the detected photons into electrical pulse signals. The digital delay pulse generator counts the electrical pulse signals within a preset time window to complete the photon counting and transmits the counting result to the data processing module (500).

7. The in-situ reactive oxygen species quantum detection device based on fluorescent nanodiamond nitrogen-vacancy color centers according to claim 4, characterized in that, The scanning confocal optical path (100) receives the first-order diffracted light from the integrated laser module (200), and the first-order diffracted light passes sequentially through the aspherical lens (108), the beam splitter (106), and the dichroic mirror (105). The scanning confocal optical path (100) is also provided with an LED light source (107) and multiple lenses (110). The white light generated by the LED light source (107) is reflected by the first beam splitter (106) and enters the optical path, and together with the first-order diffracted light, is reflected by the first reflector (104) to the objective lens (103). The objective lens (103) focuses the first-order diffraction light onto the sample to be tested and collects the fluorescence signal emitted by the stimulated emission of the color center and the white light reflected from the surface of the sample to be tested; The fluorescence signal and the white light are reflected by the first reflector (104), passed through the dichroic mirror (105) and the filter (109), and then split into two paths at the second beam splitter (112); One of the light beams is focused by one of the lenses (110) onto an industrial camera (111) to acquire a white light reflection image and a fluorescent spot image; The other beam, in conjunction with the other three lenses (110) and the pinhole filter structure (113), is filtered and collimated and focused before being coupled into the multimode fiber connected to the single-photon detector. The LED light source (107) must be turned off when performing the photon counting. The scanning confocal optical path (100) also includes a manual three-dimensional micrometer displacement stage (102), which is used to support the three-dimensional piezoelectric nanometer displacement stage (101) and the sample coupling module (300) to be tested, and to realize the positioning of the sample to be tested relative to the objective lens (103).

8. The in-situ reactive oxygen species quantum detection device based on fluorescent nanodiamond nitrogen-vacancy color centers according to claim 1, characterized in that, The integrated laser module (200) generates continuous laser light using a laser diode (201). The continuous laser light is then injected into an acousto-optic modulator (204) after its power and polarization state are adjusted by a beam shaping element comprising a rotating half-wave plate (202) and a polarized beam splitter (203). The acousto-optic modulator (204) responds to the driving signal to modulate the continuous laser into the first-order diffraction light. The first-order diffraction light is guided by multiple mirrors (205) and passes through the aperture (206), and is then coupled out by the aspherical lens (207).

9. The in-situ reactive oxygen species quantum detection device based on fluorescent nanodiamond nitrogen-vacancy color centers according to claim 7, characterized in that, The data processing module (500) runs an automatic tracking program to set an initial fluorescence count value and a percentage threshold. The data processing module (500) monitors the current fluorescence count in real time. When the current fluorescence count is lower than the product of the initial fluorescence count and the percentage threshold, the data processing module (500) sends an instruction to the integrated circuit module (400) to drive the three-dimensional piezoelectric nanostage (101) to perform a three-dimensional scan and updates the spatial coordinates of the fluorescent nanodiamond based on the Gaussian fitting result of the three-dimensional scan data.

10. The in-situ reactive oxygen species quantum detection device based on fluorescent nanodiamond nitrogen-vacancy color centers according to claim 7, characterized in that, The sample coupling module (300) is composed of a substrate. The surface of the substrate is provided with a rectangular groove (700) for positioning a glass slide and a circular groove (800) for positioning a culture dish. The glass slide and the culture dish are fixed by a mechanical clamping assembly (600) provided on the surface of the substrate. The substrate has a light-transmitting hole at its center, which is aligned with the optical axis of the objective lens (103) to allow the first-order diffracted light to pass through.

Images