Diamond fluorescent non-contact cell temperature measurement system and construction method thereof

Abstract

The invention belongs to the crossing field of biomedical detection technology and optical detection technology, and particularly relates to a diamond fluorescent non-contact cell temperature measurement system and a construction method thereof, and the diamond fluorescent non-contact cell temperature measurement system comprises a hardware system, a signal processing algorithm and a specific targeting probe. Compared with the prior art with the problems of low spatial resolution, long measurement time, poor signal-to-noise ratio, large temperature error and the like, a hardware system composed of a pulse laser source, a confocal light path, a high NA objective lens and an EMCCD is arranged, and a five-layer db4 wavelet denoising algorithm and an SV40 nuclear localization peptide modified nano-diamond probe are combined, so that the measurement accuracy is improved. The technical problem that rapid, accurate and high-signal-to-noise-ratio temperature measurement is difficult to achieve in a nanoscale area in living cells is solved. The spatial resolution lt is realized; the wavelength is 200 nm, and the result of single measurement; 2s, signal-to-noise ratio gt; the temperature error is less than or equal to + / -0.08 DEG C, and the tiny temperature difference between nucleoplasm of 0.3 DEG C is successfully captured.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical detection technology and optical detection technology, and particularly relates to a non-contact cell temperature measurement system based on diamond fluorescence and a construction method thereof. BACKGROUND

[0002] Cells are the basic units of life activities, and their internal biochemical reactions are complex and intricate, often accompanied by small heat changes. Accurate measurement of the local temperature of the cell interior, especially the nanoscale structure of organelles, is of great significance to reveal the molecular mechanisms of cell metabolism, signal transduction, energy conversion, and even pathological states. However, traditional contact temperature measurement methods (such as micro-thermocouples) are difficult to apply to the fine study of life systems due to their inherent millimeter-level spatial resolution and potential physical damage and interference to cells. Although non-contact optical temperature measurement techniques based on rare earth materials, quantum dots, and other fluorescent materials to some extent avoid the contact problem, they have the defects of biological toxicity and poor fluorescence stability. Existing temperature measurement techniques based on diamond NV color centers have good biological compatibility, but are greatly disturbed by magnetic fields, with a spatial resolution of more than 200 nm (such as the resolution of 510 nm ± 30 nm reported in the foreign literature Nat Commun 2022), a measurement time of 10.5 s ± 1 s, and a signal-to-noise ratio of only 28 dB ± 3 dB, which cannot meet the rapid and accurate temperature measurement needs of nanoscale structures (such as ribosomes) in cells. Although the domestic related patent (such as CN114235678A) optimizes the optical path, the temperature measurement error is still ± 0.22℃, which cannot capture the small temperature difference of about 0.3℃ between the nucleus and the cytoplasm.

[0003] In summary, there is an urgent need for a non-contact temperature measurement scheme in the field of cell temperature measurement that has high spatial resolution, high temperature sensitivity, high temporal resolution, excellent biological compatibility, and strong anti-interference ability. SUMMARY

[0004] The present application aims to provide a non-contact cell temperature measurement system based on diamond fluorescence and a construction method thereof, and aims to achieve the following objectives: 1. The spatial resolution is reduced to within 200 nm; 2. The single measurement time is shortened to within 2 s; 3. The signal-to-noise ratio is improved to more than 40 dB; 4. The temperature measurement error is controlled within ± 0.08℃, while ensuring biological compatibility and avoiding damage to cells.

[0005] To solve the above technical problems, the present application provides the following technical solutions: A non-contact cell temperature measurement system based on diamond fluorescence, comprising a hardware system, a signal processing algorithm, and a targeted probe; The hardware system comprises a constant-temperature stage, a signal processing module, a signal detection module, a signal collection module, a cell carrier module, an optical path adjustment module, and a laser source module. The signal processing algorithm is used for denoising and processing the fluorescent signal to extract temperature information. The targeting probe is used for specifically labeling cell substructures and responding to temperature changes through diamond fluorescence.

[0006] Preferably, the laser source module comprises a pulsed laser with an output wavelength of 532 nm and a pulse width of 10 ns, which is used for exciting nanodiamond fluorescence and realizing fluorescent lifetime measurement.

[0007] Preferably, the optical path adjustment module comprises a laser shaper, a beam expander, and a mirror, which are used for shaping, expanding, and focusing the laser on the sample, ensuring that the excitation spot is confocal with the signal collection light path, and the coaxiality error is ≤0.1 mm.

[0008] Preferably, the cell carrier module comprises a culture dish and a constant-temperature stage, and the constant-temperature stage provides a stable external temperature environment with a temperature control accuracy of ±0.05℃.

[0009] Preferably, the signal collection module comprises an objective lens with a numerical aperture ≥0.75 and a magnification of 20 times, which is used for collecting the fluorescent signal emitted by the targeting probe.

[0010] Preferably, the signal detection module comprises an EMCCD detector with an electron multiplication function and a single-photon-level sensitivity, which is used for converting the fluorescent signal into an electrical signal and digitizing it.

[0011] Preferably, the signal processing module comprises a computer processor, which is used for running the signal processing algorithm. The signal processing algorithm adopts a 5-layer db4 wavelet denoising algorithm and applies an adaptive Birgé-Massart threshold. The noise elimination rate is ≥90%, the effective signal retention rate is ≥95%, and the processing time is ≤0.5 s / frame.

[0012] Preferably, the targeting probe comprises nanodiamond particles and a targeting peptide. The particle size of the nanodiamond particles is 50 nm, and the targeting peptide is SV40 nuclear localization peptide. The cell nucleus labeling efficiency is ≥90%.

[0013] Preferably, the targeting peptide can be replaced by a mitochondrial targeting peptide or a cell membrane targeting peptide for targeting different cell substructures.

[0014] A method for constructing a non-contact cell temperature measurement system comprises the following steps: Hardware connection: Set the distance between the laser source module and the optical path adjustment module to 15 cm, connect the objective lens to the EMCCD detector through the C interface, the coaxial error is ≤0.1 mm, and connect the EMCCD detector to the computer through the USB3.0 interface; System calibration: Use standard temperature calibration pieces to calibrate the temperature in the range of 25-40℃, collect the fluorescence signal and fit the calibration curve, the fitting degree R²≥0.999, so that the temperature calculation accuracy reaches ±0.08℃.

[0015] Beneficial effects: 1. Resolution improvement: The spatial resolution reaches 100nm±5nm, which can clearly measure the temperature of nanoscale structures such as ribosomes (20-30nm) and endoplasmic reticulum; 2. Speed and accuracy optimization: The single measurement time is within 2s, the dynamic imaging speed is 30 frames / s±1, and the temperature change of zebrafish embryo cleavage process can be captured in real time; 3. Anti-interference and biocompatibility: The signal-to-noise ratio is improved to more than 40dB, and it is not affected by magnetic field interference; the diamond probe has excellent biocompatibility. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiment description. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual proportion.

[0017] Figure 1 The hardware architecture diagram of the hardware system of a non-contact cell temperature measurement system based on diamond fluorescence of the present application patent; Figure 2 The flowchart of the signal processing algorithm of a non-contact cell temperature measurement system based on diamond fluorescence of the present application patent; Figure 3 The zebrafish embryo cell graph without wavelet denoising in the application case of the present application patent in zebrafish embryo temperature measurement; Figure 4 The zebrafish embryo cell graph with three-layer wave denoising in the application case of the present application patent in zebrafish embryo temperature measurement; Figure 5 The zebrafish embryo cell graph with five-layer wave denoising in the application case of the present application patent in zebrafish embryo temperature measurement. DETAILED DESCRIPTION

[0018] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative effort fall within the protection scope of the present application.

[0019] Embodiment 1 The present application provides a non-contact cell temperature measurement system based on diamond fluorescence, aiming to realize a non-contact temperature measurement scheme with high spatial resolution, high temperature sensitivity, high time resolution, excellent biocompatibility and strong anti-interference capability. The core idea of the present system is: through a targeted probe, a nanometer thermometer (nanometer diamond) is accurately delivered to a specific part of a cell, a pulsed laser is used to excite the fluorescence thereof, a high-sensitivity detector is used to capture the fluorescence lifetime signal, and finally a special algorithm is used to extract high-precision temperature information from noise. The present system includes a hardware system, a signal processing algorithm and a targeted probe. The hardware system includes a constant-temperature sample stage, a signal processing module, a signal detection module, a signal collection module, a cell sample module, an optical path adjustment module and a laser source module, as shown in Figure 1 The hardware architecture diagram of the hardware system is shown in The laser source module: a pulsed semiconductor laser (model: LD-532-10ns) is used, with an output wavelength of 532 nm, a pulse width of 10 ns and a repeatable frequency (such as 10-80 MHz). The light outlet is connected to the optical path adjustment module through a standard SM1 threaded interface, with a distance of about 15 cm to ensure that the light beam has enough space for beam expansion.

[0020] The optical path adjustment module: all are installed on an optical breadboard or a cage system to ensure coaxiality and stability, used to accurately focus the laser on the sample to form a spot near the diffraction limit, which is the key to realizing high spatial resolution, and sequentially includes the following elements: Laser shaper: used to purify the laser mode to obtain an ideal Gaussian beam.

[0021] Beam expander: expands the diameter of the laser beam, so that a smaller focused spot can be obtained after passing through the objective lens.

[0022] Dichroic mirror: with a center wavelength of 532 nm, placed at an angle of 45 degrees. It reflects the 532 nm excitation light, making it vertically downward into the objective lens.

[0023] Objective lens: as the end point of the excitation light path and the starting point of the collection light path.

[0024] Cell holder module: provides a stable, undisturbed, physiological temperature environment for live cell samples, ensuring that the measured temperature change is originated from the cell itself, including a holder for a standard cell culture dish. The holder is integrated in a temperature-controlled stage, which uses the Peltier effect for heating / cooling, with a temperature control range from room temperature to 45°C, and a stability of ±0.05°C.

[0025] Signal collection module is used to collect extremely weak diamond fluorescence signals efficiently. High numerical aperture (NA = 0.75) is the core to improve signal intensity and spatial resolution of the system. The core is a high numerical aperture objective (model: OBJ-20x-0.75). This objective is used to focus the excitation light and collect the emission fluorescence simultaneously; the collected fluorescence (wavelength range about 650-800 nm) passes through the dichroic mirror mentioned above (because it is transmissive to long-wave fluorescence). After that, the light path passes through an emission filter (e.g. 650 nm long-pass filter) to further filter out the residual excitation light and cellular autofluorescence. The objective is connected to the subsequent detector directly or through a relay lens through a standard C interface.

[0026] Signal detection module converts the weak optical fluorescence image into a digital image sequence (i.e. fluorescence lifetime image sequence) with single-photon level sensitivity and transmits it to the computer. An electron multiplying CCD (model: EM-1024-60) with 1024x1024 pixels is used, equipped with a thermoelectric cooler (-60°C to -80°C) to greatly reduce the dark current. It is rigidly connected to the signal collection module through a C interface to ensure the flatness of the imaging surface. It is connected to the signal processing module (computer) through a USB3.0 data line (or CameraLink interface).

[0027] Signal processing module: runs the system control software and dedicated signal processing algorithms, controls the hardware to collect data, and performs complex image processing and temperature calculation. The main body is a high-performance computer workstation.

[0028] The signal processing algorithms are integrated in the computer software and process each frame or sequence of fluorescence images, as shown in Figure 2 The flowchart of the signal processing algorithm is as follows: 1. Data input: receives the fluorescence intensity-time sequence images from the EMCCD.

[0029] 2. Wavelet Denoising: A 5-level discrete wavelet transform was employed, using the 'db4' (Daubechies4) wavelet basis. An adaptive Birgé-Massart thresholding strategy was applied to filter high-frequency coefficients. This strategy dynamically adjusts the threshold based on the local characteristics of the signal, which is superior to a fixed threshold. This effectively distinguishes and filters out cell autofluorescence (short lifetime, different spectrum) and EMCCD readout noise, while preserving the intrinsic signal of diamond fluorescence lifetime. Testing showed a noise reduction rate ≥90% and an effective signal retention rate ≥95%.

[0030] 3. Fluorescence lifetime fitting: For the fluorescence decay curve of each pixel after noise reduction, the least squares method is used to perform exponential fitting to extract the fluorescence lifetime value (τ).

[0031] 4. Temperature conversion: Substitute the fitted fluorescence lifetime value (τ) into the pre-stored "temperature-lifetime" calibration curve to directly calculate the absolute temperature value of the pixel.

[0032] 5. Image Generation and Output: Combines the temperature values ​​of all pixels into a pseudo-color temperature distribution map, which can be displayed in real time on the software interface. The entire processing time is ≤0.5 seconds / frame.

[0033] Example 2 The following are application examples of this device in zebrafish embryo thermometry: I. Preparations before the experiment 1. System status confirmation: The system has been calibrated at three points: 35.0℃, 37.0℃, and 40.0℃ using a standard temperature calibration piece (25-40℃, accuracy ±0.01℃), yielding a fluorescence lifetime-temperature calibration curve τ=-0.025*T+5.8 (R²=0.9995). This curve is built into the software, and the temperature calculation accuracy has been verified to be ±0.08℃.

[0034] 2. Main materials and reagents: AB strain zebrafish embryos (24 hours post-fertilization, 24 hpf), 50 nm nanodiamond powder (with carboxyl groups on the surface), SV40 large T antigen nuclear localization peptide (NLS), EDC, NHS activator, phosphate buffered saline (PBS, pH=7.4), zebrafish embryo culture medium.

[0035] II. Experimental Procedure Step 1: Preparation of Target Probes Composition: 50nm diameter nanodiamond particles (with carboxyl groups, -COOH on the surface) and SV40 large T antigen nuclear localization peptide (NLS).

[0036] 1. Take 1 mg of nanodiamond powder and dissolve it in 1 mL of phosphate buffer (PBS, pH=7.4).

[0037] 2. Sonicate for 30 minutes to fully disperse the particles.

[0038] 3. Add EDC (carbodiimide) and NHS (N-hydroxysuccinimide) activators, and incubate with shaking at room temperature for 30 minutes to activate the carboxyl groups on the diamond surface.

[0039] 4. Add an excess of SV40 nuclear localization peptide and react in a shaker at 37°C for 2 hours.

[0040] 5. Remove unreacted peptides by ultracentrifugation (14000 rpm, 15 min) and resuspend the precipitate with PBS to obtain a target probe solution with a concentration of 0.1 mg / mL.

[0041] Step 2: Zebrafish embryo sample processing 1. Incubation probe: Take 10 healthy 24hpf zebrafish embryos and place them in an EP tube containing 1mL of probe working solution (0.1mg / mL). Incubate for 30 minutes in the dark at the normal zebrafish culture temperature (28.5℃).

[0042] 2. Washing: Carefully aspirate the probe solution with a pipette and gently wash the embryo three times with fresh embryo culture medium to thoroughly remove free probes attached to the surface of the embryo and those that have not entered the cells.

[0043] 3. Loading: Transfer the cleaned embryos to a special glass-bottomed culture dish and add an appropriate amount of culture medium to submerge them.

[0044] Step 3: System Measurement Operation 1. Placement and Stabilization: Place the culture dish on the system's thermostatic stage. To simulate a mammalian environment, set the stage temperature to 37.0℃ ± 0.05℃ and wait 10 minutes to allow the sample temperature to equilibrate with the ambient temperature.

[0045] 2. Software settings: Open the system control and data processing software → select "continuous acquisition" mode → set the acquisition frame rate to 30 frames / second → set the total acquisition time to 10 minutes.

[0046] 3. Begin measurement and real-time analysis: Click the "Start" button. The system will automatically execute the following process: a. Excitation and Collection: A pulsed laser (LD-532-10ns) emits a laser beam, which is focused onto the level of a single embryonic cell after optical path adjustment. An objective lens (OBJ-20×-0.75) collects fluorescence signals from the cell nucleus and cytoplasm.

[0047] b. Signal detection: The EMCCD (EM-1024-60) converts the optical signal into a digital image sequence (fluorescence lifetime image sequence) and transmits it to the computer in real time via a USB 3.0 interface.

[0048] c. Algorithm Processing: The signal processing algorithm within the computer processes each frame of data in real time. - Noise reduction was achieved using a 5-level db4 wavelet transform and adaptive Birgé-Massart thresholding (noise reduction rate ≥90%). Figures 3-5 The images shown are zebrafish embryonic cell images without wavelet denoising, with three layers of wavelet denoising, and with five layers of wavelet denoising, respectively.

[0049] - Perform fluorescence lifetime fitting on the denoised data.

[0050] - Based on the pre-stored calibration curve, the fluorescence lifetime value is converted into a temperature value.

[0051] 4. Data Recording: The software interface displays a pseudo-color temperature distribution map in real time. The operator uses the software's region selection tool to select the nucleus region and the adjacent cytoplasm region. The software automatically records and outputs the average temperature values ​​of these two regions over the entire 10-minute period.

[0052] A method for constructing a non-contact cell thermometry system includes the following steps: Hardware settings: Setting the distance between the laser source module and the optical path adjustment module to 15cm results in a relatively small beam diameter and potentially unsatisfactory divergence angle when the pulsed laser is emitted. After approximately 15cm of transmission, the beam expander effectively enlarges the laser beam diameter, thereby reducing the beam divergence angle. The expanded, collimated beam, after being focused by a high numerical aperture objective lens, forms a smaller, more concentrated spot on the sample, approaching the diffraction limit. This is the physical prerequisite for achieving a spatial resolution below 200nm. Too short a distance results in insufficient beam expansion; too long a distance leads to a less compact system and susceptibility to vibration and dust interference.

[0053] The objective lens is connected to the EMCCD detector via a USB-C interface, with a coaxiality error ≤0.1mm. The EMCCD detector is then connected to the computer via a USB 3.0 interface. Using a USB-C interface and strictly controlling coaxiality ensures that the intermediate image plane formed by the objective lens is accurately and flatly projected onto the photosensitive surface of the EMCCD. This avoids image blurring and reduced edge resolution caused by image plane tilt or offset, ensuring high spatial resolution throughout the field of view. Only when the focal point of the objective lens perfectly coincides with the imaging plane of the detector can the signal collected be from the laser focal point, effectively suppressing stray light outside the focal plane and significantly improving the signal-to-noise ratio and axial resolution. An error of ≤0.1mm is an engineering specification that guarantees this performance.

[0054] System Calibration: Temperature calibration is performed within the range of 25-40℃ using a standard temperature calibration strip. This strip is a known and precise temperature source with minimal inherent temperature error. Using it as a "ruler" eliminates systematic calibration errors. Calibration is performed within the 25-40℃ range because cellular physiological activities primarily occur within this temperature range. Intensive calibration within this range most accurately reflects the functional relationship (typically highly linear) between the fluorescence lifetime of nanodiamonds and temperature within this range. This ensures the system has the highest conversion accuracy within the most commonly used physiological temperature range, directly supporting the technical goal of controlling temperature measurement errors within ±0.08℃.

[0055] Fluorescence signals are collected and a calibration curve is fitted, with a goodness of fit R² ≥ 0.999, achieving a temperature calculation accuracy of ±0.08℃. R² (coefficient of determination) is an indicator of goodness of fit; the closer it is to 1, the better the linear relationship between fluorescence lifetime and temperature, and the smaller the deviation between the data points and the fitted curve. R² ≥ 0.999 means that the two exhibit an extremely excellent linear relationship within the measurement range. This indicates that the fluorescence lifetime-based temperature measurement method itself is very stable and reliable, and not easily affected by other factors. A high goodness of fit means that in actual measurements, any measured fluorescence lifetime value can be uniquely and accurately mapped to a temperature value through this highly determined curve. This is the direct mathematical guarantee for achieving ultra-high accuracy of ±0.08℃. If the R² value is low (e.g., 0.99), it means that the data dispersion is large, and the uncertainty of the calculated temperature value will also increase, failing to meet the accuracy requirements of this invention.

[0056] The foregoing description of specific exemplary embodiments of the present invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is obvious that many changes and variations can be made based on the above teachings. Although embodiments of the invention have been shown and described, these specific embodiments are merely explanations of the invention and are not intended to limit it. The specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. The purpose of selecting and describing exemplary embodiments is to explain the specific principles of the invention and its practical application, so that those skilled in the art, after reading this specification, can make modifications, substitutions, variations, and various choices and changes to the embodiments as needed without departing from the principles and spirit of the invention, provided that such modifications, substitutions, variations, and choices and changes are within the scope of the claims of the invention and are protected by patent law.

Claims

1. A non-contact cell thermometry system based on diamond fluorescence, characterized in that, This includes hardware systems, signal processing algorithms, and target probes; The hardware system includes a constant temperature stage, a signal processing module, a signal detection module, a signal collection module, a cell loading module, an optical path adjustment module, and a laser source module. The signal processing algorithm is used to denoise and process the fluorescence signal to extract temperature information; The targeting probe is used to specifically label cellular substructures and responds to temperature changes via diamond fluorescence.

2. The non-contact cell temperature measurement system according to claim 1, characterized in that, The laser source module includes a pulsed laser with an output wavelength of 532 nm and a pulse width of 10 ns, used to excite the fluorescence of nanodiamonds and to measure fluorescence lifetime.

3. The non-contact cell temperature measurement system according to claim 1, characterized in that, The optical path adjustment module includes a laser shaper, a beam expander, and a reflector, used to shape, expand, and focus the laser beam onto the sample, ensuring that the excitation spot and the signal collection optical path are confocal, with a coaxiality error ≤0.1mm.

4. The non-contact cell temperature measurement system according to claim 1, characterized in that, The cell carrier module includes a culture dish and a thermostatic stage, which provides a stable external temperature environment with a temperature control accuracy of ±0.05℃.

5. The non-contact cell temperature measurement system according to claim 1, characterized in that, The signal collection module includes an objective lens with a numerical aperture ≥0.75 and a magnification of 20x, used to collect the fluorescence signal emitted by the target probe.

6. The non-contact cell temperature measurement system according to claim 1, characterized in that, The signal detection module includes an EMCCD detector, which has an electron multiplication function and a sensitivity at the single-photon level, used to convert fluorescence signals into electrical signals and digitize them.

7. The non-contact cell temperature measurement system according to claim 1, characterized in that, The signal processing module includes a computer processor for running the signal processing algorithm. The signal processing algorithm uses a 5-layer db4 wavelet denoising algorithm and applies an adaptive Birgé-Massart threshold. The noise cancellation rate is ≥90%, the effective signal retention rate is ≥95%, and the processing time is ≤0.5s / frame.

8. The non-contact cell temperature measurement system according to claim 1, characterized in that, The targeting probe comprises nanodiamond particles and a targeting peptide. The nanodiamond particles have a particle size of 50 nm, and the targeting peptide is an SV40 nuclear localization peptide with a nuclear labeling efficiency of ≥90%.

9. The non-contact cell temperature measurement system according to claim 8, characterized in that, The targeting peptide can be replaced with a mitochondrial targeting peptide or a cell membrane targeting peptide to target different cellular substructures.

10. A method for constructing the non-contact cell temperature measurement system of claim 1, characterized in that, Includes the following steps: Hardware connection: Set the distance between the laser source module and the optical path adjustment module to 15cm, connect the objective lens to the EMCCD detector via the C interface, ensure the coaxiality error is ≤0.1mm, and connect the EMCCD detector to the computer via the USB 3.0 interface; System calibration: Temperature calibration is performed in the range of 25-40℃ using standard temperature calibration strips. Fluorescence signals are collected and calibration curves are fitted with a fit degree R²≥0.999, so that the temperature calculation accuracy reaches ±0.08℃.

Images