Cell temperature imaging device and method based on single molecule quantum coherence effect

Abstract

The invention relates to the technical field of biomedical imaging, in particular to a cell temperature imaging device and method based on a single-molecule quantum coherence effect. Comprising an excitation module, a microscopic module and a detection processing module, the excitation module is connected with the microscopic module, the microscopic module is connected with the detection processing module, the excitation module generates an excitation light beam and regulates parameters of the excitation light beam so as to excite a quantum state of fluorescent molecules, and the microscopic module is used for realizing high-resolution fluorescence imaging and signal acquisition. And the detection processing module is used for detecting a fluorescence signal and processing the signal. The device can be directly used for researching the energy exchange process in cells, the temperature effect of ATP generation and the thermodynamic characteristics of the cells, and a new tool is provided for cell biology and medical research. The method is mainly applied to the aspect of cell temperature imaging.

Description

Technical Field

[0001] The present invention relates to the field of biomedical imaging technology, and more specifically, to a cell temperature imaging device and method based on single-molecule quantum coherence effect. Background Art

[0002] The accurate measurement of cell temperature is of great significance in cell metabolism, cancer research, and disease diagnosis. In recent years, cell temperature imaging technology has received extensive attention. Especially in cell metabolism research, the metabolic process of cells is often accompanied by small but detectable temperature changes. However, there are still some challenges in existing temperature measurement technologies, mainly reflected in the following aspects: Limitations in spatial resolution and temperature resolution: Most existing cell temperature imaging technologies use fluorescence probe systems. Their temperature response signals are easily affected by inherent defects such as photobleaching and lifetime fluctuations, resulting in insufficient temperature measurement sensitivity (usually <0.5°C). The spatial resolution of typical fluorescence lifetime thermometry is mostly limited to the micron level, making it difficult to accurately analyze the temperature field of subcellular structures. At the same time, the non-specific response of the probe in the complex intracellular environment will introduce significant measurement errors; Poor anti-interference ability of temperature measurement technology: Fluorescence probes and other technologies based on temperature-sensitive materials are often easily interfered by changes in environmental conditions (such as pH, ion concentration, etc.), which poses a huge challenge to accurately measuring small temperature changes inside cells. Existing temperature imaging technologies usually cannot completely eliminate these interferences, resulting in large errors in temperature data; Limited imaging accuracy and time resolution: Most existing technologies rely on traditional fluorescence imaging or infrared imaging technologies, and the spatial resolution of these methods is generally at the micron level. In addition, the time resolution of existing technologies is usually low, and it is impossible to accurately monitor the temperature changes within a short time during the cell metabolism process. The temperature changes during the cell metabolism process are often very small, so higher-resolution imaging technologies are required to accurately capture these changes; The technical requirements for high sensitivity and high precision are not met: In cell biology and cancer research, especially for the study of abnormal metabolism of cancer cells, the accurate measurement and real-time imaging of cell temperature have important scientific research and clinical significance. Existing technologies often have certain defects in terms of sensitivity and precision and cannot meet the technical requirements of high resolution, low noise, anti-interference, and real-time monitoring. Summary of the Invention

[0003] To overcome the above deficiencies in the existing technology, the present invention provides a cell temperature imaging device and method based on single-molecule quantum coherence effect. This solution realizes real-time temperature imaging of cell substructures by precisely controlling the delay and phase of femtosecond laser pulses and combining high-resolution microscopy imaging and fluorescence detection technology. It solves the problems of insufficient spatial resolution, low temperature measurement sensitivity, weak anti-interference ability, and poor real-time performance existing in the existing technology.

[0004] To solve the above technical problems, the technical solution adopted by the present invention is as follows: A cell temperature imaging device based on single-molecule quantum coherence effect, comprising an excitation module, a microscopy module, and a detection and processing module. The excitation module is connected to the microscopy module, and the microscopy module is connected to the detection and processing module. The excitation module generates an excitation beam and regulates its pulse phase to excite the quantum state of fluorescent molecules. The microscopy module is used to achieve high-resolution cell fluorescence imaging and signal acquisition. The detection and processing module is used to detect the fluorescence signal and process the signal.

[0005] The excitation module includes a femtosecond laser, a delay line, and a phase modulator. The femtosecond laser has a pulse width of 100 femtoseconds, a repetition frequency of 80 MHz, and a wavelength adjustable in the range of 700 - 1000 nm. The femtosecond laser divides the beam into a pump optical path and a probe optical path through a beam splitter. The delay line is used to precisely adjust the time delay of the probe optical path. The phase modulator is used to control the relative phase difference between the two beams to optimize the evolution process of the single-molecule quantum state.

[0006] The microscopy module includes a high-resolution microscope, a sample platform, and a temperature control device. The high-resolution microscope is equipped with a high-performance objective lens with a numerical aperture of 1.49. Through total internal reflection fluorescence excitation technology, a spatial resolution of 200 nm is achieved, which is suitable for precise imaging of the temperature of cell substructures. The sample platform is used to fix cell samples. The temperature control device can control the sample environment temperature in the range of -10°C to 80°C to ensure a stable external environment.

[0007] The detection and processing module includes a single-photon avalanche diode detector, a multi-channel time counter, and a data processing and temperature imaging module. The single-photon avalanche diode detector highly sensitively detects the fluorescence signal of the target molecule, records the arrival time of photons, and supports high-time-resolution fluorescence data acquisition. The multi-channel time counter records the time delay of the fluorescence signal and extracts the decoherence time of single molecules as the basic parameter for temperature measurement.

[0008] The data processing unit calibrates the functional relationship between the single-molecule decoherence time and temperature through experiments and converts the decoherence time data into temperature. The temperature imaging unit generates a visual image of the measured temperature data and combines dynamic monitoring technology to display the temperature distribution and changes of cell substructures in real time.

[0009] A method for a cell temperature imaging device based on single-molecule quantum coherence effect includes the following steps: S1. The femtosecond laser emits laser light, which is split into two beams by a beam splitter. The detection optical path adjusts the time delay through a delay line, and the phase modulator controls the phase difference. The two beams act on the target molecules respectively and regulate the quantum state evolution process; S2. The high-resolution microscope focuses the excitation light on the target area of the cell sample. The fluorescence signal is collected by the SPAD detector, the time counter records the signal delay, and the decoherence time data is extracted; S3. Combining the functional relationship between the decoherence time and temperature calibrated in the experiment, the temperature of the target area is calculated. The temperature imaging module visualizes the temperature data and generates a real-time dynamic temperature image.

[0010] In step S1, the light generated by the laser passes through a quarter-wave plate to adjust the polarization state. The beam is split into two paths by a beam splitter, and one of the paths enters the electro-optic modulator for frequency modulation.

[0011] In step S2, the modulated beam is focused through a lens and irradiated onto the sample through a microscope objective. The fluorescence signal of the sample is separated by a dichroic mirror and a polarization beam splitter to eliminate the background interference signal.

[0012] In step S3, the fluorescence signal from the sample is detected by a single-photon avalanche diode, and the signal is transmitted to the multi-channel time counting processing unit for time-correlated counting analysis. The result is processed by the computer processing module to generate a cell temperature distribution map.

[0013] Compared with the prior art, the beneficial effects of the present invention are: By combining femtosecond laser pulses with a high numerical aperture microscope, temperature resolution at the subcellular scale, approximately 200 nm, has been achieved, enabling precise differentiation of the temperature distributions of subcellular structures such as mitochondria and cytoplasm. Compared with traditional fluorescence lifetime imaging techniques, it can more clearly display the minute temperature changes inside complex cells; using the decoherence time of single-molecule quantum states as a parameter for temperature measurement, it avoids the influence of fluorescence intensity fluctuations and environmental factors on the measurement results, improving the temperature resolution to below 0.1 K. By precisely controlling the delay and phase of the laser pulses, the sensitivity of temperature measurement is further enhanced, enabling it to detect extremely weak temperature changes during cell metabolism; the device can record the temperature changes of single molecules in real time and generate dynamic temperature distribution maps, providing support for the real-time study of cell metabolism processes, drug reactions, and pathological changes. Compared with traditional techniques, it can capture the temperature changes of rapidly occurring cell activities, such as transient responses caused by accelerated cell metabolism or drug effects; by measuring the single-molecule decoherence time and not relying on fluorescence intensity signals, it overcomes the interference of factors such as fluorescence lifetime, pH value, and ion concentration on the measurement, and the measurement results are more stable and reliable; not relying on specific fluorescent molecules, it can select targeting molecules suitable for different subcellular structures according to research needs, with good versatility and adaptability; through the correlation analysis of cell temperature changes and metabolic activities, it can provide precise thermometric evidence for abnormal cancer cell metabolism, drug reactions, and pathological research. This device can be directly used to study the energy exchange process inside cells, the temperature effect of ATP generation, and the thermokinetic characteristics of cells, providing a new tool for cell biology and medical research. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is a schematic structural diagram of the present invention; In the figure: 1 is the excitation module, 2 is the microscopic module, and 3 is the detection and processing module. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0015] In order to more clearly understand the above-mentioned objects, features, and advantages of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be noted that, without conflict, the embodiments of the present application and the features in the embodiments can be combined with each other.

[0016] In the following description, many specific details are set forth in order to fully understand the present invention. However, the present invention can also be implemented in other ways different from those described herein. Therefore, the protection scope of the present invention is not limited by the specific embodiments disclosed below.

[0017] Such as Figure 1As shown, a cell temperature imaging device based on single-molecule quantum coherence effect includes an excitation module 1, a microscopy module 2, and a detection and processing module 3. The excitation module 1 is connected to the microscopy module 2, and the microscopy module 2 is connected to the detection and processing module 3. The excitation module 1 generates an excitation beam and regulates its pulse phase to excite the quantum state of fluorescent molecules. The microscopy module 2 is used to achieve high-resolution cell fluorescence imaging and signal acquisition. The detection and processing module 3 is used to detect the fluorescence signal and process the signal.

[0018] Preferably, the excitation module 1 includes a femtosecond laser, a delay line, and a phase modulator. The femtosecond laser has a pulse width of 100 femtoseconds, a repetition frequency of 80 MHz, and a wavelength adjustable in the range of 700 - 1000 nm to adapt to different targeted fluorescent molecules. The femtosecond laser divides the beam into a pump optical path and a probe optical path through a beam splitter; the delay line is used to precisely adjust the time delay of the probe optical path; the phase modulator is used to control the relative phase difference between the two beams of light to optimize the evolution process of the single-molecule quantum state.

[0019] Through the excitation module 1, two pulsed laser beams act on the target molecules respectively, and through the adjustment of delay and phase, the decoherence process of the molecules is induced.

[0020] Femtosecond laser: Use the InSightX3 femtosecond laser of Spectra-Physics company. Beam splitter: Use the 50:50 non-polarizing beam splitter of Newport company to divide a single beam of laser into two beams of equal intensity laser, forming the basis of interference structured light. The excitation module 1 generates a laser beam with controllable time delay and high spatial coherence, and measures the decoherence time of single molecules by adjusting the pulse time delay.

[0021] Preferably, the microscopy module 2 includes a high-resolution microscope, a sample platform, and a temperature control device. The high-resolution microscope is equipped with a high-performance objective lens with a numerical aperture of 1.49. Through total internal reflection fluorescence excitation technology, a spatial resolution of 200 nm is achieved, which is suitable for precise imaging of the temperature of cell substructures; the sample platform is used to fix cell samples; the temperature control device can control the sample environment temperature in the range of -10°C to 80°C to ensure a stable external environment and avoid experimental errors. The microscopy module 2 focuses the laser beam on the sample area and simultaneously collects the fluorescence signal emitted by the target molecules to provide high-resolution data for subsequent temperature analysis.

[0022] The sample stage uses the high-precision XYZ-axis motorized sample stage of Prior Scientific company to position and scan the sample to be measured. Specific targeted fluorescent molecules, such as dye molecules, quantum dots, etc., are added to the sample. The selection of fluorescent molecules can be tuned according to the target subcellular structure and research needs.

[0023] Preferably, the detection and processing module 3 includes a single-photon avalanche diode detector, a multi-channel time counter, and a data processing and temperature imaging module. The single-photon avalanche diode detector highly sensitively detects the fluorescence signal of the target molecule, records the arrival time of photons, and supports high-time-resolution fluorescence data acquisition. The multi-channel time counter records the time delay of the fluorescence signal and extracts the decoherence time of single molecules, which serves as the basic parameter for temperature measurement.

[0024] The single-photon avalanche diode SPAD, using the single-photon detector of Excelitas Technologies Corp., is used to detect the single-molecule fluorescence signal, with high sensitivity and high time resolution. Two SPADs are used to measure the change in the interference light intensity. The multi-channel time-correlated photon counter MCPET, using the time-correlated photon counting system of PicoQuant GmbH, realizes the time-correlated photon counting analysis of the fluorescence signal and is used to extract the single-molecule decoherence time information; Preferably, the data processing unit calibrates the functional relationship between the single-molecule decoherence time and temperature through experiments and converts the decoherence time data into temperature. The temperature imaging unit generates a visual image of the measured temperature data and, in combination with the dynamic monitoring technology, displays the temperature distribution and changes of the cell substructure in real time.

[0025] Combined with the delay and phase control of the laser pulse, the ambient temperature is measured using the single-molecule decoherence time.

[0026] A method for a cell temperature imaging device based on the single-molecule quantum coherence effect includes the following steps: S1. The femtosecond laser emits laser light, which is divided into two beams by a beam splitter. The detection optical path adjusts the delay through a delay line, and the phase modulator controls the phase difference. The two beams act on the target molecule respectively and regulate its quantum state evolution process; S2. The high-resolution microscope focuses the excitation light on the target area of the cell sample. The fluorescence signal is collected by the SPAD detector, the time counter records the signal delay, and the decoherence time data is extracted; S3. Combining the functional relationship between the decoherence time and temperature calibrated by experiments, the temperature of the target area is calculated. The temperature imaging module visualizes the temperature data and generates a real-time dynamic temperature image.

[0027] Preferably, in step S1, the light generated by the laser passes through a quarter-wave plate to adjust the polarization state, and the beam is divided into two paths by a beam splitter, and one of the paths enters an electro-optic modulator for frequency modulation.

[0028] Preferably, in step S2, the modulated beam is focused through a lens and irradiated onto the sample through a microscope objective. The fluorescence signal of the sample is separated by a dichroic mirror and a polarization beam splitter to eliminate the background interference signal.

[0029] Preferably, in step S3, the fluorescence signal from the sample is detected by a single-photon avalanche diode, and the signal is transmitted to a multi-channel time-correlated single-photon counting (TCSPC) unit for time-correlated counting analysis. The result is processed by a computer processing module to generate a cell temperature distribution map.

[0030] Workflow: The light generated by the laser passes through a quarter-wave plate to adjust the polarization state. The beam is split into two paths by a beam splitter. One path enters an electro-optic modulator for frequency modulation. The modulated beam is focused by a lens and irradiated onto the sample through a microscope objective. The fluorescence signal of the sample is separated by a dichroic mirror and a polarization beam splitter to eliminate background interference signals. The fluorescence signal from the sample is detected by a single-photon avalanche diode, and the signal is transmitted to a multi-channel time-correlated single-photon counting (TCSPC) unit for time-correlated counting analysis. The result is processed by a computer processing module to generate a cell temperature distribution map.

[0031] Laser tuning and optical path adjustment: Tune the output wavelength of the femtosecond laser according to the excitation wavelength of the fluorescent molecule; adjust the laser polarization state and time delay so that two laser beams form a spatial interference light field at the sample focus. Sample preparation: Introduce the targeted fluorescent molecule into the biological sample; fix the sample on an electric sample stage and adjust it to the objective focus. Signal acquisition and analysis: Excite the fluorescence signal and collect it through the microscope module; use a single-photon avalanche diode (SPAD) to detect the fluorescence signal and a multi-channel plate electron multiplier time-of-flight (MCPET) to record time-correlated data; use a computer to fit the signal to obtain the single-molecule decoherence time and the corresponding temperature information. Temperature imaging: Generate a subcellular-level temperature distribution map through a specific algorithm based on the relationship between the decoherence time and temperature.

[0032] The above only describes the preferred embodiments of the present invention in detail, but the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those of ordinary skill in the art, various changes can be made without departing from the gist of the present invention, and all such changes should be included within the protection scope of the present invention.

Claims

1. A cell temperature imaging device based on single molecule quantum coherence effect, characterized in that: The invention comprises an excitation module (1), a microscopy module (2) and a detection and processing module (3), wherein the excitation module (1) is connected to the microscopy module (2), and the microscopy module (2) is connected to the detection and processing module (3). The excitation module (1) generates an excitation light beam and adjusts its pulse phase to excite the quantum state of fluorescent molecules. The microscopy module (2) is used to achieve high-resolution cell fluorescence imaging and signal acquisition, and the detection and processing module (3) is used to detect fluorescent signals and process the signals.

2. The cell temperature imaging device based on single molecule quantum coherence effect according to claim 1, characterized in that: The excitation module (1) comprises a femtosecond laser, a delay line and a phase modulator. The femtosecond laser has a pulse width of 100 femtoseconds, a repetition frequency of 80 MHz, and a wavelength that can be adjusted within the range of 700-1000 nm. The femtosecond laser divides a light beam into a pump light path and a detection light path through a beam splitter. The delay line is used to accurately adjust the time delay of the detection light path. The phase modulator is used to control the relative phase difference between the two light beams and optimize the evolution process of the single-molecule quantum state.

3. The cell temperature imaging device based on single molecule quantum coherence effect according to claim 1, characterized in that: The microscopy module (2) comprises a high-resolution microscope, a sample platform and a temperature control device. The high-resolution microscope is equipped with a high-performance objective lens with a numerical aperture of 1.49, and achieves a spatial resolution of 200 nm through total internal reflection fluorescence excitation technology, which is suitable for accurate imaging of cell substructure temperature. The sample platform is used to fix cell samples. The temperature control device can control the sample environment temperature in the range of -10°C to 80°C to ensure the stability of the external environment.

4. The cell temperature imaging device based on single molecule quantum coherence effect according to claim 1, characterized in that: The detection and processing module (3) comprises a single-photon avalanche diode detector, a multi-channel time counter and a data processing and temperature imaging module. The single-photon avalanche diode detector detects the fluorescence signal of the target molecule with high sensitivity, records the arrival time of the photons, and supports fluorescence data acquisition with high time resolution. The multi-channel time counter records the time delay of the fluorescence signal and extracts the decoherence time of a single molecule as a basic parameter for temperature measurement.

5. The cell temperature imaging device based on single molecule quantum coherence effect according to claim 4, characterized in that: The data processing unit converts the decoherence time data into temperature by experimentally calibrating the functional relationship between the single-molecule decoherence time and temperature; the temperature imaging unit generates a visual image from the measured temperature data, and combines it with dynamic monitoring technology to display the temperature distribution and changes of the cell substructure in real time.

6. A method for using the cell temperature imaging device based on single molecule quantum coherence effect as claimed in claim 1, characterized in that: The following steps are involved: S1, femtosecond laser emits laser, which is divided into two beams by beam splitter; the detection light path passes through the delay line to adjust the delay, and the phase modulator controls the phase difference. The two beams act on the target molecules respectively and regulate their quantum state evolution process; S2, a high-resolution microscope focuses the excitation light to the targeted area of ​​the cell sample; the fluorescence signal is collected by the SPAD detector, the time counter records the signal delay, and the decoherence time data is extracted; S3. Calculate the temperature of the target area based on the functional relationship between the decoherence time and temperature calibrated by the experiment; the temperature imaging module visualizes the temperature data and generates a real-time dynamic temperature image.

7. The cell temperature imaging method based on single molecule quantum coherence effect according to claim 6, characterized in that: In the step S1, the polarization state of the light generated by the laser is adjusted by a quarter wave plate, and the light beam is divided into two paths by a beam splitter, one of which enters the electro-optic modulator for frequency modulation.

8. The cell temperature imaging method based on single molecule quantum coherence effect according to claim 6, characterized in that: In step S2, the modulated light beam is focused by a lens and irradiated to the sample through a microscope objective lens, and the fluorescence signal of the sample is separated by a dichroic mirror and a polarization beam splitter to eliminate background interference signals.

9. The cell temperature imaging method based on single molecule quantum coherence effect according to claim 6, characterized in that: In step S3, the fluorescence signal from the sample is detected by a single photon avalanche diode, and the signal is transmitted to a multi-channel time counting processing unit for time-correlated counting analysis. The result is processed by a computer module to generate a cell temperature distribution map.

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