A biological microscopic measurement system and measurement method

Abstract

The present invention discloses a biological microscopic measurement system and a measurement method. The biological microscopic measurement system includes: a sample carrying module, a microwave radiation module and an optical path module; the sample carrying module is used to carry biological samples, wherein nanodiamonds are arranged in the biological samples; the microwave radiation module is located between the sample carrying module and the optical path module, and the microwave radiation module is used to emit microwaves to the biological samples under multiple different set frequency intervals, wherein the microwave radiation module corresponds to a resonant frequency in each set frequency interval and the resonant frequency is located in the corresponding set frequency interval; the optical path module is used to emit lasers to the biological samples, and is also used to obtain the fluorescence signals generated by the nanodiamonds under the action of the environmental parameters to be measured, the laser and the microwave. The present invention provides a biological microscopic measurement system and a measurement method, which has a simple structure, can accurately measure physical quantities such as the microscopic magnetic field and temperature of the biological samples, and can also perform biological microscopic imaging.

Description

Technical Field

[0001] The present invention relates to the field of precision measurement technology, and in particular to a biological microscopic measurement system and a measurement method. Background Art

[0002] Fluorescent nanodiamond (FND) refers to nanodiamond containing NV color centers. FND can be spontaneously internalized by cells and has very low toxicity. The spin properties of the NV color center combined with the optically detected magnetic resonance (ODMR) technology can realize fluorescence imaging and measurement in biological cells or other organisms. The fluorescence intensity of the NV color center is adjusted by alternating microwave radiation, while the background fluorescence remains constant. Based on the above-mentioned microwave modulation technology combined with the phase-locked amplification technology, the background spontaneous fluorescence signal can be effectively removed, thereby improving the signal-to-noise ratio of the FND fluorescence signal and improving the contrast of the fluorescence signal.

[0003] The lifetime of spontaneous fluorescence of cells and tissues is about 3ns, while the lifetime of fluorescence of NV color centers is about 20ns. Therefore, the fluorescence brightness and stability of FND make it suitable for single-particle tracing in cells. Based on confocal microscopy technology, super-resolution imaging can be achieved, and single FND particles (about 40nm) in cells can be detected. Therefore, FND has good spatial resolution structural details in living biological systems, making FND an ideal biological probe for molecular imaging and cell labeling, and can obtain high spatial resolution measurements of physical quantities such as magnetic field and temperature. The above-mentioned biological imaging and sensing technology based on FND has great application prospects in the field of biological microscopic detection.

[0004] However, the existing measurement system cannot provide sufficient microwave radiation power for FND, which reduces the contrast of the fluorescence detection signal. In addition, the measurement of physical quantities such as magnetic field and temperature in the environment where FND is located also has the problem of low accuracy. Summary of the invention

[0005] The present invention provides a biological microscopic measurement system and a measurement method, which have a simple structure and can accurately measure physical quantities such as the microscopic magnetic field and temperature of a biological sample, and can also perform biological microscopic imaging.

[0006] According to one aspect of the present invention, a biological microscopic measurement system is provided, the biological microscopic measurement system comprising: a sample carrying module, a microwave radiation module and an optical path module;

[0007] The sample carrying module is used to carry a biological sample, wherein a nanodiamond is arranged in the biological sample, and the nanodiamond contains an NV color center;

[0008] The microwave radiation module is located between the sample holding module and the optical path module, and is used to emit microwaves to the biological sample under a plurality of different set frequency intervals, wherein the microwave radiation module corresponds to a resonant frequency in each of the set frequency intervals and the resonant frequency is located in the corresponding set frequency interval; the minimum frequency in the plurality of different set frequency intervals is less than or equal to the first set frequency, and the maximum frequency in the plurality of different set frequency intervals is greater than or equal to the second set frequency, the second set frequency is greater than the first set frequency, and the first set frequency and the second set frequency are microwave resonant frequencies that cause the ground state electron spin of the NV color center in the nanodiamond to transition from the |0> state to the |±1> state under the environmental parameters to be measured;

[0009] The optical path module is used to emit laser light to the biological sample, and is also used to obtain the fluorescence signal generated by the nano-diamond under the action of the environmental parameter to be measured, the laser light and the microwave.

[0010] Optionally, the biological microscopic measurement system provided in this embodiment further includes: a frequency determination module and a threshold determination module;

[0011] The threshold determination module is used to determine the set threshold corresponding to the microwave radiation module;

[0012] The frequency determination module is used to determine the frequency interval from the first set frequency to the second set frequency, and determine the microwave scanning interval based on the frequency interval. It is also used to divide the microwave scanning interval into the multiple different set frequency intervals according to the set threshold, so that the setting coefficient of the microwave radiation module at each frequency in each of the set frequency intervals is less than the set threshold.

[0013] Optionally, the microwave radiation module is also used to control a set coefficient at the endpoint frequencies corresponding to each of the set frequency intervals to be greater than or equal to a set threshold, and is also used to control the resonant frequency of each of the set frequency intervals to be the average value of the two endpoint frequencies corresponding to the set frequency interval; wherein the set coefficient is a reflection coefficient or a transmission coefficient.

[0014] Optionally, the microwave radiation module includes a microwave source, a substrate, a microstrip line structure located on one side of the substrate, a microwave transmission line, a port structure and an impedance adjustable unit;

[0015] The microwave source is connected to the port structure, and the microwave source is used to generate microwaves;

[0016] The microstrip line structure is connected to the port structure and the microwave transmission line, and the microstrip line structure is used to transmit the microwaves generated by the microwave source to the microwave transmission line;

[0017] The substrate comprises an objective lens light transmission port, the microwave emission line crosses the objective lens light transmission port, and the microwave emission line is used to emit microwaves to the biological sample;

[0018] The impedance adjustable unit is used to adjust the resonant frequency of the microwave radiation module.

[0019] Optionally, the microwave radiation module further includes a vector network analyzer;

[0020] The port structure includes a first port and a second port;

[0021] The microstrip line structure comprises a first microstrip line and a second microstrip line, wherein a first end of the first microstrip line is connected to the first port, and a second end of the first microstrip line is connected to a first end of the microwave transmission line; a first end of the second microstrip line is connected to the second port, and a second end of the second microstrip line is connected to a second end of the microwave transmission line;

[0022] The first port is also connected to the microwave source, and the second port is also connected to the vector network analyzer.

[0023] Optionally, the microwave radiation module further includes a vector network analyzer;

[0024] The port structure is a single port;

[0025] The vector network analyzer and the microwave wave source are both connected to the port structure, and the vector network analyzer is connected to the microwave wave source in parallel.

[0026] Optionally, the microwave transmitting line includes a metal wire or a flexible flat cable;

[0027] The flexible flat cable comprises a flexible base layer and a conductive layer located in the flexible base layer; the conductive layer comprises a conductive wire and a conductive Ω ring.

[0028] Optionally, a vertical projection of the microwave emission line on the light transmission port of the objective lens coincides with a symmetry axis of the light transmission port of the objective lens.

[0029] Optionally, the optical path module includes an objective lens and a wide-field optical path sub-module;

[0030] The microwave radiation module is located between the objective lens and the sample holding module;

[0031] The wide-field optical path submodule comprises a first light source unit, a first dichroic mirror and a light collection unit;

[0032] The first light source unit is used to generate laser light and transmit the laser light to the biological sample through the first dichroic mirror and the objective lens;

[0033] The light collection unit is used to receive the fluorescent signal transmitted through the objective lens and the first dichroic mirror.

[0034] Optionally, the optical path module further includes a reflecting prism and a confocal optical path sub-module;

[0035] The confocal optical path submodule includes a second light source unit, a second dichroic mirror and a photon detection unit;

[0036] The second light source unit is used to generate laser light and transmit the laser light to the biological sample through the second dichroic mirror, the reflecting prism and the objective lens;

[0037] The photon detection unit is used to receive the fluorescence signal transmitted through the objective lens, the reflection prism and the second dichroic mirror;

[0038] The reflecting prism is located between the objective lens and the first dichroic mirror, and between the objective lens and the second dichroic mirror.

[0039] Optionally, the sample carrying module includes a sample dish, a sample cover, a translation stage, a translation screw, a translation base and a reset spring;

[0040] The sample dish is used to contain the biological sample; the sample cover is used to cover the sample dish; the translation stage is used to drive the sample dish to move relative to the translation base;

[0041] The optical path module also includes an objective lens mounting column, a scanning imaging module base, a lifting platform, a prism platform, a lifting screw and a lifting base;

[0042] The prism stand is used to support the reflecting prism;

[0043] The objective lens is arranged on the lifting platform, the lifting platform is connected to the lifting screw rod, and the lifting platform moves relative to the lifting base.

[0044] According to another aspect of the present invention, a biological microscopic measurement method is provided, and the biological microscopic measurement method is applied to the biological microscopic measurement system provided by any embodiment of the present invention;

[0045] The biological microscopic measurement method comprises:

[0046] The sample carrying module carries a biological sample, wherein nanodiamonds are arranged in the biological sample, and the nanodiamonds contain NV color centers;

[0047] The microwave radiation module emits microwaves to the biological sample in a plurality of different set frequency intervals, wherein the microwave radiation module corresponds to a resonant frequency in each of the set frequency intervals and the resonant frequency is located in the corresponding set frequency interval; the minimum frequency in the plurality of different set frequency intervals is less than or equal to the first set frequency, and the maximum frequency in the plurality of different set frequency intervals is greater than or equal to the second set frequency, the second set frequency is greater than the first set frequency, and the first set frequency and the second set frequency are microwave resonant frequencies that cause the ground state electron spin of the NV color center in the nanodiamond to transition from a |0> state to a |±1> state under the environmental parameters to be measured;

[0048] The optical path module emits laser light to the biological sample, and obtains the fluorescence signal generated by the nano-diamond under the action of the environmental parameter to be measured, the laser light and the microwave.

[0049] The embodiment of the present invention provides a biological microscopic measurement system, which includes a sample carrying module, a microwave radiation module and an optical path module. The sample carrying module is used to carry a biological sample provided with nanodiamonds, wherein the nanodiamonds contain NV color centers. The optical path module is used to emit lasers to the biological sample, and can also obtain the fluorescence signal generated by the biological sample. The microwave radiation module is located between the sample carrying module and the optical path module. In this way, the position of the microwave radiation module can be kept unchanged when the sample carrying module moves, thereby avoiding the measurement error caused by the movement of the microwave radiation module. The microwave radiation module can emit microwaves to the biological sample at multiple different set frequency intervals, and each set frequency interval corresponds to a resonant frequency, that is, the microwave radiation module emits microwaves to the biological sample at multiple resonant frequencies, ensuring that the microwaves received by the biological sample can meet the microwave power requirements of the nanodiamonds in the biological sample, thereby ensuring that the optical path module can accurately determine the physical quantities such as the microscopic magnetic field or temperature of the biological sample according to the fluorescence signal, and also ensuring that the optical path module can accurately perform marker imaging, temperature imaging, magnetic imaging, etc. according to the fluorescence signal. A resonant frequency is set in each set frequency interval, and the resonant frequency is not set to change at any time with the change of microwave frequency, so that the microwave radiation module does not need to be set with a complex structure. In summary, the biological microscopic measurement system provided by the embodiment of the present invention has a simple structure, and can accurately measure physical quantities such as the microscopic magnetic field and temperature of the biological sample, and can also perform biological microscopic imaging.

[0050] It should be understood that the contents described in this section are not intended to identify the key or important features of the embodiments of the present invention, nor are they intended to limit the scope of the present invention. Other features of the present invention will become easily understood through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0052] Figure 1 is a schematic structural diagram of a biological microscopic measurement system provided according to an embodiment of the present invention;

[0053] Figure 2 is a schematic diagram of an NV color center energy level transition provided according to an embodiment of the present invention;

[0054] Figure 3 is a schematic diagram of a corresponding relationship between a microwave frequency and a set coefficient and a CW-ODMR spectrum provided according to an embodiment of the present invention;

[0055] Figure 4 It is an imaging result diagram of a fluorescence signal determined by a biological microscopic measurement system with a fixed resonant frequency;

[0056] Figure 5 is an imaging result diagram of a fluorescence signal determined by a biological microscopic measurement system provided by an embodiment of the present invention;

[0057] Figure 6 is a schematic structural diagram of a microwave radiation module provided according to an embodiment of the present invention;

[0058] Figure 7 is a schematic structural diagram of another microwave radiation module provided according to an embodiment of the present invention;

[0059] Figure 8 for Figure 6 A schematic diagram of the three-dimensional structure of the microwave radiation module shown;

[0060] Fig. 9 is a schematic structural diagram of another biological microscopic measurement system provided according to an embodiment of the present invention;

[0061] Fig.10 is a schematic diagram of the three-dimensional structure of a biological microscopic measurement system provided according to an embodiment of the present invention;

[0062] Fig.11 is a schematic diagram of the three-dimensional structure of another biological microscopic measurement system provided according to an embodiment of the present invention;

[0063] Fig.12 It is a flowchart of a biological microscopic measurement method provided according to an embodiment of the present invention. DETAILED DESCRIPTION

[0064] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0065] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0066] Figure 1 is a schematic diagram of the structure of a biological microscopic measurement system provided according to an embodiment of the present invention, with reference to Figure 1 The biological microscopic measurement system provided in this embodiment includes a sample holding module 110, a microwave radiation module 210 and an optical path module 310; the sample holding module 110 is used to hold a biological sample 400, wherein the biological sample 400 is provided with nanodiamonds, and the nanodiamonds contain NV color centers; the microwave radiation module 210 is located between the sample holding module 110 and the optical path module 310, and the microwave radiation module 210 is used to emit microwaves to the biological sample 400 in a plurality of different set frequency intervals, wherein the microwave radiation module 210 corresponds to a resonant frequency in each set frequency interval and the resonant frequency is The vibration frequency is within the corresponding set frequency interval; the minimum frequency in the multiple set frequency intervals is less than or equal to the first set frequency, and the maximum frequency in the multiple set frequency intervals is greater than or equal to the second set frequency, the second set frequency is greater than the first set frequency, and the first set frequency and the second set frequency are microwave resonance frequencies that make the ground state electron spin of the NV color center in the nanodiamond transition from the |0> state to the |±1> state under the environmental parameters to be measured; the optical path module 310 is used to emit laser to the biological sample, and is also used to obtain the fluorescence signal generated by the nanodiamond under the action of the environmental parameters to be measured, laser and microwave.

[0067] Specifically, the sample holding module 110 includes a sample dish 111, which is used to hold the biological sample 400 and has light transmittance. The optical path module 310 includes an objective lens 311, and the laser generated by the optical path module 310 is transmitted through the objective lens 311 through the sample dish 111 to the biological sample 400.

[0068] The microwave radiation module 210 in this embodiment is located between the sample supporting module 110 and the optical path module 310. When the sample supporting module 110 drives the sample dish 111 to move relative to the optical path module 310 for scanning imaging or measurement, the positions of the microwave radiation module 210 and the objective lens 311 in the optical path module 310 are relatively fixed, that is, when the sample dish 111 moves, the relative positions of the microwave radiation module 210 and the objective lens 311 in the optical path module 310 are fixed, thereby avoiding measurement errors caused by the movement of the microwave radiation module 210.

[0069] The microwave radiation module 210 is used to provide a modulated microwave field of a resonant frequency for the nanodiamonds in the biological sample 400. The purpose of the modulation is to change the fluorescence intensity of the fluorescence signal of the nanodiamonds in the biological sample 400 according to a preset waveform (a sine wave or square wave pulse of a fixed frequency), thereby facilitating the demodulation of the fluorescence signal in the nanodiamonds in the biological sample 400 from the complex noise background.

[0070] The first set frequency is the microwave resonance frequency that causes the ground state electron spin of the NV color center in the nanodiamond to transition from the |0> state to the |-1> state under the environmental parameters to be tested, and the second set frequency is the microwave resonance frequency that causes the ground state electron spin of the NV color center in the nanodiamond to transition from the |0> state to the |+1> state under the environmental parameters to be tested. Alternatively, the first set frequency is the microwave resonance frequency that causes the ground state electron spin of the NV color center in the nanodiamond to transition from the |0> state to the |+1> state under the environmental parameters to be tested, and the second set frequency is the microwave resonance frequency that causes the ground state electron spin of the NV color center in the nanodiamond to transition from the |0> state to the |-1> state under the environmental parameters to be tested.

[0071] Figure 2 is a schematic diagram of an NV color center energy level transition provided according to an embodiment of the present invention, Figure 3 is a schematic diagram of a corresponding relationship between a microwave frequency and a set coefficient and a CW-ODMR spectrum provided according to an embodiment of the present invention, Figure 3 The horizontal axis in represents the frequency of microwaves. Figure 3 The vertical coordinate Γ in the corresponding relationship between the microwave frequency and the setting coefficient represents the setting coefficient. When the microwave radiation module 210 is a single port, the setting coefficient Γ is the reflection coefficient. When the microwave radiation module 210 is a dual port, the setting coefficient Γ is the transmission coefficient. Figure 3 The vertical axis V of the CW-ODMR spectrum PDIndicates fluorescence signal intensity or photon counts, reference Figure 2 and Figure 3 , the ODMR detection principle of NV color center is:

[0072] When microwaves are applied to the ground state electron spin of the NV color center, resonance occurs when the frequency of the microwaves is equal to the energy level difference of the ground state electron spin transition of the NV color center. The resonant microwaves cause the ground state electron spin of the NV color center to transition from the |0> state to the |+1> state or the |-1> state, resulting in a decrease in the fluorescence count. This is the NV color center ground state electron spin resonance (ESR). Unlike traditional ESR, since NV fluorescence is spin-dependent, lasers can be used to detect the paramagnetic resonance signal of the ground state electron of the NV color center. The resonance spectrum obtained by applying continuous laser and microwaves is called continuous wave optically detected magnetic resonance (CW-ODMR spectrum), or CW spectrum for short.

[0073] In the CW spectrum experiment, the laser makes the NV color center ground state electron spin tend to be arranged in the |0> state, while the resonant microwave makes the NV color center ground state electron spin away from the |0> state. Therefore, the contrast of the continuous resonance peak depends on the competitive relationship between the laser power and the microwave power. The contrast can reach the set contrast, which can be 30%. For example, when there is no external magnetic field, the |±1> state energy level is degenerate, and the CW spectrum has a resonance peak at 2870MHz. Figure 3 When there is a weak magnetic field, the energy level degenerates, and two peaks appear in the CW spectrum, and the symmetry center is 2870MHz (the symmetry center is related to the ambient temperature and the magnetic field strength, and the measurement of the symmetry center can be used for temperature measurement and magnetic field strength measurement), corresponding to the transition from the |0> state to the |-1> state or the |+1> state. When the external magnetic field changes, the NV energy level undergoes Zeeman shift, and the position of the CW spectrum resonance peak will also move accordingly, thereby realizing the measurement of the environmental parameters of the biological sample 400, and the environmental parameters of the biological sample 400 include physical quantities such as the magnetic field strength and temperature of the biological sample 400.

[0074] The environmental parameters to be measured may include physical quantities such as magnetic field intensity and temperature that need to be measured in the environment where the biological sample 400 is located.

[0075] Usually, the resonant frequency of the microwave radiation module (including the entire microwave transmission link) is fixed, as long as the bandwidth of the microwave radiation module (the effective working frequency range centered on its resonant frequency) is greater than the range in which the microwave needs to be swept; when the microwave radiation module 210 is arranged between the objective lens 311 and the sample dish 111, the microwaves generated by the microwave radiation module with a fixed resonant frequency cannot meet the microwave power requirements of the nanodiamonds in the biological sample 400, because the glass slide at the bottom of the sample dish 400 will absorb and block part of the microwave power, so that the microwave power that the nanodiamonds in the biological sample 400 can receive is extremely low, resulting in the NV color center ground state electron spin transition in the nanodiamond unable to reach a saturation state, the contrast of the fluorescence modulation signal is low, and it is difficult to demodulate the fluorescence signal in the nanodiamond from the complex noise background. Figure 4 This is an imaging result diagram of a fluorescence signal determined by a biological microscopic measurement system with a fixed resonant frequency. Figure 4 , Figure 4 The right side shows the imaging result of all fluorescence signals, and the left side shows the imaging result of the fluorescence signal of nanodiamond obtained by demodulating the fluorescence signal after microwave modulation. Figure 4 It can be seen that the fluorescence signal on the left is not obvious. If the environmental parameters to be measured are measured based on the fluorescence signal imaging result diagram of the nanodiamond on the left, there may be problems such as low measurement accuracy.

[0076] In order to solve the above problems, the present embodiment sets the resonant frequency of the microwave radiation module 210 to be adjustable, so that the microwave radiation module 210 corresponds to different resonant frequencies in different set frequency ranges. The specific introduction of the microwave radiation module 210 is as follows:

[0077] The microwave radiation module 210 is used to emit microwaves in a plurality of different set frequency intervals, that is, the frequencies of microwaves emitted by the microwave radiation module 210 to the biological sample 400 are all within a plurality of set frequency intervals, and the plurality of set frequency intervals may be continuous set frequency intervals. The minimum frequency in the plurality of set frequency intervals refers to the minimum frequency value in the plurality of set frequency intervals, and the maximum frequency in the plurality of set frequency intervals refers to the maximum frequency value in the plurality of set frequency intervals. For example, continue to refer to Figure 3 Schematic diagram of the corresponding relationship between the microwave frequency and the setting coefficient. The smaller the setting coefficient, the more microwave power the microwave acts on the biological sample 400. If multiple setting frequency intervals are [F W1 , F W2 ]、[F W2 , F W3 ]、[F W3 , F W4 ], then the minimum frequency among multiple set frequency intervals is F W1 , the maximum frequency among multiple set frequency intervals is FW4 .

[0078] By setting each set frequency interval to correspond to one resonant frequency instead of corresponding to multiple resonant frequencies, it is possible to avoid the resonant frequency changing multiple times with the change of the microwave frequency, thereby avoiding the microwave radiation module 210 from having a complex structure. By setting the resonant frequency corresponding to each set frequency interval to be within the set frequency interval, the microwave radiation module 210 can set a smaller coefficient at each frequency in the set frequency interval, so that the microwaves emitted by the microwave radiation module 210 to the biological sample 400 meet the microwave power requirements of the nanodiamonds in the biological sample 400. Figure 5 is an imaging result diagram of a fluorescence signal determined by a biological microscopic measurement system provided by an embodiment of the present invention, with reference to Figure 5 , Figure 5 The right side shows the imaging result of all fluorescence signals. Figure 5 The left side shows the fluorescence signal imaging result of nanodiamond obtained by demodulating the fluorescence signal after microwave modulation. Figure 5 It can be seen that the fluorescence signal on the left is obvious. According to the fluorescence signal imaging result of the nanodiamond on the left, the environmental parameters to be measured can be accurately measured.

[0079] Continue to refer Figure 3 , the first set frequency is recorded as F |-1> , the second set frequency is recorded as F |+1> , setting the minimum frequency in the multiple set frequency intervals to be less than or equal to the first set frequency, and the maximum frequency in the multiple set frequency intervals to be greater than or equal to the second set frequency, so that the first set frequency F |-1> and the second setting frequency F |+1> are all within multiple set frequency intervals, so the first set frequency F can be accurately determined based on the fluorescence signal |-1> and the second setting frequency F |+1> , and according to the first set frequency F |-1> and the second setting frequency F |+1> Determine physical quantities such as the microscopic magnetic field or temperature where the biological sample 400 is located.

[0080] After the optical path module 310 acquires the fluorescence signal, the light signal intensity or photon count can be determined based on the fluorescence signal, thereby determining the CW spectrum. Based on the CW spectrum, physical quantities such as the microscopic magnetic field or temperature of the biological sample 400 can be determined, and marker imaging, temperature imaging, magnetic imaging, etc. can also be performed based on the fluorescence signal.

[0081] The present embodiment provides a biological microscopic measurement system, which includes a sample carrying module, a microwave radiation module and an optical path module. The sample carrying module is used to carry a biological sample provided with nanodiamonds, wherein the nanodiamonds contain NV color centers. The optical path module is used to emit lasers to the biological sample, and can also obtain the fluorescence signal generated by the biological sample. The microwave radiation module is located between the sample carrying module and the optical path module. In this way, the position of the microwave radiation module can be kept unchanged when the sample carrying module moves, thereby avoiding the measurement error caused by the movement of the microwave radiation module. The microwave radiation module can emit microwaves to the biological sample at multiple different set frequency intervals, and each set frequency interval corresponds to a resonant frequency, that is, the microwave radiation module emits microwaves to the biological sample at multiple resonant frequencies, ensuring that the microwaves received by the biological sample can meet the microwave power requirements of the nanodiamonds in the biological sample, thereby ensuring that the optical path module can accurately determine the physical quantities such as the microscopic magnetic field or temperature of the biological sample according to the fluorescence signal, and also ensuring that the optical path module can accurately perform marker imaging, temperature imaging, magnetic imaging, etc. according to the fluorescence signal. A resonant frequency is set in each set frequency range, and the resonant frequency is not set to change at any time with the change of microwave frequency, so that the microwave radiation module does not need to be set with a complex structure. In summary, the biological microscopic measurement system provided by this embodiment has a simple structure, and can accurately measure physical quantities such as the microscopic magnetic field and temperature of the biological sample, and can also perform biological microscopic imaging.

[0082] Optionally, the biological microscopic measurement system provided in this embodiment also includes: a frequency determination module and a threshold determination module; the threshold determination module is used to determine the set threshold corresponding to the microwave radiation module; the frequency determination module is used to determine the frequency interval where the first set frequency to the second set frequency are located, and determine the microwave scanning interval according to the frequency interval, and is also used to divide the microwave scanning interval into a plurality of different set frequency intervals according to the set threshold, so that the set coefficient of the microwave radiation module at each frequency in each set frequency interval is less than or equal to the set threshold.

[0083] Specifically, the threshold determination module is used to determine the microwave power P0 corresponding to the fluorescence contrast requirement of the nanodiamonds in the biological sample, and determine the required set threshold Γ0 according to the microwave power P0 and the working power of the microwave radiation module.

[0084] The frequency determination module determines the frequency range [F |-1> ,F |+1> ], according to the frequency range [F |-1> ,F |+1> ] determines a microwave scanning interval, denoted as [a, b], where the microwave scanning interval includes the frequency interval [F |-1> ,F |+1>], that is, a≤F |-1> ,F |+1> ≤b. Multiple set frequency intervals can be [F W1 , F W2 ]、[F W2 , F W3 ]、[F W3 , F W4 ],a≤F W1 ,F W4 ≤b.

[0085] By setting the set coefficient at each frequency in each set frequency range to be less than or equal to the set threshold, the microwaves at each frequency generated by the microwave radiation module can meet the microwave power requirements of the nanodiamonds in the biological sample, thereby further improving the measurement accuracy of physical quantities such as microscopic magnetic fields and temperature.

[0086] Optionally, the microwave radiation module is also used to control the set coefficient at the endpoint frequencies corresponding to the two endpoint frequencies of each set frequency interval to be greater than or equal to a set threshold, and is also used to control the resonant frequency of each set frequency interval to be the average value of the two endpoint frequencies corresponding to the set frequency interval; wherein the set coefficient is a reflection coefficient or a transmission coefficient.

[0087] For example, continue to refer to Figure 3 , with multiple set frequency intervals [F W1 , F W2 ]、[F W2 , F W3 ]、[F W3 , F W4 ] is used as an example to illustrate that when the microwave radiation module performs microwave frequency sweep, the microwave frequency is [F W1 , F W2 ] interval, the microwave radiation module adjusts its resonant frequency so that the resonant frequency is And meet the microwave radiation module in F W1 and F W2 The setting coefficient Γ≥Γ0 at the microwave frequency is [F W2 , F W3 ] interval, the microwave radiation module adjusts its resonant frequency so that the resonant frequency is And meet the microwave radiation module in F W2 and F W3 The setting coefficient Γ≥Γ0 at the microwave frequency is [F W3 , F W4 ] interval, the microwave radiation module adjusts its resonant frequency so that the resonant frequency is And meet the microwave radiation module in F W3 and F W4 The setting coefficient Γ≥Γ0 at and.

[0088] In this embodiment, the microwave radiation module is arranged to control the setting coefficient of the microwave radiation module at the endpoint frequencies corresponding to each set frequency interval to be greater than or equal to the set threshold value, and the resonant frequency of each set frequency interval is controlled to be the average value of the two endpoint frequencies corresponding to the set frequency interval, so that the range of each set frequency interval can be made as large as possible, thereby satisfying the microwave power requirements of nanodiamonds in biological samples with a smaller number of set frequency intervals, reducing the number of times the resonant frequency is adjusted, and further simplifying the structure of the microwave radiation module.

[0089] Optional, Figure 6 is a schematic structural diagram of a microwave radiation module provided according to an embodiment of the present invention, Figure 7 is a schematic diagram of the structure of another microwave radiation module provided according to an embodiment of the present invention, referring to Figure 6 and Figure 7 The microwave radiation module 210 includes a microwave source, a substrate 211, a microstrip line structure 212, a microwave transmission line 213, a port structure 214 and an impedance adjustable unit located on one side of the substrate 211; the microwave source is connected to the port structure 214, and the microwave source is used to generate microwaves; the microstrip line structure 212 is connected to the port structure 214 and the microwave transmission line 213, and the microstrip line structure 212 is used to transmit the microwaves generated by the microwave source to the microwave transmission line 213; the substrate 211 includes an objective lens light-transmitting port 215, the microwave transmission line 213 crosses the objective lens light-transmitting port 215, and the microwave transmission line 213 is used to transmit microwaves to the biological sample; the impedance adjustable unit is used to adjust the resonant frequency of the microwave radiation module 210.

[0090] Specifically, the impedance adjustable unit may be a variable capacitor. For example, the frequency of the microwave is [F W1 , F W2 ] interval, set the variable capacitor to C1 so that the resonant frequency is Microwave frequency [F W2 , F W3 ] interval, set the variable capacitor to C2 so that the resonant frequency is Microwave frequency [F W3 , F W4 ] interval, set the variable capacitor value to C3 so that the resonant frequency is The variable capacitor only needs to be adjusted according to the frequency corresponding to the microwave sweep [F W1 , F W2 ]、[F W2 , F W3 ]、[F W3 , F W4 ] can be adjusted in sequence to C1, C2, and C3. It can be seen that the system structure provided in this embodiment is relatively simple and does not affect the imaging and measurement effects.

[0091] The laser emitted by the optical path module can be transmitted to the biological sample through the objective lens light transmission port 215, and the fluorescent signal can also be transmitted to the optical path module through the objective lens light transmission port 215. In this embodiment, the microwave transmission line 213 is arranged to cross the objective lens light transmission port 215, so that the microwaves emitted by the microwave transmission line 213 can be received by the biological sample as much as possible, thereby improving the effect of microwave radiation.

[0092] The microstrip line structure 212 includes a matching structure, and the shape of the matching structure can be U-shaped. The structural parameters of the matching structure (the width and depth of the U-shape) can determine the resonant frequency of the microwave radiation module 210. For example, by optimizing the structural parameters of the matching structure, the resonant frequency of the microwave radiation module 210 is set to 2870 MHz, and then according to actual measurement requirements, the resonant frequency of the microwave radiation module 210 is adjusted around 2870 MHz through the impedance adjustable unit.

[0093] Optional, continue to refer to Figure 6 and Figure 7 The microwave radiation module 210 also includes a vector network analyzer; the port structure 214 includes a first port 2141 and a second port 2142; the microstrip line structure 212 includes a first microstrip line 2121 and a second microstrip line 2122, the first end of the first microstrip line 2121 is connected to the first port 2141, and the second end of the first microstrip line 2121 is connected to the first end of the microwave transmission line 213; the first end of the second microstrip line 2122 is connected to the second port 2142, and the second end of the second microstrip line 2122 is connected to the second end of the microwave transmission line 213; the first port 2141 is also connected to the microwave source, and the second port 2142 is also connected to the vector network analyzer.

[0094] Specifically, the microwave radiation module 210 provided in this embodiment is a dual-port microwave radiation module 210, the first port 2141 is a microwave input port, the second port 2142 is a microwave output port, and the microwave output port is connected to a vector network analyzer, and the microwave transmission coefficient Γ can be monitored by the vector network analyzer.

[0095] The structure of the first microstrip line 2121 can be the same as that of the second microstrip line 2122. The first microstrip line 2121 and the second microstrip line 2122 both include a matching structure. The shape of the matching structure can be U-shaped. The structural parameters of the two matching structures (the width and depth of the U-shape) can determine the resonant frequency of the microwave radiation module 210. Exemplarily, the resonant frequency of the microwave radiation module 210 is set to 2870 MHz by optimizing the structural parameters of the two matching structures. Then, according to actual measurement requirements, the resonant frequency of the microwave radiation module 210 is adjusted around 2870 MHz through the impedance adjustable unit.

[0096] Optionally, the microwave radiation module also includes a vector network analyzer; the port structure is a single port; the vector network analyzer and the microwave wave source are both connected to the port structure, and the vector network analyzer and the microwave wave source are connected in parallel.

[0097] Specifically, the microwave radiation module provided in this embodiment includes one port, and is a single-port microwave radiation module. The vector network analyzer connected to the port structure can monitor the reflection coefficient Γ of the microwave radiation module.

[0098] Optional, continue to refer to Figure 6 , the microwave transmitting line includes a metal wire.

[0099] Specifically, the metal wire may be a copper wire. Arranging the microwave transmission line to include the metal wire can reduce the manufacturing cost of the microwave transmission line.

[0100] Optional, continue to refer to Figure 7 The microwave emitting line 213 includes a soft flat cable; the soft flat cable includes a flexible base layer and a conductive layer located in the flexible base layer; the conductive layer includes a conductive wire and a conductive Ω ring 201.

[0101] Specifically, metal patterning can be performed in the flexible base layer to form a conductive layer, and the microwave emission line 213 is provided to include a flexible flat cable, so that the microwave emission line 213 has a thinner thickness, thereby shortening the distance between the objective lens and the sample dish, which is beneficial to improving the resolution of microscopic imaging or measurement, and can be convenient for setting the shape of the conductive layer, and a conductive Ω ring 201 can be provided in the conductive layer, thereby improving the effect of microwave radiation. The conductive Ω ring 201 can be located at the center of the objective lens light transmission port 215, so that the microwave energy can be more concentrated near the vertical axis at the center of the objective lens light transmission port 215, and better provide microwave radiation energy for the nano-fluorite at the measured position. The conductive line is a structure in the conductive layer except the conductive Ω ring 201.

[0102] Optional, continue to refer to Figure 6 and Figure 7 , the vertical projection of the microwave emission line 213 on the objective lens light transmission port 215 coincides with a symmetry axis of the objective lens light transmission port 215 .

[0103] Specifically, setting the vertical projection of the microwave emission line 213 on the objective lens light transmission port 215 to coincide with a symmetry axis of the objective lens light transmission port 215 can make the microwave energy more concentrated near the vertical axis at the center of the objective lens light transmission port 215, so as to better provide microwave radiation energy for the nano-fluorescent stone at the measured position.

[0104] Optional, Figure 8 for Figure 6 The three-dimensional structure diagram of the microwave radiation module shown in FIG. Figure 8The microwave radiation module provided in this embodiment also includes a connection structure 216, and the connection structure 216 is detachably connected to the objective lens in the optical path module.

[0105] Specifically, the connection structure 216 matches the objective lens, and can be fixed on the objective lens or removed from the objective lens.

[0106] Optional, continue to refer to Figure 1 The optical path module 310 includes an objective lens 311 and a wide-field optical path sub-module 312; the microwave radiation module 210 is located between the objective lens 311 and the sample holding module 110; the wide-field optical path sub-module 312 includes a first light source unit 320, a first dichroic mirror 330 and a light collection unit 340; the first light source unit 320 is used to generate laser light and transmit the laser light to the biological sample 400 through the first dichroic mirror 330 and the objective lens 311; the light collection unit 340 is used to receive the fluorescence signal transmitted through the objective lens 311 and the first dichroic mirror 330.

[0107] Specifically, the first light source unit 320 includes a first light source 321, a collimating lens 322 and a deflection attenuator 323. The first light source 321 is used to generate laser light. The light collection unit 340 includes an image collection subunit 341, a filter 342 and a focusing lens 343. The image collection subunit 341 can be a CMOS camera. The wide-field optical path submodule 312 can perform high-speed planar scanning imaging of the nano-fluorescent stones in the biological sample 400. Specifically, the field of view radius of the wide-field imaging is d, and the sample dish 111 can be moved in steps of √2d / 2, so that the biological microscopic measurement system can scan the biological sample 400.

[0108] Optional, Fig. 9 is a schematic diagram of the structure of another biological microscopic measurement system provided according to an embodiment of the present invention, with reference to Fig. 9 , the optical path module 310 also includes a reflecting prism 313 and a confocal optical path sub-module 314; the confocal optical path sub-module 314 includes a second light source unit 350, a second dichroic mirror 360 and a photon detection unit 370; the second light source unit 350 is used to generate laser light and transmit the laser light to the biological sample 400 through the second dichroic mirror 360, the reflecting prism 313 and the objective lens 311; the photon detection unit 372 is used to receive the fluorescent signal transmitted through the objective lens 311, the reflecting prism 313 and the second dichroic mirror 360; the reflecting prism 313 is located between the objective lens 311 and the first dichroic mirror 330 (see Figure 1 ), and between the objective lens 311 and the second dichroic mirror 360.

[0109] Specifically, the laser light generated by the first light source unit 320 can be transmitted to the objective lens 311 , or the laser light generated by the second light source unit 350 can be transmitted to the objective lens 311 by moving the direction of the reflective prism 313 .

[0110] The second light source unit 350 includes a second light source 351 and a first pinhole baffle 352 , and the photon detection unit 370 includes a second pinhole baffle 371 and a single photon detector 372 .

[0111] In this embodiment, the confocal optical path submodule 314 can be used to perform high-precision stereoscopic measurement of the nano-fluorescent stone in the biological sample, and measure the magnetic field, temperature and other information of the nano-fluorescent stone with high resolution.

[0112] The optical path module 310 provided in this embodiment can use wide-field imaging to perform high-speed planar scanning imaging of the nano-fluorescent stones in the biological sample 400, and can also use confocal microscopy imaging to perform high-precision stereoscopic measurement of the nano-fluorescent stones in the biological sample.

[0113] Fig.10 is a schematic diagram of the three-dimensional structure of a biological microscopic measurement system provided according to an embodiment of the present invention, Fig.11 is a schematic diagram of a three-dimensional structure of another biological microscopic measurement system provided according to an embodiment of the present invention, with reference to Fig.10 and Fig.11 The sample carrying module also includes a sample cover 112, a translation stage 113, a translation screw 114, a translation base 115 and a reset spring 116. The optical path module also includes an objective lens mounting column 315, a scanning imaging module base 316, a lifting platform 317, a prism platform 318, a lifting screw 319 and a lifting base 301. The sample cover 112 is used to cover the sample dish 111, and the sample cover 112 can be used to protect the biological sample in the sample dish 111 from being contaminated by external objects. The prism platform 318 is used to support the reflective prism 313; the objective lens 311 is set on the lifting platform 317, the lifting platform 317 is connected to the lifting screw 319, and the lifting platform 317 moves relative to the lifting base 301.

[0114] The translation stage 113 can move horizontally relative to the translation base 115, thereby driving the sample dish 111 to move horizontally relative to the objective lens 311; there are two translation screws 114, corresponding to the x and y coordinates respectively, and a reset spring 116 is provided to enable the translation stage 113 to always be close to the free end of the translation screw 114, thereby improving the displacement accuracy of the translation stage 113; the objective lens 311 is arranged below the translation stage 113, and the microwave radiation module 210 is arranged on the upper end of the objective lens 311. The objective lens 311 has a liftable structure, which is convenient for disassembling and assembling the sample dish 111 and the microwave radiation module 210, so that the objective lens 311 and the microwave radiation module 210 are as close to the sample dish 111 as possible; the lifting structure of the objective lens 311 includes a lifting base 301, a lifting platform 317 that is matched with the lifting base 301 through a track and can move up and down, the objective lens 311 is fixedly arranged on the lifting platform 317, and the lifting platform 317 is connected to the free end of the lifting screw 319.

[0115] The reflecting prism 313 is arranged below the objective lens 311. By changing the position of the scanning imaging module or changing the orientation of the reflecting prism 313, the sample dish 111, the microwave radiation module 210, the objective lens 311, the reflecting prism 313 and the wide-field optical path sub-module 312 can form a wide-field imaging measurement system, or the sample dish 111, the microwave radiation module 210, the objective lens 311, the reflecting prism 313 and the confocal optical path sub-module 314 can form a confocal microscopic imaging measurement system.

[0116] This embodiment provides a biological microscopic measurement method, which is applied to the biological microscopic measurement system provided by any embodiment of the present invention. Fig.12 is a schematic diagram of a biological microscopic measurement method according to an embodiment of the present invention, with reference to Fig.12 , the biological microscopic measurement method includes the following steps:

[0117] S110, the sample carrying module carries the biological sample.

[0118] Among them, nanodiamonds are arranged in the biological sample, and the nanodiamonds contain NV color centers.

[0119] S120, the microwave radiation module emits microwaves to the biological sample at a plurality of different set frequency ranges.

[0120] Among them, the microwave radiation module corresponds to a resonant frequency in each set frequency interval and the resonant frequency is located in the corresponding set frequency interval; the minimum frequency in multiple set frequency intervals is less than or equal to the first set frequency, and the maximum frequency in multiple set frequency intervals is greater than or equal to the second set frequency, the second set frequency is greater than the first set frequency, and the first set frequency and the second set frequency are microwave resonant frequencies that make the ground state electron spin of the NV color center in the nanodiamond transition from the |0> state to the |±1> state under the environmental parameters to be tested.

[0121] S130, the optical path module emits laser to the biological sample, and obtains the fluorescence signal generated by the nanodiamond under the action of the environmental parameters to be measured, laser and microwave.

[0122] The biological microscopic measurement method provided in this embodiment has corresponding beneficial effects with the biological microscopic measurement system provided in any embodiment of the present invention. For technical details not elaborated in this embodiment, please refer to the biological microscopic measurement system provided in any embodiment of the present invention.

[0123] It should be understood that the various forms of processes shown above can be used to reorder, add or delete steps. For example, the steps described in the present invention can be executed in parallel, sequentially or in different orders, as long as the desired results of the technical solution of the present invention can be achieved, and this document does not limit this.

[0124] The above specific implementations do not constitute a limitation on the protection scope of the present invention. It should be understood by those skilled in the art that various modifications, combinations, sub-combinations and substitutions can be made according to design requirements and other factors. Any modification, equivalent substitution and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A biological microscopic measurement system, characterized in that: include: Sample holding module, microwave radiation module and optical path module; The sample carrying module is used to carry a biological sample, wherein a nanodiamond is arranged in the biological sample, and the nanodiamond contains an NV color center; The microwave radiation module is located between the sample holding module and the optical path module, and is used to emit microwaves to the biological sample under a plurality of different set frequency intervals, wherein the microwave radiation module corresponds to a resonant frequency in each of the set frequency intervals and the resonant frequency is located in the corresponding set frequency interval; the minimum frequency in the plurality of different set frequency intervals is less than or equal to the first set frequency, and the maximum frequency in the plurality of different set frequency intervals is greater than or equal to the second set frequency, the second set frequency is greater than the first set frequency, and the first set frequency and the second set frequency are microwave resonant frequencies that cause the ground state electron spin of the NV color center in the nanodiamond to transition from the |0> state to the |±1> state under the environmental parameters to be measured; The optical path module is used to emit laser light to the biological sample, and is also used to obtain the fluorescence signal generated by the nano-diamond under the action of the environmental parameter to be measured, the laser light and the microwave.

2. The biological microscopic measurement system according to claim 1, characterized in that: Also includes: A frequency determination module and a threshold determination module; The threshold determination module is used to determine the set threshold corresponding to the microwave radiation module; The frequency determination module is used to determine the frequency interval from the first set frequency to the second set frequency, and determine the microwave scanning interval based on the frequency interval. It is also used to divide the microwave scanning interval into the multiple different set frequency intervals according to the set threshold, so that the setting coefficient of the microwave radiation module at each frequency in each of the set frequency intervals is less than the set threshold.

3. The biological microscopic measurement system according to claim 1, characterized in that: The microwave radiation module is also used to control the set coefficient at the endpoint frequencies corresponding to each of the set frequency intervals to be greater than or equal to a set threshold, and is also used to control the resonant frequency of each of the set frequency intervals to be the average value of the two endpoint frequencies corresponding to the set frequency interval; wherein the set coefficient is a reflection coefficient or a transmission coefficient.

4. The biological microscopic measurement system according to claim 1, characterized in that: The microwave radiation module comprises a microwave source, a substrate, a microstrip line structure located on one side of the substrate, a microwave transmission line, a port structure and an impedance adjustable unit; The microwave source is connected to the port structure, and the microwave source is used to generate microwaves; The microstrip line structure is connected to the port structure and the microwave transmission line, and the microstrip line structure is used to transmit the microwaves generated by the microwave source to the microwave transmission line; The substrate comprises an objective lens light transmission port, the microwave emission line crosses the objective lens light transmission port, and the microwave emission line is used to emit microwaves to the biological sample; The impedance adjustable unit is used to adjust the resonant frequency of the microwave radiation module.

5. The biological microscopic measurement system according to claim 4, characterized in that: The microwave radiation module also includes a vector network analyzer; The port structure includes a first port and a second port; The microstrip line structure comprises a first microstrip line and a second microstrip line, wherein a first end of the first microstrip line is connected to the first port, and a second end of the first microstrip line is connected to a first end of the microwave transmission line; a first end of the second microstrip line is connected to the second port, and a second end of the second microstrip line is connected to a second end of the microwave transmission line; The first port is also connected to the microwave source, and the second port is also connected to the vector network analyzer.

6. The biological microscopic measurement system according to claim 4, characterized in that: The microwave radiation module also includes a vector network analyzer; The port structure is a single port; The vector network analyzer and the microwave wave source are both connected to the port structure, and the vector network analyzer is connected to the microwave wave source in parallel.

7. The biological microscopic measurement system according to claim 4, characterized in that: The microwave transmission line includes a metal wire or a flexible flat cable; The flexible flat cable comprises a flexible base layer and a conductive layer located in the flexible base layer; the conductive layer comprises a conductive wire and a conductive Ω ring.

8. The biological microscopic measurement system according to claim 4, characterized in that: The vertical projection of the microwave emission line on the light transmission port of the objective lens coincides with a symmetry axis of the light transmission port of the objective lens.

9. The biological microscopic measurement system according to claim 1, characterized in that: The optical path module includes an objective lens and a wide-field optical path sub-module; The microwave radiation module is located between the objective lens and the sample holding module; The wide-field optical path submodule comprises a first light source unit, a first dichroic mirror and a light collection unit; The first light source unit is used to generate laser light and transmit the laser light to the biological sample through the first dichroic mirror and the objective lens; The light collection unit is used to receive the fluorescent signal transmitted through the objective lens and the first dichroic mirror.

10. The biological microscopic measurement system according to claim 9, characterized in that: The optical path module also includes a reflecting prism and a confocal optical path sub-module; The confocal optical path submodule includes a second light source unit, a second dichroic mirror and a photon detection unit; The second light source unit is used to generate laser light and transmit the laser light to the biological sample through the second dichroic mirror, the reflecting prism and the objective lens; The photon detection unit is used to receive the fluorescence signal transmitted through the objective lens, the reflection prism and the second dichroic mirror; The reflecting prism is located between the objective lens and the first dichroic mirror, and between the objective lens and the second dichroic mirror.

11. The biological microscopic measurement system according to claim 10, characterized in that: The sample carrying module comprises a sample dish, a sample cover, a translation stage, a translation screw, a translation base and a reset spring; The sample dish is used to contain the biological sample; the sample cover is used to cover the sample dish; the translation stage is used to drive the sample dish to move relative to the translation base; The optical path module also includes an objective lens mounting column, a scanning imaging module base, a lifting platform, a prism platform, a lifting screw and a lifting base; The prism stand is used to support the reflecting prism; The objective lens is arranged on the lifting platform, the lifting platform is connected to the lifting screw rod, and the lifting platform moves relative to the lifting base.

12. A biological microscopic measurement method, characterized in that: The biological microscopic measurement method is applied to the biological microscopic measurement system according to any one of claims 1 to 11; The biological microscopic measurement method comprises: The sample carrying module carries a biological sample, wherein nanodiamonds are arranged in the biological sample, and the nanodiamonds contain NV color centers; The microwave radiation module emits microwaves to the biological sample in a plurality of different set frequency intervals, wherein the microwave radiation module corresponds to a resonant frequency in each of the set frequency intervals and the resonant frequency is located in the corresponding set frequency interval; the minimum frequency in the plurality of different set frequency intervals is less than or equal to the first set frequency, and the maximum frequency in the plurality of different set frequency intervals is greater than or equal to the second set frequency, the second set frequency is greater than the first set frequency, and the first set frequency and the second set frequency are microwave resonant frequencies that cause the ground state electron spin of the NV color center in the nanodiamond to transition from a |0> state to a |±1> state under the environmental parameters to be measured; The optical path module emits laser light to the biological sample, and obtains the fluorescence signal generated by the nano-diamond under the action of the environmental parameter to be measured, the laser light and the microwave.

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