Multipath infrared fluorescence detection system and detection method for living body flow cytometry

Abstract

The invention relates to a multi-channel infrared fluorescence detection system and a multi-channel infrared fluorescence detection method for living body flow cytometry. The multi-path infrared fluorescence detection system comprises a light source, an infrared fluorescence probe, an incident light modulation light path, an emergent light modulation light path and a superconducting nanowire single-photon detector, after exciting light emitted by the light source passes through the incident light modulation light path, the exciting light is guided to the direction of a sample; infrared fluorescence emitted after a sample is irradiated enters different modulation light paths after being subjected to light splitting through the light splitter and then is guided to the superconducting nanowire single-photon detectors in different detection wave band ranges for signal collection, and collected signals are transmitted to the computer to generate a real-time multi-path signal intensity graph. The invention provides a new technical means for in-vivo typing and counting of circulating tumor cells, immune cells and the like of living small animals.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescence detection, and in particular to a multi-channel infrared fluorescence detection system and a detection method for in vivo flow cytometry. Background Art

[0002] Analyzing the dynamic behavior of cells in vivo is crucial for understanding the development of diseases and understanding the microscopic cellular events during disease treatment. In the development of diseases such as inflammation, tumors, and cardiovascular diseases, a deep understanding of the body's own immune cells or therapeutic cells injected back into the body can help to effectively assist the treatment process in real time. However, to date, real-time non-invasive detection of cell dynamic behavior in vivo (in vivo) remains a huge challenge.

[0003] At present, in basic research and clinical research, the quantitative analysis of cells such as immune cells, blood cells and tumor cells in the body mainly relies on flow cytometry. However, despite its many advantages such as high speed, high precision, multi-parameter analysis and sorting functions, traditional flow cytometry is still limited to in vitro analysis. Most of the cell objects analyzed by traditional flow cytometry are obtained through in vitro blood collection, tissue biopsy post-processing and other methods, and the post-processing processes such as sample collection, separation and preservation make them separated from the original physiological environment. Changes in blood or tissue parameters during sample collection may cause changes in the parameters of the object being measured. In order to solve this problem, researchers have successively developed a series of in vivo flow cytometry to quantify and analyze circulating cells in the body. However, currently, most in vivo flow cytometry can only detect circulating cells in the ear blood vessels (0.2-0.5 mm) or tail blood vessels (0.5-1 mm) of mice (Optics letters, 2004, 29 (1): 77-79.; Biomedical Optics Express, 2023, 14 (11): 5555-5568.), which often requires the use of commercial visible light or short-wave infrared dyes, and requires the use of tissue transparency technology; there are still great difficulties in analyzing cell information in deeper tissue blood vessels. When analyzing cells in deep tissue blood vessels, it is often necessary to artificially construct a monitoring window through surgery. The process of window construction will cause trauma to the body, thereby destroying the body's original physiological environment and causing changes in cell behavior in the body, which inevitably interferes with the accurate detection of cell behavior in the body. Summary of the invention

[0004] In order to overcome the defects of existing flow cytometry, especially the shortcomings of existing in vivo flow cytometry in deep tissue detection in vivo, the present invention proposes a multi-channel infrared fluorescence detection system and detection method for in vivo flow cytometry to achieve real-time non-invasive in vivo detection of flow cell detection in deep tissue blood vessels.

[0005] In order to achieve the above-mentioned purpose, the first aspect of the present invention provides a multi-channel infrared fluorescence detection system for in vivo flow cytometry, the multi-channel infrared fluorescence detection system comprising: a light source, an infrared fluorescent probe, an incident light modulated optical path, an outgoing light modulated optical path and a superconducting nanowire single-photon detector; the excitation light emitted by the light source is guided to the target cell of the in vivo sample marked by the infrared fluorescent probe via the incident light modulated optical path, the infrared fluorescence emitted by the in vivo sample after being irradiated by the excitation light is guided to the superconducting nanowire single-photon detector via the outgoing light modulated optical path for signal collection, and the collected signal is transmitted to a computer to generate a real-time signal intensity graph, wherein

[0006] The light source is a near-infrared laser, and the wavelength of the excitation light emitted is 700-2000nm; the infrared fluorescent probe is an inorganic or organic infrared fluorescent probe, and the wavelength of the infrared fluorescence emitted after being irradiated by the excitation light is 1000-2500nm.

[0007] Furthermore, the near-infrared laser emits an excitation light with a wavelength of 730 nm, 760 nm, 808 nm, 915 nm, 980 nm, 1064 nm, 1280 nm, 1310 nm, 1550 nm or 1710 nm.

[0008] Furthermore, the target cells are labeled with infrared fluorescent probes through in vivo labeling or in vitro labeling. The in vivo labeling is performed by injecting antibodies and / or polypeptides coupled with infrared fluorescent probes into a living body; the in vitro labeling is performed by co-incubating the infrared fluorescent probes with the target cells in vitro and then returning the labeled target cells to the body.

[0009] Furthermore, according to the direction of travel of the excitation light, the incident light modulated optical path includes a first beam splitter, a galvanometer and a microscope objective in sequence; according to the direction of travel of the infrared fluorescence, the outgoing light modulated optical path includes the microscope objective, the galvanometer and the first beam splitter in the incident light modulated optical path, as well as one or more second beam splitters and a filter behind each second beam splitter, and a corresponding superconducting nanowire single-photon detector is arranged on the light output side of each filter.

[0010] Furthermore, the first beam splitter and the second beam splitter are dichroic mirrors or prisms in the infrared band of 1000-2000nm, and the filter is an infrared long-pass, short-pass or band-pass filter.

[0011] Furthermore, the microscope objective lens is a water lens or an air lens, and the magnification is 10×, 20×, 25×, 40× or 50×.

[0012] Furthermore, the wavelength detection range of the superconducting nanowire single-photon detector is 1000nm-10μm, and the optical fiber used is single-mode optical fiber or multi-mode optical fiber coupling.

[0013] A second aspect of the present invention provides a multi-channel infrared fluorescence detection method for in vivo flow cytometry using any of the multi-channel infrared fluorescence detection systems described above, the method comprising the following steps:

[0014] (1) labeling target cells of a living sample with an infrared fluorescent probe, wherein the infrared fluorescent probe emits infrared fluorescence having a wavelength of 1000-2500 nm after being irradiated with excitation light;

[0015] (2) placing a living sample in which target cells are labeled with infrared fluorescent probes under the microscope objective of the multi-channel infrared fluorescence detection system;

[0016] (3) causing a near-infrared laser light source to emit excitation light with a wavelength of 700-2000 nm, and directing the excitation light to the living sample via an incident light modulation optical path;

[0017] (4) collecting an infrared fluorescence signal of a corresponding wavelength derived from the output light modulation optical path through at least one superconducting nanowire single-photon detector; and

[0018] (5) The collected signals are transmitted to a computer to generate a real-time signal strength graph.

[0019] Furthermore, the target cells are labeled with infrared fluorescent probes by in vivo labeling or in vitro labeling. The in vivo labeling is performed by injecting antibodies and / or polypeptides coupled with infrared fluorescent probes into a living body; the in vitro labeling is performed by co-incubating the infrared fluorescent probes with the target cells in vitro and then returning the labeled target cells to the body.

[0020] The third aspect of the present invention provides an application of the above-mentioned multiplex infrared fluorescence detection system for in vivo flow cytometry, and the application includes the following aspects:

[0021] (1) In vivo typing and counting of endogenous circulating cells in blood vessels of normal living animals or living animals with disease models; the diseases include inflammation, tumors and / or cardiovascular diseases, and the cells include tumor cells, immune cells and / or blood cells;

[0022] (2) dynamics and therapeutic evaluation of therapeutic cells in vivo and in vivo blood vessels after reinfusion into living small animals, wherein the therapeutic cells include stem cells and / or immune cells; and

[0023] (3) In vivo evaluation of the dynamics of micron-sized particles of the same order of magnitude as cells within the blood vessels of living small animals, wherein the micron-sized particles include microplastics.

[0024] Beneficial effects of the present invention:

[0025] The technical solution of the present invention adopts infrared fluorescence (1000-2500nm) imaging technology, which greatly overcomes the problems of strong tissue absorption, scattering and autofluorescence interference faced by traditional fluorescence (400-900nm) imaging, and can achieve higher tissue penetration depth and time and spatial resolution in living imaging. It has the characteristics of low autofluorescence, deep tissue penetration, small light scattering, high signal-to-noise ratio, etc. It can perform high-sensitivity and specific real-time imaging of biological systems, and has multi-spectral and multiplexing capabilities, which greatly broadens the application scope of in vivo flow cytometry.

[0026] The system and method for infrared live flow cytometry provided by the present invention provide a new technical means for in vivo detection of immune cells, circulating tumor cells, and reinfused therapeutic cells in deep tissue blood vessels. Using a multi-channel infrared fluorescence detection system, the cell behavior in deep tissue blood vessels is dynamically monitored in real time, which provides an effective means for deeply understanding the dynamic changes of immune cells and assisting in immunotherapy evaluation. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a simplified optical path diagram of the multi-channel infrared fluorescence detection system provided in Example 1 of the present invention when used for infrared in vivo flow single-channel cell signal detection.

[0028] Figure 2 This is a simplified optical path diagram of the multi-channel infrared fluorescence detection system provided in Example 2 of the present invention when used for infrared in vivo flow multi-channel cell signal detection.

[0029] Figure 3 This is a signal peak diagram of the infrared in vivo flow single-channel cell signal detection provided in Example 1 of the present invention.

[0030] Figure 4 This is a signal peak diagram of the infrared in vivo flow multi-channel cell signal detection provided in Example 2 of the present invention.

[0031] [Description of Reference Numerals]

[0032] 1-light source; 2-first spectrometer; 3-galvanometer; 4-microscope objective; 5-second spectrometer a; 6-filter a; 7-superconducting nanowire single-photon detector a; 8-filter z; 9-superconducting nanowire single-photon detector z; 10-second spectrometer b; 11-filter b; 12-superconducting nanowire single-photon detector b. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments. However, the present invention is by no means limited to these drawings and embodiments, and they cannot be understood as limiting the scope of protection of the present invention. Any modification, substitution or improvement made within the spirit and principle of the present invention should be included in the scope of protection of the present invention.

[0034] In the description of the present invention, it should be understood that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of the present invention, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0035] In the description of this specification, the description of the terms "one embodiment", "some embodiments", "embodiment", "example", "specific example" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

[0036] Example 1

[0037] Infrared live flow cytometry single-channel cell signal detection of in vivo labeled target cells. Figure 1 The optical path diagram of the multi-channel infrared fluorescence detection system is as follows:

[0038] (1) Construct a multi-channel infrared fluorescence detection system for the optical path of single-channel cell signal detection. The detection optical path includes a light source 1, an incident light modulation optical path (a first beam splitter 2, a galvanometer 3 and a microscope objective 4), an outgoing light modulation optical path (a microscope objective 4, a galvanometer 3, a first beam splitter 2, a second beam splitter a-5, a filter a-6), and a superconducting nanowire single-photon detector a-7, which are used for the detection of single-channel cell signals. In addition, a filter z-8 and a superconducting nanowire single-photon detector z-9 can also be optionally provided to selectively analyze the light additionally separated by the second beam splitter a-5. Of course, for the detection of single-channel cell signals only, the filter z-8 and the superconducting nanowire single-photon detector z-9 can be omitted.

[0039] Among them, the light source 1 is a 980nm near-infrared laser, the incident light is completely reflected by the first beam splitter 2 and the galvanometer 3, and is guided to the sample stage (not shown) through the 25× water mirror 4. The sample output light is infrared fluorescence above 1000nm. After the output light is completely reflected by the galvanometer 3, it passes through the first beam splitter 2 with high transmittance, is split by the 1180nm dichroic mirror 5, and is then filtered by the 1500nm long-pass filter 6. The infrared fluorescence with a wavelength greater than 1500nm can be detected by the superconducting nanowire single-photon detector 7 with a central collection wavelength of 1550nm. The superconducting nanowire single-photon detector 7 uses multi-mode optical fiber coupling.

[0040] (2) Preparation of infrared fluorescent labeling probe. The infrared fluorescent labeling probe used is a cubic rare earth nanoprobe α-NaYbF4: 2% Er, 2% Ce@NaYF4, and is modified with the neutrophil targeting antibody Ly6G. The specific synthesis and modification steps are as follows:

[0041] a. Preparation of rare earth precursors.

[0042] Preparation of ytterbium trifluoroacetate Yb(TFA)3: Take a 100mL three-necked round-bottom flask as the reaction container, add 0.1mmol ytterbium oxide (Yb2O3) and 50mL trifluoroacetic acid (TFA) in sequence, heat to 70℃ under vacuum and magnetic stirring conditions and maintain for 12h, and finally obtain a clear and transparent Yb(TFA)3 solution with a concentration of 2mmol / L.

[0043] Preparation of yttrium trifluoroacetate Y(TFA)3: Take a 100mL three-necked round-bottom flask as the reaction container, add 0.1mmol yttrium oxide (Y2O3) and 50mL trifluoroacetic acid (TFA) in sequence, heat to 70℃ under vacuum and magnetic stirring conditions and maintain for 12h, and finally obtain a clear and transparent Y(TFA)3 solution with a concentration of 2mmol / L.

[0044] Preparation of trifluoroacetic acid bait Er(TFA)3: Take a 100mL three-necked round-bottom flask as the reaction container, add 0.1mmol oxidized bait (Er2O3) and 50mL trifluoroacetic acid (TFA) in sequence, heat to 70℃ under vacuum and magnetic stirring conditions and maintain for 12h, and finally obtain a clear and transparent Er(TFA)3 solution with a concentration of 2mmol / L.

[0045] Preparation of cerium trifluoroacetate Ce(TFA)3: Take a 100mL three-necked round-bottom flask as the reaction container, add 0.1mmol cerium oxide (Ce2O3) and 50mL trifluoroacetic acid (TFA) in sequence, heat to 70℃ under vacuum and magnetic stirring conditions and maintain for 12h, and finally obtain a clear and transparent Ce(TFA)3 solution with a concentration of 2mmol / L.

[0046] b. Preparation of luminescent core layer α-NaYbF4: 2% Er, 2% Ce.

[0047] Take a 100mL three-necked round-bottom flask as a reaction vessel, add 1mmol sodium trifluoroacetate (NaTFA), 0.96mmol ytterbium trifluoroacetate Yb(TFA)3, 0.02mmol trifluoroacetic acid Er(TFA)3, 0.02mmol trifluoroacetic acid Ce(TFA)3, 10mmol oleic acid OA, 10mmol oleylamine OM and 20mmol 1-octadecene ODE to the reaction vessel. Stir and heat the reactants to 100°C under vacuum dehydration and deoxygenation conditions until a transparent solution is formed. Then, under argon protection, the reaction system is heated to 310°C at a heating rate of 15°C / min, and the heating is maintained for 25min. After the reactants are cooled to room temperature, ethanol is added to precipitate the product from the solution, and then the supernatant is removed by centrifugation. After repeated washing with anhydrous ethanol for 3 times, it is dissolved in 2mL cyclohexane (C6H 12 ) for future use.

[0048] c. Preparation of inert shell α-NaYbF4:2%Er,2%Ce@NaYF4.

[0049] Here, the luminescent core layer and the inert shell layer are epitaxially grown in a ratio of 1:2. Take a 100mL three-necked round-bottom flask as a reaction vessel, add 1mmol of luminescent core layer α-NaYbF4:2% Er, 2% Ce nanocrystals dissolved in cyclohexane in the previous step, and then add 2mmol sodium trifluoroacetate (NaTFA), 2mmol yttrium trifluoroacetate Y (TFA) 3, 10mmol oleic acid OA, 10mmol 1-octadecene ODE. The reactants are stirred and heated to 100°C under vacuum dehydration and deoxygenation conditions until a transparent solution is formed. Then, under argon protection, the reaction system is heated to 290°C at a heating rate of 10°C / min, and the heating is maintained for 70min. After the reactants are cooled to room temperature, ethanol is added to precipitate the product from the solution, and then the supernatant is removed by centrifugation. After repeated washing with anhydrous ethanol for 3 times, it is dissolved in 10mL chloroform for use.

[0050] d. Preparation of active ester modified α-NaYbF4:2%Er,2%Ce@NaYF4.

[0051] Take a 250mL eggplant-shaped bottle as a reaction container, add 100μL α-NaYbF4:2%Er,2%Ce@NaYF4 chloroform dispersion, 5mL chloroform, 12mg DSPE-PEG2000-OCH3 and 3mg DSPE-PEG2000-NHS. Under heating conditions at 40℃, the solvent in the mixed system is evaporated, and then vacuum dried for 1h under negative pressure to form a film. Then, 5mL of 100℃ deionized water is added, and the film dispersion is promoted by ultrasound at 100℃. Finally, the obtained water dispersion system is transferred to an ultrafiltration centrifuge tube and centrifuged at 7012g for 25min. The final active ester-modified α-NaYbF4:2%Er,2%Ce@NaYF4 is fixed to 200μL.

[0052] e. Preparation of antibody-labeled α-NaYbF4:2% Er, 2% Ce@NaYF4.

[0053] 200 μL of active ester-modified α-NaYbF4:2%Er,2%Ce@NaYF4 and 10 μL Ly6G antibody (0.5 μg / μL) were added to 2 mL Tris-HCl buffer solution (pH 8.5). After stirring overnight at room temperature, the mixed system was transferred to an ultrafiltration centrifuge tube and centrifuged at 7012g for 25 min. Finally, the volume was adjusted to 200 μL with Tris-HCl buffer solution of pH 7.0 for use, and the infrared fluorescent probe of α-NaYbF4:2%Er,2%Ce@NaYF4 labeled with Ly6G antibody (referred to as α-ErNPs-Ly6G) was obtained.

[0054] (3) Infrared live flow single-channel signal detection. SPF-grade Balb / c mice were used as experimental animals. In this embodiment, the in vivo detection of cells in the mouse ear blood vessels was demonstrated as an example of shallow tissue. Since the mouse ear skin is relatively thin, the mouse ear blood vessels can be clearly seen under the bright field by only a simple hair removal process, which is used for in vivo flow cell detection in blood vessels. After constructing a mouse inflammation model, the prepared infrared fluorescent probe α-ErNPs-Ly6G was injected into the inflammation model mouse through the tail vein. The probe will actively target and label the neutrophils in the inflammation model mouse. The fluorescently labeled mouse is placed on the sample stage of the constructed infrared live flow single-channel signal detection optical path, and clear focus of the mouse ear blood vessels can be achieved by simple epi-illumination. By placing the laser spot on the vascular area of ​​the mouse ear, infrared live flow single-channel cell signal detection can be performed. When the α-ErNPs-Ly6G fluorescently labeled cells flow through the detected vascular area, they can emit 1532nm infrared fluorescence under 980nm laser excitation. The superconducting nanowire single-photon detector can detect the fluorescence signal in the living cells flowing through the blood vessels in real time. The real-time signal is synchronously presented on the computer in the form of a peak graph, such as Figure 3 The peak graph can intuitively reflect the number of neutrophils flowing through the blood vessels per unit time.

[0055] Example 2

[0056] Infrared live flow multi-channel cell signal detection of in vitro labeled reinfused cells. Figure 2 The optical path diagram of the multi-channel infrared fluorescence detection system is as follows:

[0057] (1) Build a multi-channel infrared fluorescence detection system for the optical path of multi-channel cell signal detection. The detection optical path includes a light source 1, an incident light modulation optical path (a first beam splitter 2, a galvanometer 3 and a microscope objective 4), an outgoing light modulation optical path (a microscope objective 4, a galvanometer 3, a first beam splitter 2, a second beam splitter a-5, a filter a-6, a second beam splitter b-10, a filter b-11), a superconducting nanowire single photon detector a-7 (first channel), and a superconducting nanowire single photon detector b-12 (second channel), which are used for the detection of two-channel cell signals. In addition, a filter z-8 and a superconducting nanowire single photon detector z-9 can also be optionally provided to selectively analyze the light separated by the second beam splitter b-10, thereby realizing the detection of three-channel cell signals.

[0058] Among them, the light source 1 is a 980nm near-infrared laser, the incident light is completely reflected by the first beam splitter 2 and the galvanometer 3, and is guided to the sample stage (not shown) through the 50× air mirror 4. The sample emits infrared fluorescence above 1000nm. After the emittance light is completely reflected by the galvanometer 3, it passes through the first beam splitter 2 with high transmittance, is split by the 1580nm dichroic mirror 5, and then is filtered by the 1500nm long-pass filter 6. The infrared fluorescence with a wavelength greater than 1500nm can be detected by the superconducting nanowire single-photon detector 7 with a central collection wavelength of 1550nm. The fluorescence reflected by the dichroic mirror 5 is split by the 1780nm dichroic mirror 10, and then is filtered by the 1900nm short-pass filter 11. The infrared fluorescence with a wavelength less than 1900nm can be detected by the superconducting nanowire single-photon detector 12 with a central collection wavelength of 1850nm. Both the superconducting nanowire single-photon detectors 7 and 12 are coupled with multimode optical fibers.

[0059] (2) Preparation of infrared fluorescent labeling probes. The infrared fluorescent labeling probes are cubic rare earth nanoprobes α-NaYbF4:2% Er,2% Ce@NaYF4, β-NaYbF4:5% Tm@NaYbF4@NaYF4, and commercial infrared fluorescent probe ICG. Among them, the excitation wavelength of α-NaYbF4:2% Er,2% Ce@NaYF4 (denoted as α-ErNPs-1532) is 980nm, and the emission peak is at 1532nm; the excitation wavelength of β-NaYbF4:5% Tm@NaYbF4@NaYF4 (denoted as β-TmNPs-1852) is 980nm, and the emission peak is at 1852nm. α-ErNPs-1532 and β-TmNPs-1852 can be used to label two types of cells cultured in vitro, respectively, and ICG can be used to label blood flow in vivo to achieve vascular focusing in living mice. The specific synthesis steps of α-ErNPs-1532 refer to ac in step (2) of Example 1, and the specific synthesis steps of β-TmNPs-1852 are as follows:

[0060] a. Preparation of rare earth precursors.

[0061] Preparation of yttrium trifluoroacetate Y(TFA)3: Take a 100mL three-necked round-bottom flask as the reaction container, add 0.1mmol yttrium oxide (Y2O3) and 50mL trifluoroacetic acid (TFA) in sequence, heat to 70℃ under vacuum and magnetic stirring conditions and maintain for 12h, and finally obtain a clear and transparent Y(TFA)3 solution with a concentration of 2mmol / L.

[0062] Preparation of ytterbium trifluoroacetate Yb(TFA)3: Take a 100mL three-necked round-bottom flask as the reaction container, add 0.1mmol ytterbium oxide (Yb2O3) and 50mL trifluoroacetic acid (TFA) in sequence, heat to 70℃ under vacuum and magnetic stirring conditions and maintain for 12h, and finally obtain a clear and transparent Yb(TFA)3 solution with a concentration of 2mmol / L.

[0063] b. Preparation of luminescent core layer β-NaYbF4:5% Tm.

[0064] Take a 100mL three-necked round-bottom flask as the reaction vessel, add 0.05mmol thulium chloride (TmCl3), 0.95mmol ytterbium chloride (YbCl3), and then add 6mL oleic acid OA and 15mL 1-octadecene ODE. The reactants are stirred and heated to 140°C under vacuum dehydration and deoxygenation conditions, and maintained for 60min. After cooling to room temperature, a transparent mixed solution is finally obtained. 2.5mmol sodium hydroxide (NaOH) and 4mmol ammonium fluoride (NH4F) are dissolved in 10mL methanol (CH3OH), mixed and added to the above reaction solution for reaction. After continuous stirring for 1h, the reactants are heated to 300°C at a rate of 10℃ / min under the protection of high-purity argon and then kept warm for 60min. After the reactants are cooled to room temperature, ethanol is added to precipitate the product from the solution, and then the supernatant is removed by centrifugation. After repeated washing with anhydrous ethanol for 3 times, it is dissolved in 10mL cyclohexane (C6H 12 ) for future use.

[0065] c. Preparation of energy-sensitizing shell β-NaYbF4: 5% Tm@NaYbF4.

[0066] Take a 100mL three-necked round-bottom flask as a reaction vessel, add 2mL of nanocrystals dissolved in cyclohexane in step b, then add 4mL of oleic acid OA and 6mL of 1-octadecene ODE, and then add 0.4mmol of sodium trifluoroacetate NaTFA, 0.4mmol of ytterbium trifluoroacetate Yb(TFA)3 in sequence. The reactants were stirred and heated to 100°C under vacuum dehydration and deoxygenation conditions and maintained for 20min. The reaction system was then heated to 290°C at a rate of 10°C / min and kept warm for 70min. After the reactants were cooled to room temperature, ethanol was added to precipitate the product from the solution, and then the supernatant was removed by centrifugation. After repeated washing with anhydrous ethanol for 3 times, the mixture was dissolved in 2mL of cyclohexane (C6H 12 ) for future use.

[0067] d. Preparation of inert shell β-NaYbF4: 5% Tm@NaYbF4@NaYF4.

[0068] Take a 100mL three-necked round-bottom flask as a reaction vessel, add all the nanocrystals dissolved in cyclohexane in step c, then add 4mL oleic acid OA and 6mL 1-octadecene ODE, and then add 0.4mmol sodium trifluoroacetate NaTFA and 0.4mmol yttrium trifluoroacetate Y(TFA)3 in sequence. The reactants were stirred and heated to 100°C under vacuum dehydration and deoxygenation conditions and maintained for 20min. The reaction system was then heated to 290°C at a rate of 10°C / min and kept warm for 60min. After the reactants were cooled to room temperature, ethanol was added to precipitate the product from the solution, and then the supernatant was removed by centrifugation. After repeated washing with anhydrous ethanol for 3 times, it was dissolved in 2mL cyclohexane (C6H 12 ) for later use to obtain an infrared fluorescent probe β-NaYbF4:5% Tm@NaYbF4@NaYF4 (denoted as β-TmNPs-1852) with an emission peak at 1852nm.

[0069] (3) Infrared fluorescent probes are used to label cells cultured in vitro. All cells were cultured in a standard 5% CO2, 37°C incubator. The cell culture medium used DMEM high-glucose culture medium, and all cell experiments were performed in a standard clean bench. In this embodiment, the infrared fluorescent probe α-ErNPs-1532 was used to label tumor cells, and β-TmNPs-1852 was used to label immune cells. When the cells covered 85%-90% of a 100mm diameter cell culture dish, 5μM infrared fluorescent probe α-ErNPs-1532 or β-TmNPs-1852 solution was prepared with cell culture medium. After the probe solution was incubated with the cells for 1-3h, the cell culture supernatant was removed, the cells were washed 2-3 times with PBS buffer solution, and the cells labeled with the infrared fluorescent probe were collected by centrifugation to prepare a fluorescently labeled cell suspension.

[0070] (4) Infrared in vivo streaming multi-channel signal detection. The experimental animals used were SPF-grade C57 mice. In this embodiment, in vivo detection of cells in mouse brain blood vessels was used as a deep tissue example demonstration. Since the skull of the mouse scatters fluorescence very seriously, the depth of the mouse meningeal blood vessels is about 0.5-1mm, and the depth of a specific brain area can reach 1-4mm. Selecting brain blood vessels with skull preservation as in vivo detection objects can better reflect the outstanding advantages of the present invention in in vivo detection of cells in deep tissue blood vessels in vivo. In this example, an immunotherapy model for mouse metastasis was constructed. The mouse was placed on the sample stage, a 50× air mirror was used to aim at the mouse brain, and the laser spot was placed in the mouse brain blood vessel area. The commercial infrared fluorescent probe ICG was injected into the mouse tail vein, and an 808nm laser was used for excitation and a 1000nm long-pass filter was used for filtering, thereby collecting infrared fluorescence above 1000nm, and clear focus on the mouse brain blood vessels can be achieved. Tumor cell suspensions labeled with infrared fluorescent probe α-ErNPs-1532 were injected into mice through the tail vein to construct a tumor metastasis model. At the same time, immune cell suspensions labeled with infrared fluorescent probe β-TmNPs-1852 were injected into mice through the tail vein to construct an immune cell transfusion treatment model for mouse metastatic tumors. Using the infrared in vivo streaming multi-channel signal detection optical path, when tumor cells labeled with α-ErNPs-1532 and immune cells labeled with β-TmNPs-1852 flow through the detected blood vessel area, they can emit 1532nm or 1852nm infrared fluorescence respectively under 980nm laser excitation, and the intracellular fluorescence signals flowing through the blood vessels are detected in real time by different superconducting nanowire single-photon detectors. The real-time signals are synchronously presented on the computer in the form of dual-channel peak graphs, such as Figure 4 The dual-channel peak graph can intuitively reflect the number of tumor cells and therapeutic immune cells flowing through the blood vessels per unit time.

[0071] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may alter, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A multi-channel infrared fluorescence detection system for in vivo flow cytometry, characterized in that: The multi-channel infrared fluorescence detection system comprises: a light source, an infrared fluorescence probe, an incident light modulation optical path, an outgoing light modulation optical path and a superconducting nanowire single-photon detector; the excitation light emitted by the light source is guided to a living sample whose target cells are marked by the infrared fluorescence probe via the incident light modulation optical path, the infrared fluorescence emitted by the living sample after being irradiated by the excitation light is guided to the superconducting nanowire single-photon detector via the outgoing light modulation optical path for signal collection, and the collected signal is transmitted to a computer to generate a real-time signal intensity graph, wherein The light source is a near-infrared laser, and the wavelength of the excitation light emitted is 700-2000nm; the infrared fluorescent probe is an inorganic or organic infrared fluorescent probe, and the wavelength of the infrared fluorescence emitted after being irradiated by the excitation light is 1000-2500nm.

2. The multi-channel infrared fluorescence detection system according to claim 1, characterized in that: The near-infrared laser emits an excitation light with a wavelength of 730 nm, 760 nm, 808 nm, 915 nm, 980 nm, 1064 nm, 1280 nm, 1310 nm, 1550 nm or 1710 nm.

3. The multi-channel infrared fluorescence detection system according to claim 1, characterized in that: The target cells are labeled with infrared fluorescent probes through in vivo labeling or in vitro labeling. The in vivo labeling is performed by injecting antibodies and / or polypeptides coupled with infrared fluorescent probes into a living body; the in vitro labeling is performed by co-incubating the infrared fluorescent probes with the target cells in vitro and then returning the labeled target cells to the body.

4. The multi-channel infrared fluorescence detection system according to claim 1, characterized in that: According to the direction of travel of the excitation light, the incident light modulation optical path includes a first beam splitter, a galvanometer and a microscope objective in sequence; according to the direction of travel of the infrared fluorescence, the outgoing light modulation optical path includes the microscope objective, the galvanometer and the first beam splitter in the incident light modulation optical path, as well as one or more second beam splitters and a filter behind each second beam splitter, and a corresponding superconducting nanowire single-photon detector is arranged on the light output side of each filter.

5. The multi-channel infrared fluorescence detection system according to claim 4, characterized in that: The first beam splitter and the second beam splitter are dichroic mirrors or prisms in the infrared band of 1000-2000nm, and the filter is an infrared long-pass, short-pass or band-pass filter.

6. The multi-channel infrared fluorescence detection system according to claim 4, characterized in that: The microscope objective lens is a water lens or an air lens, and the magnification is 10×, 20×, 25×, 40× or 50×.

7. The multi-channel infrared fluorescence detection system according to claim 1, characterized in that: The wavelength detection range of the superconducting nanowire single-photon detector is 1000nm-10μm, and the optical fiber used is single-mode optical fiber or multi-mode optical fiber coupling.

8. A method for multi-channel infrared fluorescence detection of in vivo flow cytometry using the multi-channel infrared fluorescence detection system according to any one of claims 1 to 7, characterized in that: The method comprises the following steps: (1) labeling target cells of a living sample with an infrared fluorescent probe, wherein the infrared fluorescent probe emits an infrared fluorescence wavelength of 1000-2500 nm after being irradiated with excitation light; (2) placing a living sample in which target cells are labeled with infrared fluorescent probes under the microscope objective of the multi-channel infrared fluorescence detection system; (3) causing a near-infrared laser light source to emit excitation light with a wavelength of 700-2000 nm, and directing the excitation light to the living sample via an incident light modulation optical path; (4) collecting an infrared fluorescence signal of a corresponding wavelength derived from the output light modulation optical path through at least one superconducting nanowire single-photon detector; and (5) The collected signals are transmitted to a computer to generate a real-time signal strength graph.

9. The multi-channel infrared fluorescence detection method according to claim 8, characterized in that: In step (1), the target cells are labeled with an infrared fluorescent probe by in vivo labeling or in vitro labeling. The in vivo labeling is performed by injecting antibodies and / or polypeptides coupled with infrared fluorescent probes into a living body; the in vitro labeling is performed by incubating the infrared fluorescent probes with the target cells in vitro and then returning the labeled target cells to the body.

10. Application of the multi-channel infrared fluorescence detection system according to any one of claims 1 to 7, the application comprising the following aspects: (1) In vivo typing and counting of endogenous circulating cells in blood vessels of normal living animals or living animals with disease models; the diseases include inflammation, tumors and / or cardiovascular diseases, and the cells include tumor cells, immune cells and / or blood cells; (2) dynamics and therapeutic evaluation of therapeutic cells in vivo and in vivo blood vessels after reinfusion into living small animals, wherein the therapeutic cells include stem cells and / or immune cells; and (3) In vivo evaluation of the dynamics of micron-sized particles of the same order of magnitude as cells within the blood vessels of living small animals, wherein the micron-sized particles include microplastics.

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