CN113390844A

CN113390844A - Multi-scale optical fiber fluorescence microscopic imaging system

Info

Publication number: CN113390844A
Application number: CN202110670806.1A
Authority: CN
Inventors: 顾月清; 林巧; 屈军乐; 钱志余
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2021-06-17
Filing date: 2021-06-17
Publication date: 2021-09-14

Abstract

The invention discloses a multi-scale optical fiber fluorescence microscopic imaging system. The invention is applied to research the concentration dynamic change of the drug in blood/tissue, the distribution of main metabolic organs and the transport characteristics in target cells by a multi-scale multi-wavelength fluorescence microscopic system with three optical fiber fluorescence probes with different resolutions, and particularly realizes the high-resolution microscopic imaging of the transport and release characteristics in the target cells. The invention applies the multi-scale optical fiber fluorescence microscopic imaging system to the in vivo pharmacokinetics research, can eliminate the influence of various random errors, more accurately reflects the pharmacokinetics behavior, and ensures that the correlation among multiple parameters is more reliable, thereby truly and systematically disclosing the transport mechanism of the medicine in the small animal body.

Description

Multi-scale optical fiber fluorescence microscopic imaging system

Technical Field

The invention belongs to the field of microscopic imaging, and particularly relates to a multi-scale optical fiber fluorescence microscopic imaging system.

Background

The research and development period of the medicine is long, the clinical transformation efficiency is low, and the root cause is the lack of a pre-clinical systematic patent medicine evaluation system. The pharmacokinetic characteristics of the drug are closely related to the curative effect, toxic and side effects and the like of the drug, and the key for evaluating the clinical transformation possibility of the drug is. In basic studies of the in vivo dynamic properties of drugs before clinical use, it is necessary to reveal not only the dynamic distribution properties in blood but also the accumulation of drugs in major metabolic organs (liver, kidney, etc.), the damage of organ tissues, and the dynamic distribution properties in target cells. Although various in vivo imaging systems (CT, MRI, PET, SPECT, etc.) can show more accumulation of the drug at the diseased tissue, due to their lower resolution, it is not possible to observe whether it enters or is pumped out by the target cells, nor to evaluate the damage of the major metabolic organs. Various endoscopic systems can identify pathological conditions on the surface of an organ, but cannot track drugs and have resolution that does not meet the requirements of intracellular drug monitoring. Various optical microscopic techniques have the imaging monitoring function of high-resolution intracellular drug transport, but cannot realize the pharmacokinetic characteristic research of cells in deep tissues of living animals. Meanwhile, the in vivo imaging technology visualizes the distribution of the in vivo drug, provides a powerful means for the pharmacokinetic research of the drug, can carry out continuous data analysis on the same experimental body, and avoids the data difference possibly brought by different individuals. Therefore, the optical fiber fluorescent endoscopic probe with different resolutions is used for simultaneously researching the dynamic change of the fluorescent material in blood and the distribution of the main metabolic tissue and organs to obtain synchronous pharmacokinetic parameters, researching the influence of the expression and function inhibition of the in vivo efflux transporters on the effectiveness and targeting of the medicament, and revealing the metabolic difference of blood-organs.

With the improvement of modern diagnosis and treatment level and experimental requirements, the endoscope imaging requirements are more refined, and higher diagnosis accuracy and coincidence rate are required. However, the observation of animal tissues by using a conventional standard-definition endoscope is gradually unable to meet experimental requirements, especially when slight changes of mucosal superficial blood vessels and tissue morphology occur. The optical microscopic imaging technology is the only feasible method for researching the transport of the drug in the cell or the organelle, and the problem of shallow penetration depth of the biological tissue of overcoming the optical imaging technology is a hot spot of recent domestic and foreign research. The system with the most potential application is the laser confocal micro-endoscope, which integrates a confocal laser micro-imaging system on the basis of the traditional optical fiber endoscope, not only exerts the advantage that the optical fiber can be flexibly inserted into deep tissues, but also keeps the characteristics of high resolution and high contrast of the confocal microscope, and realizes the micro-imaging of the deep tissues and cells. Fiber-optic in vivo endoscopic imaging systems are generally divided into fiber-optic bundle-based systems and single-fiber scanning-based systems. The system scanning mechanism based on the optical fiber bundle is arranged at the near end of the optical fiber bundle and is close to the main body of the optical system, the image transmission optical fiber bundle can be in a small diameter of hundreds of micrometers to several millimeters and comprises tens of thousands of optical fibers or even more than one hundred thousands of optical fibers, and when the scanning component couples laser into each optical fiber point by point, each optical fiber simultaneously plays the roles of a point light source and a confocal pinhole. The theoretical resolution of the product of Cellvizo of Mauna Kea of France can reach 1.4 μm at most, but in practical application, the single cell structure is difficult to distinguish (we have performed experiments by using the system), and the single cell structure can be seen by our target cell imaging system (FIG. 10A). The system based on single optical fiber scanning is a confocal endoscope system which realizes imaging by using a single optical fiber to scan at a far end, and the method generally adopts a piezoelectric wafer or piezoelectric ceramics to drive an optical fiber cantilever to vibrate so as to realize scanning. The single optical fiber is used as a point light source, is used for collecting returned signal light and can play a role in detecting the small holes. This scanning approach can achieve very high optical resolution, but due to the limitations of the scanning mechanism, it is difficult to achieve a small physical size at the distal end of the scanning probe. Taking a laser confocal endoscope which is jointly researched by Pentax corporation and Optiscan corporation in Australia as a representative (the laser confocal endoscope is sold in the market), Goetz and the like use the confocal microscope to investigate different expression levels of Epidermal Growth Factor Receptors (EGFR) of colon cancer in vivo, which suggests that the confocal microscope can carry out predictive diagnosis of tumor marker molecule imaging in vivo, is helpful for predicting lesion risks and suggests targeted therapy.

Conventional confocal microscopy is typically used for imaging tissue sections or cells in vitro. Meanwhile, most laser confocal systems are fixed on an optical platform, and a conventional objective lens has a large volume and a small operable working distance, and is limited by the volume size of living animal tissues, so that the internal tissues and organs of a living body are difficult to image. The optical fiber endoscopic technology is combined with a laser scanning confocal microscope to realize in-vivo imaging without being restricted by an animal cavity. Therefore, it is necessary to develop a fiber optic endoscope technology in combination with a laser scanning confocal microscope to realize in vivo imaging without being constrained by an animal cavity, check the pathological condition of a living tissue, and provide a new idea and technology for clinical medical disease diagnosis and longitudinal medical development.

The liver and the kidney are the largest metabolic organs of a living body, and the normal metabolism of the body is maintained. For example: the liver not only plays roles of de-oxidation, storage of glycogen, synthesis of secretory protein and the like, but also has a detoxifying function on toxic substances in the body, and even has an important regulating role in maintaining dynamic balance of blood flow and blood coagulation. The kidney has great significance for the generation of urine, the endocrine of hormone, the removal of wastes in vivo and the like. Therefore, pathological diseases of the liver and the kidney have great influence on normal life activities of organisms, physiological characteristics of the organs can be examined in a timely and nondestructive manner, and whether pathological changes occur or not is very important. The system can nondestructively detect the pathological condition of the living tissue through a specific fluorescent probe, and provides a new idea and technology for clinical medical disease diagnosis and longitudinal medical development. The system can also be applied to adipose tissue, intestinal tract, spleen, bladder, muscle, and virtually any tissue organ other than the thoracic cavity, by exposing the organ of interest through minimally invasive surgery. In addition, the disease progress of the same mouse can be continuously tracked after surgical suture, so that more long-term and high-reliability data can be obtained.

Regardless of the route of administration, the drug enters the blood and then is distributed to various tissues of the body along with the blood. The drug is first distributed to tissues with fast blood flow rate, and then distributed to tissues with slow blood flow rate, such as muscle, skin or fat. The type of drug distribution depends on physiological factors and the physicochemical properties of the drug, including tissue blood flow rate, the presence of a physiological barrier, affinity of the drug to the tissue, lipid solubility of the drug, binding of the drug to plasma proteins, and the like. Classical pharmacokinetics holds that the pharmacological effect is proportional to the plasma concentration of the drug after the drug is balanced in vivo. However, more and more researches show that the traditional pharmacokinetics can not well explain the pharmacological effects of some medicines, especially for some special tissues and organs (such as tumors), how the medicines exert the pharmacodynamic action is not clear, and the blood concentration is difficult to comprehensively and accurately reflect the pharmacodynamic action. In spite of the current research situation at home and abroad, no instrument can meet the requirement of multi-scale pharmacokinetic research in living animals. Therefore, a real-time imaging system for the multi-scale drug transport characteristics of the living bodies of the small animals is provided, which not only can monitor the distribution and transport characteristics of the drugs in target cells at a microscopic level, but also can inspect the local blood changes of the drugs at a macroscopic level and inspect the distribution and damage of main metabolic organs and the like at a mesoscopic level. The method is characterized in that extension type optical fiber probes with different resolutions and different visual field sizes are constructed, and the extendable detection probe is directly contacted with a tissue to be detected in a minimally invasive surgery organ exposure mode, so that the size of the probe is not limited by the size of a cavity, the method is used for multi-scale synchronous real-time imaging of a medicament in a small living animal, and high-resolution imaging of local blood flow tissues, metabolic organs and target cells on a living body is realized.

Disclosure of Invention

The invention provides a multi-scale optical fiber fluorescence microscopic imaging system aiming at the problems in the background technology.

The technical scheme is as follows:

a multi-scale optical fiber fluorescence microscopic imaging system mainly comprises a fluorescence intensity monitoring channel, a tissue imaging channel and a target cell imaging channel, wherein:

the laser fusion and light splitting light path comprises a 488nm laser, a 650nm laser, a light shutter, a long-pass dichroic mirror and a light path system. The light emitted by the 488nm laser sequentially passes through the first light shutter SH1 and the first reflector M1 to enter the first long-pass dichroic mirror DM1, and the light emitted by the 650nm laser passes through the second shutter SH2 to enter the first long-pass dichroic mirror DM 1; the fused light of the first long-pass dichroic mirror DM1 is divided into three paths through a light path system and is respectively sent to a fluorescence intensity monitoring channel, a tissue imaging channel and a target cell imaging channel;

the fluorescence intensity monitoring channel comprises a fluorescence intensity monitoring optical fiber bundle FB1 and a fluorescence spectrometer SD, wherein: one path of laser is coupled into an excitation light port of a fluorescence intensity monitoring fiber bundle FB1 through a first lens L1, and the fiber bundle FB1 transmits a fluorescence signal to a fluorescence spectrometer SD;

the tissue imaging channel comprises a tissue imaging fiber bundle FB2, a filter conversion wheel FW and an EMCCD, wherein: one path of laser is coupled to an excitation light guide beam organized into an image fiber beam FB2 through a lens L4, and the fiber beam FB2 enables the excitation light to be uniformly irradiated on tissues on one hand and transmits fluorescent signals to an EMCCD (electron-multiplying charge coupled device) for imaging through the lens and a filter conversion wheel FW on the other hand;

the target cell imaging channel comprises a dichroic mirror, a small hole, a scanning galvanometer GM, a target cell imaging optical fiber beam FB3 and a detection part, wherein: the laser sequentially passes through a long-pass dichroic mirror DM4, an objective OBJ1, a pinhole MP, an objective OBJ2, a scanning galvanometer GM and a LENS group, is coupled to a target cell imaging optical fiber beam FB3 after passing through the objective OBJ3, and is coupled to a self-focusing LENS GRIN LENS through an 8 th LENS L8 to realize scanning excitation of fluorescent dye in the tissue sample; the generated fluorescence signal is collected by a self-focusing LENS GRIN LENS and returns along the original optical path to enter the detection part of the target cell imaging channel.

Specifically, the detection part of the target cell imaging channel comprises a dichroic mirror, an optical filter, a photomultiplier and a data acquisition card, and the detection light path comprises three paths, wherein:

first detection light path: the generated fluorescence signal is reflected to a detection light path by a third dichroic mirror DM3, filtered to non-signal light by a first optical filter F1, enters a first photomultiplier tube PMT1 for photoelectric conversion, and finally is extracted from the fluorescence signal of each scanning point on a focal plane according to the corresponding time of laser scanning and fluorescence signal acquisition, and a shell fluorescence microscopic image is obtained through image reconstruction; in the optical path, the fluorescent substance 1 has an excitation light wavelength Ex: 488nm, emission center wavelength Em: 500 nm-560 nm;

a second detection light path: the generated fluorescence signal is reflected to a detection light path by a second dichroic mirror DM2, filtered to non-signal light by a second optical filter F2, enters a second photomultiplier PMT2 for photoelectric conversion, and finally, the generation of a cell membrane high-resolution fluorescence image is realized by system software; in the optical path, the fluorescent substance 2 has an excitation light wavelength Ex: 488nm, emission center wavelength Em: 570 nm-640 nm;

the third detection light path: the generated fluorescent signal enters a detection light path through a second dichroic mirror DM2, is filtered to non-signal light through a third filter F3, enters a third photomultiplier PMT3 for photoelectric conversion, and is finally guided into an upper computer system to obtain a high-resolution microscopic image of the encapsulated drug; in the optical path, the fluorescent substance 3 has an excitation light wavelength Ex: 650nm, an emission center wavelength Em >665 nm.

Preferably, the fluorescence intensity monitoring fiber bundle FB1 includes two paths: the first path transmits system laser to blood vessels/tissues of animals to excite drugs of the animals to emit fluorescence; the second path transmits the fluorescence back to the fluorescence spectrometer SD for detecting and analyzing the fluorescence signal.

Specifically, the central optical fiber is an optical fiber with the diameter of 200-600 μm; the outer ring of the optical fiber bundle is formed by 1-12 optical fibers with the diameter of 100-400 mu m surrounding the center optical fiber bundle at an equal angle.

Specifically, the tissue image fiber bundle FB2 includes a light guide bundle and an image transmission bundle: the light guide beam transmits system laser to organ tissues of the animal, and excites the medicine in the tissues to emit fluorescence; the image transmission beam transmits the fluorescence to the EMCCD for imaging.

Specifically, the image transmission bundle is formed by arranging 6000-30000 optical fibers in a regular hexagon, and the diameter of the image transmission bundle is 500-1500 μm; the light guide beam is 100 and 500 optical fibers with the diameter of 10-50 mu m which surround the image transmission beam.

Specifically, the target cell imaging fiber bundle FB3 includes a green lens and an image transmission bundle: transmitting the system laser to the cell of the animal by the image transmitting beam; the Green lens is used as a probe to obtain a high-resolution image, the image beam transmits the image to a photomultiplier tube PMT, the photomultiplier tube PMT converts a returned fluorescent signal into an electric signal, and the electric signal is further amplified, so that a weak fluorescent signal is detected.

Specifically, 20000-.

Specifically, the optical path system includes a first one-half wave plate HW1, a second one-half wave plate HW2, a first beam splitter prism SP1, and a second beam splitter prism SP2, the fused light of the first long-pass dichroic mirror DM1 sequentially passes through the first one-half wave plate HW1 and the first beam splitter prism SP1, and is divided into two paths in the first beam splitter prism SP 1: one path is connected to a fluorescence intensity monitoring channel; the other path of light sequentially passes through a second half-wave plate HW2 and a second beam splitter prism SP2 and is divided into two paths in the second beam splitter prism SP 2: one path is connected to the tissue imaging channel; the other path to the target cell imaging channel.

Preferably, the fluorescence intensity monitoring channel, the tissue imaging channel and the target cell imaging channel form a fluorescence signal acquisition cycle in a time-sharing acquisition working mode, and each scale works independently; the interval between sampling periods is adjusted within a few seconds to a few minutes according to experimental requirements; the fluorescence intensity monitoring channel and the tissue imaging channel are used for collecting shell fluorescence signals and drug fluorescence signals in a time-sharing manner, namely, a spectrometer or an EMCCD is used in a time-sharing manner; the target cell imaging module has high resolution and small imaging field of view, and the slight drift of the probe has great influence on fluorescence imaging and fusion, so that the experimental operation needs to be carried out after the animal breathes stably. According to the actual biological experiment needs, three PMTs are adopted to be matched with a dichroic mirror and a filter plate to simultaneously acquire three fluorescence signals (the reason that the three PMTs acquire the signals simultaneously is that the time for each PMT to acquire a clear picture is at least 1.024s, for a living animal, the time for acquiring the three fluorescence signals in a time-sharing way is too long, the images acquired by the three fluorescence signals are not the same image (position), and the fusion images cannot be completed), so that the distribution and the intensity change of the three fluorescence signals in the animal body are analyzed;

the fluorescence intensity monitoring channel works, the 488nm laser light shutter is opened, the 650nm laser light shutter is closed, the fluorescence spectrometer collects the fluorescence spectrum of the

fluorescent substance

1 or 2, after the fluorescence intensity detection is finished, the 488nm laser light shutter is closed, the 650nm laser light shutter is opened, and the fluorescence spectrometer collects the fluorescence spectrum of the fluorescent substance 3.

The tissue imaging channel works, the 488nm laser light shutter is opened, the 650nm laser light shutter is closed, the filter conversion wheel is turned to a high-pass filter with the cut-off wavelength of 500nm, and the EMCCD carries out fluorescence microscopic imaging on the

fluorescent substance

1 or 2 in the tissue to observe the distribution condition of the fluorescent substance. The 488nm laser light shutter is closed, the 650nm laser light shutter is opened, the filter conversion wheel rotates to the high-pass filter with cut-off wavelength of 665nm, and the EMCCD carries out fluorescence microscopic imaging on the fluorescent substance 3 in the tissue to observe the distribution condition of the fluorescent substance;

the target cell imaging channel works, a 488nm laser optical shutter is opened, a 650nm laser optical shutter is opened, a scanning galvanometer GM is scanned, three PMTs in the target cell imaging channel work simultaneously, and fluorescent signals generated by

fluorescent substances

1, 2 and 3 are collected respectively;

and after one sampling period is finished, waiting for a next sampling period according to the set sampling frequency and the instruction of the control software. The upper computer controls the laser light shutter, the optical filter conversion wheel, the fluorescence spectrometer, the EMCCD, the galvanometer and the PMT according to the time sequence, and because the response time of the laser light shutter and the optical filter conversion wheel is extremely short, the problem of collection point drift caused by factors such as heartbeat, respiration and the like of animals when fluorescence is collected in vivo can be solved, and therefore fluorescence data with different wavelengths are accurately fused together.

The invention has the advantages of

The invention discloses a multi-scale (dynamic change of fluorescent substances, damage of main metabolic organs and transport in target cells) optical fiber fluorescence microscopic imaging system applied to synchronously monitoring the transport characteristics of a medicament in vivo small animal, which can eliminate the influence of various random errors, more accurately reflect the pharmacokinetic behavior and ensure that the correlation among multiple parameters is more reliable, thereby truly and systematically disclosing the transport mechanism of the medicament in the small animal. Meanwhile, the method explores and establishes a drug property evaluation system.

Drawings

FIG. 1 is a block diagram of the optical path of the system of the present invention

FIG. 2 is a schematic view of a fluorescence intensity monitoring fiber bundle

FIG. 3 is a schematic view of a tissue imaging fiber bundle

FIG. 4 is a schematic view of a target cell imaging spot scanning system

FIG. 5 is a timing diagram of system hardware device operation

FIG. 6 is a diagram illustrating the overall system hardware configuration

FIG. 7 is a graph showing the kinetics of fluorescein and fluorescein sodium in liver and kidney of a mouse living body

FIG. 8 is a graph of the dynamic study of fluorescein liposome with different particle sizes on liver and kidney of mouse living body

FIG. 9 is a graph of kidney imaging and in vivo kinetics studies in normal and kidney-injured mice

FIG. 10 is the liver imaging and in vivo dynamics study of normal mice and liver fibrosis mice

FIG. 11 is an image of a mouse tissue in vivo

In the formula:

(1) a 488nm laser; (2) a 650nm laser; (3) the optical shutter SH 1; (4) the optical shutter SH 2; (5) a mirror M1; (6) a long-pass dichroic mirror DM 1; (7) a beam splitter prism SP 1; (8) a beam splitter prism SP 2; (9) a lens L1; (10) fluorescence intensity monitoring fiber bundle FB 1; (11) a spectrometer; (12) a lens L2; (13) a lens L3; (14) tissue imaging fiber bundle FB 2; (15) an optical filter conversion wheel FW; (16) an EMCCD; (17) a beam splitter prism SP 3; (18) objective OBJ 1; (19) a small hole MP; (20) objective OBJ 2; (21) scanning a galvanometer GM; (22) a lens group; (23) objective OBJ 3; (24) target cell fiber bundle FB 3; (25) a beam splitter prism SP 4; (26) photomultiplier tube PMT 1; (27) a beam splitter prism SP 5; (28) photomultiplier tube PMT 2; (29) photomultiplier tube PMT 3; (30) a scanning galvanometer power supply; (31) a filter switching wheel controller; (32) secondary gain of the photomultiplier; (33) an animal gas anesthesia apparatus; (34) an animal experiment platform; (35) an experimental mouse; (36) an air duct; (37) a computer.

Detailed Description

The invention is further illustrated by the following examples, without limiting the scope of the invention:

1. and (5) product structure. With reference to fig. 1, a multi-scale fiber fluorescence microscopic imaging system includes a laser fusion and light splitting path, a fluorescence intensity monitoring channel, a tissue imaging channel, and a target cell imaging channel, wherein:

the laser fusion and light splitting light path comprises a 488nm laser, a 650nm laser, a light shutter, a long-pass dichroic mirror and a light path system, light emitted by the 488nm laser sequentially passes through a first light shutter SH1 and a first reflector M1 and then is emitted into a first long-pass dichroic mirror DM1, and light emitted by the 650nm laser passes through a second shutter SH2 and then is emitted into a first long-pass dichroic mirror DM 1; the fused light of the first long-pass dichroic mirror DM1 is divided into three paths by a light path system and is respectively sent to the fluorescence intensity monitoring channel, the tissue imaging channel and the target cell imaging channel. The laser fusion and light splitting optical path adopts two lasers with different wavelengths, an optical shutter, a dichroic mirror and a light splitting prism, and realizes dual-wavelength laser switch control, dual-wavelength laser fusion, light splitting and light intensity adjustment. And distributing the fused laser to a fluorescence intensity monitoring channel, a tissue imaging channel and a target cell imaging channel according to any proportion, wherein the three channels are used for exciting different fluorescent dyes to emit fluorescence. The dual wavelength Laser light is emitted by 488nm and 650nm continuous lasers, which correspond to Laser1 and Laser2 in the figure respectively. SH1 and SH2 before the two lasers are optical shutters, and the on and off of the laser beams with the two wavelengths in the optical path are respectively controlled. The light of the Laser1 passes through M1 (a reflective mirror) and DM1(491nm long-pass dichroic mirror) to be fused with the Laser2 and enter the optical path system together. Light splitting and light intensity adjustment are realized by combining two groups of half-wave plates (HW1 and HW2) and light splitting prisms (SP1 and SP2), and the fused laser is divided into three paths.

The fluorescence intensity monitoring channel comprises a fiber bundle FB1 and a fluorescence spectrometer SD, wherein: one path of laser light is coupled into the excitation light port of the fluorescence intensity monitoring fiber bundle FB1 through the first lens L1, and the fiber bundle FB1 transmits the fluorescence signal to the fluorescence spectrometer SD. The fluorescence intensity monitoring channel monitors the fluorescence spectrum of the fluorescent substance in blood/tissue respectively mainly from a macroscopic level. The basic working principle of the scale channel is as follows: the two-path wavelength laser is provided with electric baffles (SH1 and SH2) in front, SH2 is in a closed state when SH1 is opened (SH1 is in a closed state when SH2 is opened), and only one path of exciting light (488nm or 650nm) exists in a light path when a fluorescence intensity monitoring scale is used. The excitation light is coupled into the excitation light port of the Y-shaped fiber bundle FB1 through a lens L1. The excitation light reaches the tissue through the optical fiber bundle FB1 to realize the excitation of the fluorescence sample, the generated fluorescence signal is collected through the receiving port of the optical fiber probe and transmitted to the SD (fluorescence spectrometer) along the optical fiber bundle FB1, and finally the spectrum of the emitted fluorescence is detected. Because the excitation light wavelength and the emission light wavelength of the fluorescent dye are different, the intensity of fluorescence emitted by the fluorescent dye can be easily extracted from a spectrometer, and an optical filter does not need to be added.

The fluorescence intensity monitoring fiber bundle FB1 comprises two paths: the first path transmits system laser to the blood vessel/tissue of the animal to excite the fluorescent drug therein to emit fluorescence; the second path transmits the fluorescence back to the fluorescence spectrometer SD for detecting and analyzing the fluorescence signal. Referring to fig. 2, the optical fiber cable comprises a probe part (101), a handheld/holding part (102), a first optical fiber bundle (103) and a second optical fiber bundle (104), wherein the probe part (101) is connected with the first optical fiber bundle (103) and the second optical fiber bundle (104), the handheld/holding part (102) is fixed at the tail end of the probe part (101), and a plastic-coated hose (105) is wrapped outside the first optical fiber bundle (103) and the second optical fiber bundle (104).

The tissue imaging channel comprises a tissue imaging fiber bundle FB2, a filter conversion wheel FW and an EMCCD, wherein: one path of laser light is reflected by the second reflecting mirror M2 and is coupled into the excitation light guide beam organized into an image optical fiber beam FB2 through the fourth lens L4, the optical fiber beam FB2 enables the excitation light to be uniformly irradiated on tissues on the one hand, and on the other hand, the fluorescence signal returns through the fifth lens L5 and is transmitted to the EMCCD for imaging through the third lens L3, the optical filter conversion wheel FW and the second lens L2 in sequence. The tissue imaging channel is used for tissue mesoscopic fluorescence microscopic imaging, and the distribution of fluorescent substances in the tissue is observed in real time in the form of images. The excitation light comes from two lasers of Laser1 and Laser2, SH2 is in a closed state when an optical shutter SH1 is opened (or SH1 is in a closed state when SH2 is opened), and only one type of excitation light (488nm or 650nm) exists in the light path. The excitation light is reflected by the mirror M2 and coupled into the excitation light guide fiber bundle of the fiber bundle FB2 by the lens L4. At the detection port, the light guide fiber surrounds the image transmission beam in the middle, so that the exciting light is uniformly irradiated on the tissue to excite the fluorescence. The fluorescence signal from the drug in the tissue returns along the image beam through lens L5 and finally through lens L3. The lenses L5 and L3 are matched with the image transmission beam, and the fluorescence image signals emitted by the tissues are subjected to microscopic amplification and transmitted to the detection light path. FW in the detection light path is an optical filter conversion wheel, and 500nm long-pass and 665nm long-pass optical filters are arranged on the optical filter conversion wheel and used for filtering 488nm exciting light and 650nm exciting light respectively. The optical shutter SH1 is opened, and the filter conversion wheel adopts a 500nm long-pass filter. The optical shutter SH2 is opened, and the filter conversion wheel adopts a 665nm long-pass filter. The fluorescence signal passes through the filter, and the scattered fluorescence signal is imaged by a lens L2 (optical lens), so that a clear fluorescence image can be collected by the EMCCD at the rear end. Through the action of the tissue imaging channel, the system observes the enrichment, distribution and leakage of the drug in the target organ tissues and the damage condition of the tissues in a microscopic imaging mode. Accordingly, the targeting effect, stability and drug effect of the drug are analyzed from the mesoscopic scale.

The tissue image fiber bundle FB2 includes a light guide bundle and an image transmission bundle: the light guide beam transmits system laser to organ tissues of the animal, and excites the medicine in the tissues to emit fluorescence; the image transmission beam transmits the fluorescence to the EMCCD for imaging. With reference to fig. 3, the device comprises an eyepiece (201), a light guide beam (202), an image transmission beam (203), and an objective lens (204), wherein the eyepiece (201) is connected with the objective lens (204) through the image transmission beam (203), and the light guide beam (202) is connected with the objective lens (204).

The target cell imaging channel comprises a dichroic mirror, a small hole, a scanning galvanometer GM, a target cell imaging optical fiber beam FB3 and a detection part, wherein: the two paths of laser sequentially pass through a third reflector M3 and a fourth reflector M4 and then enter a fourth long-pass dichroic mirror DM4, reflected light sequentially passes through a first objective OBJ1, a pinhole MP, a second objective OBJ2, a scanning vibrating mirror GM, a sixth LENS L6, a seventh LENS L7 and a third objective OBJ3 and then is coupled to a target cell imaging optical fiber bundle FB3, and laser after passing through the optical fiber bundle is coupled to a self-focusing LENS GRIN LENS through an 8 th LENS L8 to realize scanning excitation of fluorescent dye in a tissue sample; the generated fluorescence signal is collected by a self-focusing LENS GRIN LENS and returns along the original optical path to enter the detection part of the target cell imaging channel. The target cell imaging channel is mainly used for microscopic imaging, and can be used for high-resolution imaging of cells in a target organ area. When the light path works, SH1 and SH2 need to be opened simultaneously, two paths of laser light enter a dichroic mirror DM4 after being reflected by reflectors M3 and M4, and DM4 reflects exciting light with two wavelengths of 488nm and 650 nm. The objective OBJ1, the 50-micron pinhole MP and the OBJ2 are combined to block stray light outside an imaging focal plane, enhance the contrast of an image and improve the resolution of the image. Then the two paths of laser reach a scanning galvanometer GM to carry out beam scanning, the two paths of laser enter a 4f system consisting of lenses L6 and L7 to adjust the scanning beam and then enter an objective OBJ3, the objective OBJ3 couples the scanned laser beam to an image transmission beam (FB3), the laser after the optical fiber beam is coupled to a self-focusing Lens GRIN Lens through a micro Lens L8 to further compress a light spot, the GRIN Lens realizes the scanning excitation of fluorescent substances in a tissue sample, and the generated fluorescent signal returns along an original light path through the GRIN Lens collection and enters a detection part of a target cell imaging channel. The three detection light paths of the target cell imaging scale are respectively as follows: the fluorescence generated by the fluorescent substance 1 is reflected to a detection light path by a dichroic mirror DM3, non-signal light is filtered by a light filter F1, and then enters a photomultiplier tube PMT1 for photoelectric conversion, and finally, the extraction of the fluorescence signal of each scanning point on a focal plane is realized according to the corresponding time of laser scanning and fluorescence signal acquisition, and a shell fluorescence microscopic image is obtained through image reconstruction; the fluorescent signal generated by the fluorescent substance 2 is reflected to a detection light path by the dichroic mirror DM2, and is converted into a photoelectric signal by the PMT2 after passing through the optical filter F2, and the generation of a cell membrane high-resolution fluorescent image is realized by system software; fluorescence generated by the fluorescent substance 3 enters a detection light path through a dichroic mirror DM2, a filter F3 filters non-fluorescence interference signals, and then the fluorescence is converted into an electric signal through a photomultiplier tube PMT3 and is led into an upper computer system to obtain a high-resolution microscopic image of the fluorescent drug. The system can be used for microscopically imaging the target cells, and fluorescent images of the distribution of three fluorescent substances (medicines) in the cells can be respectively displayed in an image mode.

The target cell imaging fiber bundle FB3 includes a Green lens and an image transmission bundle: transmitting the system laser to the cell of the animal by the image transmitting beam; the Green lens is used as a probe to obtain a high-resolution image, the image beam transmits the image to a photomultiplier tube PMT, the photomultiplier tube PMT converts a returned fluorescent signal into an electric signal, and the electric signal is further amplified, so that a weak fluorescent signal is detected. Referring to fig. 4, the laser beam sequentially passes through the sixth lens L6(301), the seventh lens L7(302), and the objective lens OBJ3(23) and is incident on the target cell optical fiber bundle FB3(24), and the target cell optical fiber bundle FB3(24) transmits the laser beam to the cells of the animal through the self-focusing lens (303).

By monitoring the changes of the three fluorescence images in real time, the transmembrane, leakage and release control processes of the drug in the target cell can be analyzed.

In order to make the system compact, reduce the occupied area and facilitate the use of operators, the light path of the system is designed into a three-layer three-dimensional structure, and the finally constructed system hardware is an integral entity as shown in fig. 6. Laser fusion and beam split light path, fluorescence intensity monitoring channel and tissue imaging channel are built to the bottom, include: the optical shutter SH1 (3); the optical shutter SH2 (4); a mirror M1 (5); a long-pass dichroic mirror DM1 (6); a beam splitter prism SP1 (7); a beam splitter prism SP2 (8); a lens L1(9), a fluorescence intensity monitoring fiber bundle FB1 (10); a spectrometer (11); a lens L2 (12); a lens L3 (13); tissue imaging fiber bundle FB2 (14); a filter conversion wheel FW (15); an EMCCD (16). Use optics to climb the frame and promote laser source to the intermediate level from the bottom platform, the target cell imaging channel is built to the intermediate level, and top layer placement system's power and controller include: a beam splitter prism SP3 (17); objective OBJ1 (18); a pinhole MP (19); objective OBJ2 (20); a scanning galvanometer GM (21); a lens group (22); objective OBJ3 (23); target cell fiber bundle FB3 (24); a beam splitter prism SP4 (25); photomultiplier tube PMT1 (26); a beam splitter prism SP5 (27); photomultiplier tube PMT2 (28); the photomultiplier tube PMT3 (29). The right side of the system hardware (the middle part of figure 6) is provided with an animal experiment platform and an animal gas anesthesia device, and the animal gas anesthesia device comprises: a 488nm laser (1); a 650nm laser (2); a scanning galvanometer power supply (30); a filter switching wheel controller (31); photomultiplier tube secondary gain (32). The rightmost side of fig. 6 is a computer display screen and a host, including: an animal gas anesthesia apparatus (33); an animal experiment platform (34); laboratory mice (35); a vent conduit (36); a computer (37).

2. And controlling a multi-scale optical fiber fluorescence microscope system.

Multiple scale acquisition sequences and corresponding hardware action sequences are provided. The software realizes the functions of driving, parameter setting, on-off control and the like of the optical shutter aiming at the laser fusion and light splitting subsystem; aiming at the fluorescence intensity monitoring subsystem, the functions of driving a spectrometer, setting parameters, collecting spectra, extracting average intensity of an interested wavelength band and the like are realized; aiming at the wide-field tissue fluorescence imaging subsystem, the driving of an optical filter conversion wheel, parameter setting, optical filter switching, the driving of an EMCCD (electron-multiplying charge coupled device), parameter setting and image acquisition are realized, and the fluorescence images with two wavelengths are added with pseudo colors and fused; matlab control is realized for a target cell microscopic endoscopic imaging subsystem, so that ScanImage software is controlled to acquire target cell image data, acquired image data is acquired, and acquired fluorescent images with 3 wavelengths are subjected to pseudo-color addition and fusion. The storage format of the experimental data is designed, and the system software can completely and clearly store all the experimental data.

With reference to fig. 5, in order to implement the monitoring and imaging timings of modules with different scales, an action timing of corresponding hardware is designed, a rectangle or a triangle in the diagram indicates hardware operation, and a width indicates hardware operation duration. The 488nm laser and the 650nm laser are connected to the 220V power supply and are kept normally open. The light shutters in front of the 488nm laser and the 650nm laser are respectively connected with the upper computer through respective controllers, and the opening or closing of the light shutters is controlled by the upper computer. The fluorescence spectrometer is connected with an upper computer through a USB, the upper computer controls the acquisition starting time and the acquisition time (integral time), and the rectangular width represents the acquisition time. In one acquisition, the acquisition time of the fluorescence spectrometer controls the opening and closing duration of the optical shutter of the laser. The EMCCD is connected with an upper computer through a USB, the upper computer controls the acquisition starting time and the acquisition time (integral time), and the rectangular width represents the acquisition time. The optical filter conversion wheel is connected with an upper computer through a controller, the upper computer controls the rotating and stopping positions of the optical filter conversion wheel, corresponding triangles in the graph show fluorescence emitted by 488nm laser, and corresponding rectangles show fluorescence emitted by 650nm laser. In one acquisition, the acquisition time of the electron multiplying EMCCD controls the duration of the laser shutter opening and closing, and controls the position of the filter switching wheel. The galvanometer and the PMT are connected with an upper computer through a data acquisition card, the galvanometer starts scanning, the three PMTs start working simultaneously, the positions of the galvanometers correspond to the positions of acquisition points in the target cells, and images of three fluorescent substances in the target cells can be combined according to the fluorescence intensity acquired by the PMT. And after one sampling period is finished, waiting for the next sampling period according to the set sampling frequency.

The time sequence control method designed by the system ensures the durability of the system, simultaneously realizes orderly detection of the fluorescent samples with different wavelengths by the three submodules with different scales, provides guarantee for the correct fusion of later-period fluorescent data, is a control core of system software, and is the basis for multi-scale analysis of drug effect.

3. The multi-scale optical fiber fluorescence microscope system is applied to the dynamics research of fluorescent substances with different dissolubility on the liver and the kidney of a mouse living body.

After a rat is anesthetized by isoflurane gas and a fat-soluble fluorescent substance (fluorescein) is injected into a tail vein, a spectrum scanning channel fiber-optic probe living body monitors the change of relative fluorescence intensity value of jugular vein blood of the rat, and simultaneously, blood is collected at multiple points in vitro. By fitting the fluorescence intensity curve with time and calculating the relevant parameters of pharmacokinetics, the following can be obtained: the in vivo and in vitro hemodynamic curves are approximately coincident, which shows that the relative fluorescence value acquired by the system can be used for representing the concentration of the in vivo fluorescent substance, and the pharmacokinetics detection of the living body of the small animal has the advantage of avoiding the generation of large experimental errors caused by in vitro blood sampling. Meanwhile, the abdominal cavity of a rat is minimally invasive, the liver and the kidney are exposed, the optical fiber probe of the laser scanning endoscope is fixed right above the rat, continuous imaging is carried out, and the average fluorescence intensity of the images collected at each time point is recorded. It can be found that after the fat-soluble fluorescent substance (fluorescein) enters the liver cells from the liver blood sinuses, the fat-soluble fluorescent substance is metabolized out of the liver through the liver blood sinuses; the fat-soluble fluorescent substance is distributed in the renal tubule lumen and the wall. The gene is determined by the physicochemical properties of fluorescein, the fat solubility is strong, the metabolism is mainly liver and intestine, the metabolism is slow, the reabsorption of renal tubules is more, the excretion is slow, the fluorescein in the lumen is gathered, the fluorescence intensity is high (figure 7AB), and the pharmacokinetic parameters of each group are shown in figure 7C.

In the same operation process and rule, the water-soluble fluorescent substance (fluorescein sodium) has strong water solubility, and is hardly distributed or slightly distributed in the liver; in the kidney, the renal tubules reabsorb less, excrete more rapidly, and are mainly absorbed by the renal tubule wall, and the intraluminal urea contains less sodium fluorescein (fig. 7D). Further, the collected curves of the change of the in vivo fluorescein sodium relative to the average fluorescence intensity with time at different times were fitted (fig. 7E), and a non-compartmental model was used for calculation to obtain each group of pharmacokinetic parameters (fig. 7F).

4. The multi-scale optical fiber fluorescence microscope system is applied to the dynamics research of fluorescein liposome with different particle sizes on the liver and the kidney of a mouse living body.

After a rat is anesthetized by isoflurane gas, tail vein is injected with fluorescein liposome with small particle size (200nm), jugular vein is exposed through minimally invasive surgery, a spectrum scanning channel fiber probe is placed right above the jugular vein, a laser aperture covers the width of a blood vessel, and the change of the jugular vein blood relative to the fluorescence intensity value of the rat is monitored. Meanwhile, the carotid artery is inserted into the tube to take blood at multiple points in vitro. And cutting open the abdominal cavity of the rat through minimally invasive surgery to expose the liver and the kidney, fixing the optical fiber probe of the laser scanning endoscope right above the rat, performing continuous microscopic imaging, and recording the average fluorescence intensity of the images collected at each time point. It can be found that, at 10min, the fluorescein liposome with small particle size (200nm) enters into the liver cells from the liver blood sinuses; it was distributed in the hepatocytes up to 4 h. In the kidney, small particle size liposomes are mainly absorbed by the renal tubule wall, and the fluorescence intensity of luminal fluorescein liposomes is weak, because small particle size liposomes are easily captured by liver cells, and compared with the kidney, the renal tubules absorb less again and excrete quickly. By fitting the fluorescence intensity curve with time and calculating the relevant parameters of pharmacokinetics, the following can be obtained: the in vivo and in vitro hemodynamic curves are approximately coincident, which shows that the relative fluorescence value acquired by the system can be used for representing the concentration of the fluorescent substance in vivo, and the pharmacokinetics detection of the living body of the small animal has the advantage of avoiding the generation of large experimental errors caused by in vitro blood sampling (figure 8 ABC).

The same operation flow and rules, the large-particle-size (1000nm) fluorescein liposome is mainly distributed in hepatic sinus due to the larger particle size, and a small amount of the fluorescein liposome is absorbed by hepatic cells; while in the kidney, metabolism is slow, renal tubules reabsorb less, excretion is slow, and there is distribution of pro-urine and wall cells in the lumen of the renal tubules (fig. 8D). Further fitting the collected curves of the relative mean fluorescence intensity of the fluorescein liposomes in vivo at different times with time (fig. 8E), and calculating by using a non-compartmental model to obtain each group of pharmacokinetic parameters (fig. 8F).

5. The multi-scale optical fiber fluorescence microscope system is applied to the kidney disease detection and in vivo dynamics research of normal mice and kidney injury mice.

Furthermore, kidney tissues of normal mice and kidney-injured mice were imaged, and the injury of the kidney tissues of the mice was evaluated in vivo. After mouse tail vein injection with fluorescent dye R3, the mouse live kidney tissue was imaged with a laser scanning microscopy system. It can be seen that R3 is mainly distributed in the renal tubular cavity of the kidney injury mouse, but the structure is seriously damaged due to the serious shedding of renal tubular epithelial cells, and the structure is obviously different from the renal tubular structure of a normal mouse. The cryo-section, in vitro pathological staining results were consistent with the live renal tubule imaging results (fig. 9A).

The in vivo kinetics of a normal mouse and a kidney injury mouse are researched, the fluorescence intensity is shown by a time curve, and after the mouse is anesthetized by isoflurane gas and is injected with fluorescein sodium in tail vein, the in vivo mouse hemodynamics and kidney kinetics research is carried out. The optical fiber probe of the spectral scanning channel monitors the fluorescence change of the fluorescein sodium in the jugular vein blood of the mouse, and the optical fiber probe of the laser scanning endoscope monitors the fluorescence change of the fluorescein sodium in the kidney of the mouse. The time-dependent change curves of the fluorescence intensity of a normal mouse and a kidney injury mouse show that the metabolism of the kidney tissue of the mouse is slower than that of venous blood, in the normal mouse, the fluorescein sodium in the venous blood is almost completely metabolized within about 0.2h, and the fluorescein sodium in the kidney tissue is almost completely metabolized within 1 h; however, in kidney-injured mice, intravenous blood fluorescein sodium was almost completely metabolized at about 0.5h, whereas in kidney tissue it was almost completely metabolized by 2.5 h. The results also showed that the metabolism of sodium fluorescein in vivo was slower in the kidney-injured mice than in the normal mice, both in venous blood and in kidney tissue (fig. 9B); fig. 9C is the pharmacokinetic parameters for each group.

6. The multi-scale optical fiber fluorescence microscope system is applied to liver disease detection and in vivo dynamics research of normal mice and liver fibrosis mice.

The liver of a normal mouse and the liver of a liver fibrosis mouse are subjected to living cell level imaging, and it can be seen that R3 is distributed in liver blood sinuses, normal liver cells are regularly arranged and consistent in size, and liver fibrosis liver cells are in hypertrophy and disorderly arranged. The cells of the mouse liver fibrosis injected with the rhodamine B liposome in the tail are vacuolated and disorganized. HE staining also showed proliferation of hepatic fibrosis adipose-like cells, infiltration of inflammatory cells, and disorganization of the array. Masson staining can show that the collagen fibers are obviously extended, the fiber intervals are formed, the hepatic fibrosis is serious, and the hepatic lobule structure is damaged. The in vitro pathological staining results were consistent with the live liver imaging results (fig. 10A).

The in vivo kinetics of a normal mouse and a hepatic fibrosis mouse are researched, the fluorescence intensity time curve is obtained, and the hemodynamics and the liver kinetics of a living mouse are researched after the mouse is anesthetized by isoflurane gas and the tail vein is injected with fluorescein. The optical fiber probe of the spectral scanning channel monitors the fluorescence change of fluorescein in jugular vein blood of the mouse, and the optical fiber probe of the laser scanning endoscope monitors the fluorescence change of fluorescein in kidney of the mouse. The time-dependent change curves of the fluorescence intensity of a normal mouse and a liver fibrosis mouse show that the metabolism of the liver tissue of the mouse is slower than that of venous blood, in the normal mouse, the fluorescein in the venous blood is almost completely metabolized within about 0.5h, and the fluorescein in the liver tissue is almost completely metabolized within 1.5 h; however, in liver fibrosis mice, intravenous blood fluorescein was almost completely metabolized for about 1.5h, whereas in liver tissue it was almost completely metabolized by 3.5 h. At the same time, the results also showed that the in vivo fluorescein metabolism in liver fibrosis mice was slower than that in normal mice, both in venous blood and liver tissue (fig. 10B); fig. 10C is the pharmacokinetic parameters for each group.

7. The wide field fluorescence imaging system is applied to in vivo mouse tissue imaging.

By carrying out wide-field imaging on the HE stained tissue section of the heart, the liver, the spleen, the lung and the kidney of the mouse, the imaging result is complete in structure, clear in picture, distinct in level and high in contrast. Injecting CdSe as fluorescent substance into mouse tail vein, exposing liver through enterocoelia minimally invasive operation, and imaging mouse living liver. Since the system is wide field imaging, the working distance can be properly adjusted, the visual field of the first three pictures in the upper row of fig. 11 is gradually enlarged, and the image is still clear. Comparing the results of HE staining on pathological liver sections, CdSe is distributed in the hepatic lobular cell area of liver (bright color), and the central venous area is dark color due to the absorption of 500-600nm light by blood. A fluorescent substance FITC is injected into a tail vein of a mouse, the liver, spleen and kidney of the living body of the mouse are imaged through a minimally invasive surgery, and due to the non-specific staining of the FITC and the metabolism of the mouse, the imaging of each tissue is not different. The imaging of the blood vessels on the surface of the small intestine is clearly visible. In summary, wide field imaging is widely applied, and slices, living bodies, bright fields and fluorescence fields can be clearly imaged.

The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.

Claims

1. A multi-scale optical fiber fluorescence microscopic imaging system is characterized by comprising a fluorescence intensity monitoring channel, a tissue imaging channel and a target cell imaging channel, and the fluorescent substances in the living bodies of animals are continuously monitored in real time on different scales.

2. The multi-scale optical fiber fluorescence microscopic imaging system according to claim 1, wherein the system emits laser light by two lasers with different wavelengths, and the laser light is split by the optical path splitting system after being fused and sent to different channels, wherein:

the fluorescence intensity monitoring channel comprises a fluorescence intensity monitoring optical fiber bundle FB1 and a fluorescence spectrometer SD, wherein: one path of laser is coupled into an excitation light port of the optical fiber bundle FB1 through a lens, and the optical fiber bundle FB1 transmits a fluorescence signal to the fluorescence spectrometer SD;

the tissue imaging channel comprises a tissue imaging fiber bundle FB2, a filter conversion wheel FW and an EMCCD, wherein: one path of laser is coupled into an excitation light guide beam organized into an image fiber beam FB2 through a lens, the fiber beam FB2 enables the excitation light to be uniformly irradiated on tissues on one hand, and on the other hand, a fluorescence signal is transmitted to an EMCCD (electron-multiplying charge coupled device) for imaging through the lens, a filter conversion wheel FW and the lens in sequence;

the target cell imaging channel comprises an objective lens, a small hole, a scanning galvanometer GM, a target cell imaging optical fiber beam FB3 and a detection part, wherein: the two paths of laser are coupled to a target cell imaging optical fiber beam FB3 after sequentially passing through a first objective LENS OBJ1, a small hole MP, a second objective LENS OBJ2, a scanning galvanometer GM, a LENS group and a third objective LENS OBJ3, and the laser after passing through the optical fiber beam is coupled to a self-focusing LENS GRIN LENS through an 8 th LENS L8 to realize scanning excitation of fluorescent dye in a tissue sample; the generated fluorescence signal is collected by a self-focusing LENS GRIN LENS and returns along the original optical path to enter the detection part of the target cell imaging channel.

3. The multi-scale optical fiber fluorescence microscopic imaging system according to claim 2, wherein the detection portion of the target cell imaging channel comprises a dichroic mirror, a filter, a photomultiplier tube, and a data acquisition card, and the detection light path comprises three paths, wherein:

first detection light path: the generated fluorescence signal is reflected to a detection light path by a third dichroic mirror DM3, filtered to non-signal light by a first optical filter F1, enters a first photomultiplier tube PMT1 for photoelectric conversion, and finally is extracted from the fluorescence signal of each scanning point on a focal plane according to the corresponding time of laser scanning and fluorescence signal acquisition, and a fluorescent substance 1 microscopic image is obtained through image reconstruction; a second detection light path: the generated fluorescent signal is reflected to a detection light path by a second dichroic mirror DM2, filtered to non-signal light by a second optical filter F2, enters a second photomultiplier PMT2 for photoelectric conversion, and finally, the generation of a high-resolution fluorescent image of the fluorescent substance 2 is realized by system software; the third detection light path: the generated fluorescent signal enters a detection light path through a second dichroic mirror DM2, is filtered to non-signal light through a third filter F3, enters a third photomultiplier PMT3 for photoelectric conversion, and is finally guided into an upper computer system to obtain a high-resolution microscopic image of the fluorescent substance 3.

4. The multi-scale fiber fluorescence microscopy imaging system according to claim 2, characterized in that the fluorescence intensity monitoring fiber bundle FB1 is a Y-shaped fiber bundle: the central optical fiber transmits system laser to the surface of the tissue/blood vessel to be detected, and excites the medicine in the tissue/blood vessel to emit fluorescence; the outer ring optical fiber bundle transmits the fluorescence back to the fluorescence spectrometer SD for detecting and analyzing the fluorescence signal.

5. The multi-scale fiber fluorescence microscopy imaging system according to claim 2, wherein the tissue imaging fiber bundle FB2 comprises a light guide bundle and an image transmission bundle: the outer ring light guide beam transmits system laser to organ tissues of animals to excite the drugs in the tissues to emit fluorescence; the inner-ring image transmission beam transmits the fluorescence to the EMCCD for imaging.

6. The multi-scale fiber fluorescence microscopy imaging system according to claim 2, wherein the target cell imaging fiber bundle FB3 comprises a Green lens and an image transmission bundle: transmitting the system laser to the cell of the animal by the image transmitting beam; the Green lens is used as a probe to obtain a high-resolution image, the image beam transmits the image to a photomultiplier tube PMT, the photomultiplier tube PMT converts a returned fluorescent signal into an electric signal, and the electric signal is further amplified, so that a weak fluorescent signal is detected.

7. The multi-scale fiber fluorescence microscopic imaging system according to claim 2, wherein the optical path splitting system comprises a first half wave plate HW1, a second half wave plate HW2, a first beam splitter prism SP1 and a second beam splitter prism SP2, the fused light of the first long pass dichroic mirror DM1 sequentially passes through the first half wave plate HW1 and the first beam splitter prism SP1, and is split into two paths in the first beam splitter prism SP 1: one path is connected to a fluorescent substance monitoring optical path; the other path of light sequentially passes through a second half-wave plate HW2 and a second beam splitter prism SP2 and is divided into two paths in the second beam splitter prism SP 2: one path is connected to the tissue imaging channel; the other path to the target cell imaging channel.

8. The multi-scale optical fiber fluorescence microscopic imaging system according to claim 1, wherein the fluorescence intensity monitoring channel, the tissue imaging channel and the target cell imaging channel form a fluorescence signal acquisition cycle by a time-sharing acquisition working mode, and each scale works independently; the interval between sampling periods is adjusted within a few seconds to a few minutes according to experimental requirements; the fluorescence intensity monitoring channel and the tissue imaging channel are used for carrying out time-sharing collection on the fluorescence signals of the fluorescent substance 1 or 2 and the fluorescent substance 3, namely, a spectrometer or an EMCCD is used in a time-sharing mode; three fluorescence signals are simultaneously collected by adopting a mode that three PMTs are matched with a dichroic mirror and a filter plate, so that the distribution and the intensity change of the three fluorescence signals in an animal body are analyzed;

the fluorescence intensity monitoring channel works, the optical shutter of the laser1 is opened, the optical shutter of the laser2 is closed, the fluorescence spectrometer collects the fluorescence spectrum of the fluorescent substance 1 or 2, after the fluorescence intensity detection is finished, the optical shutter of the 488nm laser is closed, the optical shutter of the laser2 is opened, and the fluorescence spectrometer collects the fluorescence spectrum of the fluorescent substance 3.

The tissue imaging channel works, the optical shutter of the laser1 is opened, the optical shutter of the laser2 is closed, and the EMCCD carries out fluorescence microscopic imaging on the fluorescent substance 1 or 2 in the tissue to observe the distribution condition of the fluorescent substance. The laser light shutter is closed, the laser light shutter is opened, and the EMCCD carries out fluorescence microscopic imaging on the fluorescent substance 3 in the tissue to observe the distribution condition of the fluorescent substance;

the target cell imaging channel works, the optical shutter of the laser1 is opened, the optical shutter of the laser2 is opened, the scanning galvanometer GM is used for scanning, three PMTs in the target cell imaging channel work simultaneously, and fluorescent signals generated by the fluorescent substance 1, the fluorescent substance 2 and the fluorescent substance 3 are respectively collected;

and after one sampling period is finished, waiting for a next sampling period according to the set sampling frequency and the instruction of the control software.