CN110221051B - A dual-wavelength dual-scale nanomedicine in vivo monitoring system and timing control method - Google Patents
A dual-wavelength dual-scale nanomedicine in vivo monitoring system and timing control method Download PDFInfo
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Abstract
本发明公开了一种双波长双尺度纳米药物在体检测系统及时序控制方法，系统包括：波长为488nm的激光器、波长为650nm的激光器、488nm激光器快门、650nm激光器快门、第一反光镜、第一双色镜、二分之一玻片、分光棱镜、第一液晶可调谐滤光器、第一液晶可调谐滤光器控制器、荧光光谱仪、传光光纤束、第二液晶可调谐滤光器、第二液晶可调谐滤光器控制器、电子倍增CCD、传像光纤束、小型显微物镜和PC上位机；方法在保证系统的耐用度的同时，对不同荧光样本的探测有序进行，为后期荧光数据的正确处理提供了保证。
The invention discloses a dual-wavelength dual-scale nanometer drug in-vivo detection system and a time sequence control method. The system comprises: a laser with a wavelength of 488nm, a laser with a wavelength of 650nm, a 488nm laser shutter, a 650nm laser shutter, a first reflection mirror, a second A dichroic mirror, a half glass, a beam splitter prism, a first liquid crystal tunable filter, a first liquid crystal tunable filter controller, a fluorescence spectrometer, a light transmission fiber bundle, and a second liquid crystal tunable filter , a second liquid crystal tunable filter controller, an electron multiplying CCD, an image-transmitting fiber bundle, a small microscope objective lens and a PC host computer; the method ensures the durability of the system, and the detection of different fluorescent samples is carried out in an orderly manner. It provides a guarantee for the correct processing of the later fluorescence data.
Description
Technical Field
The invention relates to the technical field of nano-drug medical detection, in particular to a dual-wavelength dual-scale nano-drug in-vivo monitoring system and a time sequence control method.
Background
The nano-drug is a nanoparticle in pharmaceutics, has a large surface area, and has high chemical activity, strong effect and high absorption speed compared with a common preparation. The nanometer reagent has very small size and high penetrating capacity in organism, and the nanometer preparation prepared with proper material and technological process can regulate the distribution of medicine in body. Therefore, the nano-drug can improve the stability and the circulation time of the drug, enhance the targeted absorption capacity and improve the distribution and the metabolic process of the drug.
The nano-drug has unique advantages and is a hot spot of drug research, but the clinical transformation efficiency is extremely low, and the fundamental reason is the lack of a pre-clinical systematic patent drug evaluation system. The nanometer medicine is dyed with fluorescent dye, and the stability, targeting property, metabolic process and the like of the nanometer medicine in vivo are reflected by detecting fluorescence, so that the method is the most common method for evaluating the efficacy of the nanometer medicine. Usually, dye FITC (ex/em:480nm/520nm) and the like are used for marking the nanoshell, the tissue distribution of the nanoshell is observed, and the targeting effect of the nano-drug is revealed; drug labeling was performed with the cy5 series (ex/em:650/670nm) to observe drug leakage and controlled release characteristics. At present, fluorescence is mainly detected through a tissue section, and the method mainly has the following problems: on one hand, the whole data of all time points cannot be obtained in the same animal body, the difference between different experimental individuals is large, the model animal cannot be dynamically observed in real time, the tissue slices can only be reproduced when the data of different points are required to be obtained, the tissue slices can be processed after the animal is processed, and the obtained experimental result is not necessarily the real condition of the living animal; on the other hand, the method for detecting the fluorescent dye through the tissue slice has single scale, can only obtain the distribution condition of the nano-drug in the tissue, and cannot obtain the dynamic change of the nano-drug in the blood.
The traditional in vivo detection of nano-drugs is generally realized by a nuclide imaging method, and the method has low spatial resolution and can only observe the radioactive accumulation of the nano-drugs in certain organs of the animal body. In addition, only one radioactive element can be detected in a nuclide imaging experiment, and the detection of the separation condition of the nanoshell and the drug cannot be realized, so that the characteristics of drug leakage, controlled release and the like cannot be analyzed.
Disclosure of Invention
The invention aims to solve the technical problem of providing a dual-wavelength dual-scale nano-drug in-vivo monitoring system and a time sequence control method, which ensure the durability of the system and simultaneously ensure that two sub-modules detect different fluorescence samples in order, thereby ensuring the correct processing of later fluorescence data.
In order to solve the above technical problems, the present invention provides a dual-wavelength dual-scale in vivo nano-drug monitoring system, comprising: a laser 1 with the wavelength of 488nm, a laser 2 with the wavelength of 650nm, a 488nm laser shutter 3, a 650nm laser shutter 5, a first reflective mirror 7, a first dichroic mirror 8, a half glass 9, a beam splitter prism 10, a first liquid crystal tunable filter 12, a first liquid crystal tunable filter controller 13, a fluorescence spectrometer 15, a light transmission optical fiber bundle 17, a second liquid crystal tunable filter 20, a second liquid crystal tunable filter controller 21, an electron multiplying CCD23, an image transmission optical fiber bundle 25, a small microscope objective 29 and a PC upper computer 30; wherein: the laser 1 with the wavelength of 488nm and the laser 2 with the wavelength of 650nm excite two fluorescent coloring agents corresponding to the nanoshell and the medicine, the lasers emitted by the two lasers enter a main light path by using a first reflective mirror 7 and a first dichroic mirror 8, the 488nm laser shutter 3 and the 650nm laser shutter 5 jointly control the existence of the excitation light with only one wavelength in the main light path, and the lasers are divided into two sub-modules of the system by combining a half glass slide 9 and a beam splitter prism 10; the first sub-module monitors the concentration change of the plasma drug from a macroscopic angle, a light transmitting optical fiber bundle 17, a first liquid crystal tunable optical filter 12 and a fluorescence spectrometer 15 are adopted, a first liquid crystal tunable optical filter controller 13 and the fluorescence spectrometer 15 are connected with a PC upper computer 30 through a USB, and the upper computer controls a laser shutter, the liquid crystal tunable optical filter and the fluorescence spectrometer according to a time sequence, so that the fluorescence signals of the drug in main blood vessels of the small animals and the fluorescence signals of a marked nano carrier can be respectively monitored in situ, and the macroscopic metabolism condition and the stability of the drug in blood can be reflected in real time; the second submodule carries out microscopic imaging on fluorescence in the tissue structure from a microscopic angle, an image transmission optical fiber beam 25, a small microscopic objective 29, a second liquid crystal tunable filter 20 and an electron multiplication CCD23 are adopted, a second liquid crystal tunable filter controller 21 and the electron multiplication CCD23 are connected with a PC upper computer 30 through a USB, the upper computer controls a laser shutter, the liquid crystal tunable filter and the electron multiplication CCD according to a time sequence, the PC upper computer 30 adds a pseudo color to two images acquired by the electron multiplication CCD23 in a time-sharing mode and fuses the two images together, so that a nano carrier fluorescence signal and a wrapped medicine fluorescence signal in a specific organ of a small animal can be detected in situ, the conditions of enrichment, distribution, leakage and tissue damage of nano medicines in a metabolic organ are analyzed from a microscopic scale, and the sequential acquisition of the two submodules can be realized through the time sequence control of the PC upper computer.
Preferably, the 488nm laser shutter 3 and 650nm laser shutter 5 are monolithic optical shutters with high sensitivity and response time, and the switch activation time is only 4.08 ms.
Preferably, the fluorescence spectrometer 15 is ocean optics QEPoR and the electron multiplying CCD23 is ANDOR iXon Life.
Preferably, image transfer fiber bundle 25 has 30000 fibers.
Preferably, the light-transmitting fiber bundle 17 comprises a quartz protection zone 31, a stainless steel probe 32, a handle 33, a fiber splitter 34, an incident fiber 35 and a receiving fiber 36; the quartz protection zone 31 is located in front of the stainless steel probe 32, and the stainless steel probe 32 includes an incident optical fiber filament and six receiving optical fiber filaments, and the incident optical fiber filament and the receiving optical fiber filaments are divided into an incident optical fiber 35 and a receiving optical fiber 36 in the optical fiber beam splitter 34.
Correspondingly, the time sequence control method of the dual-wavelength dual-scale nano-drug in-vivo monitoring system comprises the following steps:
(1) injecting nano-drug into the animal body at the beginning of the experiment, and then monitoring the fluorescence signal;
(2) in a sampling period, the plasma drug concentration monitoring submodule works firstly, the 488nm laser shutter 3 is opened, the 650nm laser shutter 5 is closed, the first liquid crystal tunable filter 12 is set to have the passing wavelength of 520nm, and the fluorescence spectrometer 15 works to detect the fluorescence intensity of the nanoshell in the plasma; after the fluorescence intensity detection of the nanoshell in the plasma is completed, the 488nm laser shutter 3 is closed, the 650nm laser shutter 5 is opened, the wavelength of the first liquid crystal tunable filter 12 is set to be 670nm, and the fluorescence spectrometer 15 works to detect the fluorescence intensity of the nano-drug in the plasma;
(3) after the plasma drug concentration monitoring submodule finishes detecting, the tissue structure drug distribution monitoring submodule starts working; firstly, a 488nm laser shutter 3 is closed, a 650nm laser shutter 5 is opened, a second liquid crystal tunable filter 20 is set to have a passing wavelength of 670nm, an electron multiplying CCD23 is used for carrying out microscopic imaging on fluorescence of nano-drugs in a tissue to observe the distribution condition of the fluorescence, then the 488nm laser shutter 3 is opened, the 650nm laser shutter 5 is closed, the second liquid crystal tunable filter 20 is set to have a passing wavelength of 520nm, and an electron multiplying CCD23 is used for carrying out microscopic imaging on the fluorescence of nanoshells in the tissue to observe the distribution condition of the fluorescence; and after the detection of one sampling period is finished, waiting for the next sampling period according to the set sampling frequency.
Preferably, the response time of the liquid crystal tunable filter is less than 40ms, while the response time of a common filter switching wheel is generally greater than 1s, the liquid crystal tunable filter has no mechanical wear.
The invention has the beneficial effects that: the invention can collect the concentration distribution conditions of the nanoshells and the drugs in the blood vessels at all time points in the same animal body in vivo, and carry out microscopic imaging on the nanoshells and the drugs in the tissue so as to observe the distribution conditions in real time, and the pharmacodynamic characteristics of the nanometer drugs are analyzed through the two scales, so that a basis is provided for the clinical use of the nanometer drugs; the sampling points can be replaced at will, and the problem that the traditional tissue slice is complicated to observe is solved; the liquid crystal tunable filter is different from a common filter conversion wheel, the response time of the liquid crystal tunable filter is less than 40ms, while the response time of the common filter conversion wheel is generally more than 1s, so that the liquid crystal tunable filter has no mechanical wear and is more stable in long-time high-load use; the upper computer controls the laser shutter, the liquid crystal tunable filter, the fluorescence spectrometer and the electron multiplication CCD by using a time sequence, and because the response time of the laser shutter and the response time of the liquid crystal tunable filter are extremely short, the problem of collection point drift caused by factors such as tested respiration and the like during the in-vivo collection of fluorescence can be solved, so that the fluorescence data of two different wavelengths are fused together more accurately.
Drawings
FIG. 1 is a schematic diagram of the system of the present invention.
Fig. 2 is a schematic view of a structure of a light-transmitting fiber bundle according to the present invention.
Fig. 3 is an enlarged cross-sectional view of the light-transmitting fiber bundle probe of the present invention.
Fig. 4 is an enlarged cross-sectional view of an incident optical fiber according to the present invention.
FIG. 5 is an enlarged cross-sectional view of a receiver fiber according to the present invention.
FIG. 6 is a schematic diagram of the timing sequence of different scale monitoring of different fluorescence samples according to the present invention.
FIG. 7 is a timing diagram of the operation of the apparatus of the present invention.
Wherein, 1, a laser with the wavelength of 488 nm; 2. a laser with a wavelength of 650 nm; 3. a 388nm laser shutter; 4. a 488nm laser shutter controller; 5. a 650nm laser shutter; 6. a 650nm laser shutter controller; 7. a first reflective mirror; 8. a first dichroic mirror; 9. a half of glass slide; 10. a beam splitter prism; 11. a second dichroic mirror; 12. a first liquid crystal tunable filter; 13. a first liquid crystal tunable filter controller; 14. a first lens; 15. a fluorescence spectrometer; 16. a second lens; 17. a light transmitting fiber bundle; 18. a second reflective mirror; 19. a third dichroic mirror; 20. a second liquid crystal tunable filter; 21. a second liquid crystal tunable filter controller; 22. a third lens; 23. electron multiplying CCD; 24. a fourth lens; 25. an image transmission optical fiber bundle; 26. a fifth lens; 27. a sixth lens; 28. a seventh lens; 29. a small microscope objective; 30. a PC upper computer; 31. a quartz protection zone; 32. a stainless steel probe; 33. a handle; 34. an optical fiber beam splitter; 35. an incident optical fiber; 36. receiving the optical fiber.
Detailed Description
As shown in fig. 1, a dual-wavelength dual-scale in vivo nano-drug monitoring system comprises: a laser 1 with a wavelength of 488nm, a laser 2 with a wavelength of 650nm, a 488nm laser shutter 3, a 488nm laser shutter controller 4, a 650nm laser shutter 5, a 650nm laser shutter controller 6, a first reflective mirror 7, a first dichroic mirror 8, a half glass 9, a beam splitter prism 10, a second dichroic mirror 11, a first liquid crystal tunable filter 12, a first liquid crystal tunable filter controller 13, a first lens 14, a fluorescence spectrometer 15, a second lens 16, a light transmitting fiber bundle 17, a second reflective mirror 18, a third dichroic mirror 19, a second liquid crystal tunable filter 20, a second liquid crystal tunable filter controller 21, a third lens 22, an electron multiplying CCD23, a fourth lens 24, an image transmitting fiber bundle 25, a fifth lens 26, a sixth lens 27, a seventh lens 28, a miniature microscope objective 29 and a PC upper computer 30; wherein: the laser 1 with the wavelength of 488nm and the laser 2 with the wavelength of 650nm excite two fluorescent coloring agents corresponding to the nanoshell and the medicine, the lasers emitted by the two lasers enter a main light path by using a first reflective mirror 7 and a first dichroic mirror 8, the 488nm laser shutter 3 and the 650nm laser shutter 5 jointly control the existence of the excitation light with only one wavelength in the main light path, and the lasers are divided into two sub-modules of the system by combining a half glass slide 9 and a beam splitter prism 10; the first sub-module monitors the concentration change of the plasma drug from a macroscopic angle, a light transmitting optical fiber bundle 17, a first liquid crystal tunable optical filter 12 and a fluorescence spectrometer 15 are adopted, a first liquid crystal tunable optical filter controller 13 and the fluorescence spectrometer 15 are connected with a PC upper computer 30 through a USB, and the upper computer controls a laser shutter, the liquid crystal tunable optical filter and the fluorescence spectrometer according to a time sequence, so that the fluorescence signals of the drug in main blood vessels of the small animals and the fluorescence signals of a marked nano carrier can be respectively monitored in situ, and the macroscopic metabolism condition and the stability of the drug in blood can be reflected in real time; the second submodule carries out microscopic imaging on fluorescence in the tissue structure from a microscopic angle, an image transmission optical fiber beam 25, a small microscopic objective 29, a second liquid crystal tunable filter 20 and an electron multiplication CCD23 are adopted, a second liquid crystal tunable filter controller 21 and the electron multiplication CCD23 are connected with a PC upper computer 30 through a USB, the upper computer controls a laser shutter, the liquid crystal tunable filter and the electron multiplication CCD according to a time sequence, the PC upper computer 30 adds a pseudo color to two images acquired by the electron multiplication CCD23 in a time-sharing mode and fuses the two images together, so that a nano carrier fluorescence signal and a wrapped medicine fluorescence signal in a specific organ of a small animal can be detected in situ, the conditions of enrichment, distribution, leakage and tissue damage of nano medicines in a metabolic organ are analyzed from a microscopic scale, and the sequential acquisition of the two submodules can be realized through the time sequence control of the PC upper computer.
The laser 1 with the wavelength of 488nm and the laser 2 with the wavelength of 650nm are provided with a 488nm laser shutter 3 and a 650nm laser shutter 5 in front, which are respectively connected with a controller 488nm laser shutter controller 4 and a 650nm laser shutter controller 6, the two laser shutter controllers are connected with a PC upper computer 30 through USB, the PC upper computer 30 controls the opening and closing of the laser shutter according to time sequence, when the 488nm laser shutter 3 is opened, the 650nm laser shutter 5 is in a closed state (or when the 650nm laser shutter 5 is opened, the 488nm laser shutter 3 is in a closed state), at this time, only one path of exciting light 488nm (or 650nm) exists in the light path. The half slide 9 and the spectroscope 10 are matched to use to distribute laser into the plasma drug concentration monitoring submodule and the tissue structure drug distribution monitoring submodule according to any proportion.
In the plasma drug concentration monitoring submodule, excitation light is split and then first reaches the second dichroic mirror 11, and then is coupled into the light transmission optical fiber bundle 17 through the second lens 16, the excitation light is used for exciting a fluorescence sample in plasma after passing through the light transmission optical fiber bundle 17, a generated fluorescence signal returns to the second dichroic mirror 11 along an original optical path after passing through a collection port of the light transmission optical fiber bundle 17, the second dichroic mirror 11 is mainly used for separating the excitation light from the fluorescence signal, the separated fluorescence signal is reflected to a detection optical path, non-signal light is filtered through the first liquid crystal tunable optical filter 12, and finally the fluorescence signal is focused into the fluorescence spectrometer 15 through the first lens 14, the fluorescence spectrum 15 transmits the collected signal to the PC upper computer 30 through the USB, and effective detection of fluorescence spectrum intensity is finally realized. The first liquid crystal tunable filter 12 is connected with the first liquid crystal tunable filter controller 13, the first liquid crystal tunable filter controller 13 is connected with the PC upper computer 30 through a USB, and the PC upper computer 30 controls the bandpass wavelength and the bandwidth of the first liquid crystal tunable filter 12 according to the opening and closing conditions of the 488nm laser shutter 3 and the 650nm laser shutter 5 and the type of the fluorescent sample according to the time sequence.
In the tissue structure drug distribution monitoring submodule, the excitation light comes from a laser 1 with the wavelength of 488nm or a laser emitted by a laser with the wavelength of 650nm and is split by a half glass 9 and a spectroscope 10 to the part which does not enter the plasma drug concentration monitoring submodule. The laser light enters a fourth lens 24 after passing through a dichroic plate third dichroic mirror 19, wherein the fourth transmission 24 is used for coupling excitation light into an image transmission optical fiber bundle 25 (containing 30000 optical fibers), then adjusting the light beam passing through the image transmission optical fiber bundle 25 through a fifth lens 26, a sixth lens 27 and a seventh lens 28, so that the light beam just fills the pupil of a small microscope objective 29, the performance of the objective is fully exerted, and finally tissue excitation fluorescence is reached, fluorescent signals are collected by the objective and then return to the third dichroic mirror 19 along the original light path, are reflected to a detection light path, are filtered by a second liquid crystal tunable filter 20 and adjusted by a third lens 22 and then enter an electron multiplying CCD23, the electron multiplying CCD23 is connected with a PC upper computer 30 by using USB3.0, and the processing such as image false color adding and fusion is carried out in the PC upper computer 30, so as to carry out microscopic imaging. The same second liquid crystal tunable filter 20 is connected to a second liquid crystal tunable filter controller 21, the second liquid crystal tunable filter controller 21 is connected to a PC upper computer 30 through a USB, and the PC upper computer 30 controls the bandpass wavelength and the bandwidth of the second liquid crystal tunable filter 20 according to the opening and closing conditions of the 488nm laser shutter 3 and the 650nm laser shutter 5 and the type of the fluorescent sample according to the time sequence.
The invention adopts the single-chip optical shutter, the liquid crystal tunable filter and the specific time sequence control method to rapidly convert the detection of different fluorescent samples with different scales, thereby ensuring that the acquisition points and the acquisition time of the different fluorescent samples are almost the same and providing the guarantee for the correct processing of the fluorescent data.
The invention adopts a spectrometer with small volume, high sensitivity and high signal-to-noise ratio, and is provided with a refrigeration type detector to realize low-light detection and reduce spectral errors. Is very suitable for the application of low-light scene such as fluorescence detection.
The image processing and analyzing module can realize data processing and analysis while acquiring data, and analyze an experiment result so as to improve the experiment operation process in time according to requirements.
As shown in fig. 2, the light-transmitting optical fiber bundle 17 used in the plasma drug concentration monitoring submodule designed for the present invention is composed of a quartz protection area 31, a stainless steel probe 32, a handle 33, an optical fiber beam splitter 34, an incident optical fiber 35 and a receiving optical fiber 36. The thickness of the quartz protective region 31 is 3 mm, which can increase the overlap of the incident and exit regions and improve the excitation and collection efficiency of fluorescence.
As shown in FIG. 3, the cross section of the probe of the present invention is a section which needs to detect the fluorescence intensity in the blood vessel due to the special use scenario of the plasma drug concentration monitoring submodule, and the probe is thin enough. The diameter of the optical fiber filament in the probe is 0.2 mm, and by using the arrangement mode shown in fig. 3, the middle optical fiber is an incident optical fiber, and the peripheral six optical fibers are receiving optical fibers.
The probe designed by the invention can improve the excitation and collection efficiency of fluorescence while meeting the requirement that the probe can be aligned with a blood vessel.
As shown in fig. 4, which is a cross section of the incident optical fiber 35 of the present invention, the excitation light is focused on the cross section of the optical fiber by the second lens 16, and the excitation light is coupled into the incident optical fiber 35.
As shown in fig. 5, for the cross section of the receiving fiber 36 of the present invention, since the spectrometer is used for detecting through the slit, a special arrangement of six receiving fibers is designed, and when the receiving fiber is installed, the receiving fiber is properly adjusted to allow more fluorescence to pass through the slit, so as to improve the detection sensitivity of the fluorescence spectrometer 15.
As shown in FIG. 6, for different scale monitoring sequences of different fluorescence samples designed by the present invention, the injection of the nano-drug into the animal at the beginning of the experiment and then the monitoring of the fluorescence signal are started. In a sampling period, firstly, the plasma drug concentration monitoring submodule is used for detecting a fluorescence sample in plasma, firstly, the fluorescence intensity of the nanoshell is detected, and then, the fluorescence intensity of the nano drug is detected. After the plasma drug concentration monitoring submodule detects, the tissue structure drug distribution monitoring submodule starts to work. Because the 488nm laser shutter 3 and the 650nm laser shutter 5 are mechanical structures, in order to reduce the abrasion, the distribution of the fluorescence of the nano-drug in the tissue is observed by microscopic imaging firstly, and then the distribution of the fluorescence of the nano-shell in the tissue is observed by microscopic imaging. And after the detection of one sampling period is finished, waiting for the next sampling period according to the set sampling frequency.
As shown in fig. 7, the device action control timing sequence is designed for the different scale monitoring timing sequences of the different fluorescence samples. The laser 1 with wavelength of 488nm and the laser 2 with wavelength of 650nm are connected to a 220V power supply and are kept normally open. The 488nm laser shutter 3 and the 650nm laser shutter 5 are connected with the PC upper computer 30 through respective controllers, and the opening and closing of the shutters are controlled by the PC upper computer 30. The corresponding rectangle in the figure indicates that it is open, otherwise it is closed. The first liquid crystal tunable filter 12 and the second liquid crystal tunable filter 20 are connected to a PC upper computer 30 through respective controllers, the PC upper computer 30 controls the band-pass wavelength and the bandwidth thereof, the corresponding rectangle in the figure represents the fluorescence emitted by the nanoshell, and the corresponding triangle represents the fluorescence emitted by the nanopharmaceutical. The fluorescence spectrometer 15 is connected to the PC upper computer 30 via USB, and the PC upper computer 30 can control the start of collection and the collection time (integration time), where the corresponding rectangle in the figure indicates the start of collection and the width indicates the collection time. In one acquisition, the acquisition time of the fluorescence spectrometer 15 controls the opening time of the laser shutter and controls the time for the liquid crystal tunable filter to pass through the corresponding fluorescence. The electron multiplying CCD23 is connected to the PC upper computer 30 via USB, and the start of acquisition and the acquisition time (integration time) of the electron multiplying CCD23 can be controlled by the PC upper computer 30, and the start of acquisition is indicated by the corresponding rectangle in the figure and the acquisition time is indicated by the width. In one acquisition, the acquisition time of the electron multiplying CCD23 controls the opening time of the laser shutter and controls the time of the liquid crystal tunable filter passing through the corresponding fluorescence. And after the detection of 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 invention ensures the durability of the system, simultaneously leads the two sub-modules to detect different fluorescence samples in order, provides guarantee for the correct processing of later fluorescence data, is the control core of the system and is the basis of the efficacy analysis of nano-drugs.
Claims (7)
1. A dual-wavelength dual-scale in-vivo nano-drug monitoring system is characterized by comprising: the device comprises a laser (1) with the wavelength of 488nm, a laser (2) with the wavelength of 650nm, a 488nm laser shutter (3), a 650nm laser shutter (5), a first reflector (7), a first dichroic mirror (8), a half glass (9), a beam splitter prism (10), a first liquid crystal tunable filter (12), a first liquid crystal tunable filter controller (13), a fluorescence spectrometer (15), a light transmission optical fiber bundle (17), a second liquid crystal tunable filter (20), a second liquid crystal tunable filter controller (21), an electron multiplication CCD (23), an image transmission optical fiber bundle (25), a small microscope objective (29) and a PC upper computer (30); wherein: a laser (1) with the wavelength of 488nm and a laser (2) with the wavelength of 650nm excite two fluorescent coloring agents corresponding to the nano-shell and the medicine, a first reflector (7) and a first dichroic mirror (8) are used for enabling laser emitted by the two lasers to enter a main light path, a 488nm laser shutter (3) and a 650nm laser shutter (5) jointly control the existence of the excitation light with only one wavelength in the main light path, and the laser is divided into two sub-modules of the system through the combined use of a half glass (9) and a beam splitter prism (10); the first sub-module monitors the concentration change of plasma drugs from a macroscopic angle, a light transmitting optical fiber bundle (17), a first liquid crystal tunable optical filter (12) and a fluorescence spectrometer (15) are adopted, a first liquid crystal tunable optical filter controller (13) and the fluorescence spectrometer (15) are connected with a PC (personal computer) upper computer (30) through a USB (universal serial bus), and the upper computer controls a laser shutter, the liquid crystal tunable optical filter and the fluorescence spectrometer according to a time sequence, so that the fluorescence signals of drugs in main blood vessels of small animals and the fluorescence signals of marked nano carriers can be respectively monitored in situ, and the macroscopic metabolic condition and the stability of the drugs in blood can be reflected in real time; the second submodule carries out microscopic imaging on the fluorescence in the tissue structure from a microscopic angle, an image transmission optical fiber beam (25), a small-sized microscope objective (29), a second liquid crystal tunable optical filter (20) and an electron multiplication CCD (23) are adopted, a second liquid crystal tunable optical filter controller (21) and the electron multiplication CCD (23) are connected with a PC upper computer (30) through a USB, the upper computer controls a laser shutter, the liquid crystal tunable optical filter and the electron multiplication CCD according to a time sequence, the PC upper computer (30) adds a false color to two images acquired by the electron multiplication CCD (23) in a time-sharing manner and fuses the two images together, so that a nano carrier fluorescence signal and a wrapped drug fluorescence signal in a specific organ of a small animal can be detected in situ, and the conditions of enrichment, distribution, leakage and tissue damage of nano drugs in a metabolic organ are analyzed from a microscopic scale, sequential collection of the two sub-modules can be realized through sequential control of the PC upper computer.
2. The dual wavelength dual scale nano-drug in vivo monitoring system of claim 1, wherein the 488nm laser shutter (3) and the 650nm laser shutter (5) are monolithic optical shutters with high sensitivity and response time, and the switch activation time is only 4.08 ms.
3. The dual wavelength dual scale nano-drug in vivo monitoring system of claim 1, wherein the fluorescence spectrometer (15) is marine optical qerpro and the electron multiplying CCD (23) is ANDOR iXon Life.
4. The dual wavelength dual scale nano-drug in vivo monitoring system of claim 1, wherein the image transmitting fiber bundle (25) has 30000 fibers.
5. The dual-wavelength dual-scale nano-drug in-vivo monitoring system of claim 1, wherein the light-transmitting fiber bundle (17) comprises a quartz protection zone (31), a stainless steel probe (32), a handle (33), a fiber splitter (34), an incident fiber (35) and a receiving fiber (36); the quartz protection area (31) is arranged in front of the stainless steel probe (32), the stainless steel probe (32) comprises an incident optical fiber filament and six receiving optical fiber filaments, and the incident optical fiber filament and the receiving optical fiber filaments are divided into an incident optical fiber (35) and a receiving optical fiber (36) in the optical fiber beam splitter (34).
6. The timing control method of the dual-wavelength dual-scale nano-drug in-vivo monitoring system according to claim 1, comprising the steps of:
(1) injecting nano-drug into the animal body at the beginning of the experiment, and then monitoring the fluorescence signal;
(2) in a sampling period, the plasma drug concentration monitoring submodule works firstly, a 488nm laser shutter (3) is opened, a 650nm laser shutter (5) is closed, the first liquid crystal tunable filter (12) is set to have the passing wavelength of 520nm, and a fluorescence spectrometer (15) works to detect the fluorescence intensity of a nanoshell in plasma; after the fluorescence intensity detection of the nanoshell in the plasma is completed, the 488nm laser shutter (3) is closed, the 650nm laser shutter (5) is opened, the wavelength of the first liquid crystal tunable filter (12) is set to be 670nm, and the fluorescence spectrometer (15) works to detect the fluorescence intensity of the nano-drug in the plasma;
(3) after the plasma drug concentration monitoring submodule finishes detecting, the tissue structure drug distribution monitoring submodule starts working; firstly, a 488nm laser shutter (3) is closed, a 650nm laser shutter (5) is opened, a second liquid crystal tunable filter (20) is set to have a passing wavelength of 670nm, an electron multiplying CCD (23) is used for carrying out microscopic imaging on fluorescence of nano-drugs in a tissue to observe the distribution condition of the fluorescence, then the 488nm laser shutter (3) is opened, the 650nm laser shutter (5) is closed, the second liquid crystal tunable filter (20) is set to have a passing wavelength of 520nm, and the electron multiplying CCD (23) is used for carrying out microscopic imaging on the fluorescence of nanoshells in the tissue to observe the distribution condition of the fluorescence; and after the detection of one sampling period is finished, waiting for the next sampling period according to the set sampling frequency.
7. The timing control method of claim 6, wherein the liquid crystal tunable filter response time is less than 40 ms.
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