CN110464309B - Cross-scale fluorescence endoscopic imaging system - Google Patents
Cross-scale fluorescence endoscopic imaging system Download PDFInfo
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- CN110464309B CN110464309B CN201910794851.0A CN201910794851A CN110464309B CN 110464309 B CN110464309 B CN 110464309B CN 201910794851 A CN201910794851 A CN 201910794851A CN 110464309 B CN110464309 B CN 110464309B
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Abstract
The invention discloses a trans-scale fluorescence endoscopic imaging system, which comprises: an illumination unit and a microscopic imaging unit; the illumination unit is used for emitting first excitation light and/or second excitation light and transmitting fluorescence generated by the first excitation light and/or the second excitation light to the macroscopic spectrum analysis unit, the mesoscopic imaging unit and the microscopic high-resolution imaging unit; the macroscopic spectrum analysis unit is used for acquiring a first fluorescence signal and generating a fluorescence spectrogram according to the first fluorescence signal; the mesoscopic imaging unit is used for acquiring the second fluorescent signal and generating a mesoscopic distribution image according to the second fluorescent signal; the microscopic high-resolution imaging unit is used for acquiring a third fluorescence signal and generating a nano-drug fluorescence image, a nano-shell fluorescence image and a cell membrane distribution map according to the third fluorescence signal; the system realizes monitoring of drug transport in cells through a nano-drug fluorescence map, a nano-shell fluorescence map and a cell membrane distribution map, and realizes research of cell pharmacokinetic characteristics in deep tissues of living animals.
Description
Technical Field
The invention relates to the technical field of optical microscopic imaging in medicine, in particular to a cross-scale fluorescence endoscopic imaging system.
Background
With the development of medical technology, optical microscopic imaging is widely applied to medical observation, examination, detection and other projects; existing medical imaging techniques include: various endoscopic systems, such as fiber optic endoscopes, electronic endoscopes, ultrasonic endoscopes, and the like, and various optical microscopy techniques, such as laser focused microscopy, two-photon microscopy, and the like.
However, for the fundamental study of the intrinsic kinetics of the nano-drug in clinical precursor, not only the drug transport property in the target cell but also the animal distribution of the nano-drug, the dynamic distribution property in the blood vessel, the accumulation and damage of the major metabolic organs (liver, kidney, etc.), and the dynamic distribution property of the target tissue of the target organ are revealed. Although various in vivo imaging systems (CT \ US \ MRI \ PET \ SPECT, etc.) can show that the nano-drug is more accumulated at the diseased tissue/organ, it still cannot be observed whether the nano-drug enters into the target cell to act or is pumped out by the target cell, and the damage condition of the main metabolic organ cannot be evaluated.
In the prior art, various endoscopic systems can identify the tissue damage characteristics of the cavity surface, but the resolution ratio cannot meet the requirement of monitoring intracellular drugs; and various optical microscopy techniques have a function of detecting drug transport in cells with high resolution, but cannot realize the study of the cell pharmacokinetic characteristics in deep tissues of living animals. Summarizing the application range and spatial resolution of various imaging techniques available, it is clear that there is a lack of a system that can achieve simultaneous imaging of whole, organs, tissues and cells in vivo, which is required for the development of optical imaging techniques in medicine.
Therefore, there is a need for a trans-scale endoscopic imaging system.
Disclosure of Invention
The application provides a cross-scale fluorescence endoscopic imaging system, which can monitor the drug transfer in cells and solve the technical problem that the optical imaging technology in the prior art can not realize the research on the cell pharmacokinetic characteristics in deep tissues of living animals.
The invention provides a trans-scale fluorescence endoscopic imaging system, which comprises: an illumination unit and a microscopic imaging unit;
wherein the microscopic imaging unit comprises: the device comprises a macroscopic spectrum analysis unit, a mesoscopic imaging unit and a microscopic high-resolution imaging unit;
the illumination unit is used for emitting first excitation light and/or second excitation light and transmitting the first excitation light and/or the second excitation light to the macroscopic spectrum analysis unit, the mesoscopic imaging unit and the microscopic high-resolution imaging unit respectively;
the macroscopic spectrum analysis unit is used for acquiring a first fluorescence signal generated by exciting a fluorescence sample by the incident first excitation light or the incident second excitation light and generating a fluorescence spectrum of the fluorescence sample according to the first fluorescence signal;
the mesoscopic imaging unit is used for acquiring a second fluorescence signal generated by exciting the fluorescence sample by the incident first excitation light or the second excitation light based on the fluorescence spectrogram and acquiring a mesoscopic distribution image of the fluorescence sample according to the second fluorescence signal;
the microscopic high-resolution imaging unit is used for acquiring a third fluorescence signal of the fluorescence sample excited by the first excitation light and the second excitation light based on the mesoscopic distribution image, and performing high-resolution imaging according to the third fluorescence signal to obtain a nano-drug fluorescence image, a nano-shell fluorescence image and a cell membrane distribution image for observing the fluorescence sample in real time.
Optionally, the lighting unit comprises: a light source assembly and a main light path system;
the light source assembly includes: a first semiconductor continuous laser and a second semiconductor continuous laser;
the main light path system includes: the device comprises a first reflective mirror, a first dichroic mirror, a first adjusting glass sheet, a first light splitting prism, a second adjusting glass sheet, a second light splitting prism and a second reflective mirror;
the first exciting light emitted by the first semiconductor continuous laser sequentially passes through the first dichroic mirror, the first adjusting glass sheet, the first light splitting prism, the second adjusting glass sheet, the second light splitting prism and the second reflector;
the second excitation light emitted by the second semiconductor continuous laser sequentially passes through the first reflector, the first dichroic mirror, the first adjusting glass slide, the first light splitting prism, the second adjusting glass slide, the second light splitting prism and the second reflector;
the first light splitting prism is used for transmitting the first exciting light or the second exciting light to the macroscopic spectrum analysis unit in a light splitting mode, and transmitting the first exciting light or the second exciting light to the second adjusting glass slide in sequence;
the second light splitting prism is used for transmitting the first exciting light or the second exciting light to the mesoscopic imaging unit in a split mode and transmitting the first exciting light or the second exciting light to the second reflector;
wherein the second mirror is configured to transmit the first excitation light and the second excitation light to the microscopic high-resolution imaging unit.
Optionally, the light source assembly further comprises: a first electric light barrier and a second electric light barrier;
the first electric light barrier is positioned between the first semiconductor continuous laser and the first dichroic mirror;
the second electric light barrier is located between the second semiconductor continuous laser and the first mirror.
Optionally, the macro-spectrum analysis unit includes: the device comprises a second dichroic mirror, a first lens, a first optical fiber bundle, a first filter, a second lens and a fluorescence spectrometer;
the first dichroic mirror is used for transmitting the first excitation light or the second excitation light to the second dichroic mirror;
the second dichroic mirror is used for transmitting the first excitation light or the second excitation light to the first lens;
the first lens is used for coupling the first excitation light or the second excitation light with preset wavelength into the first optical fiber bundle;
the first optical fiber bundle is used for transmitting the first excitation light or the second excitation light to a fluorescent sample, collecting a first fluorescent signal obtained after the fluorescent sample is excited by the first excitation light or the second excitation light through a port of the first optical fiber bundle, and sequentially transmitting the collected first fluorescent signal to the first optical fiber bundle, the first lens and the second dichroic mirror;
the second dichroic mirror is used for separating the fluorescent signal from the first exciting light or the second exciting light and transmitting the first fluorescent signal to the first filter sheet in a total reflection manner;
the first filter is used for filtering the first fluorescent signal and transmitting the filtered first fluorescent signal to the second lens;
the second lens is used for transmitting the filtered first fluorescence signal to the fluorescence spectrometer to obtain a fluorescence spectrogram.
Optionally, the mesoscopic imaging unit includes: the third dichroic mirror, a third lens, a second optical fiber bundle, a micro-lens component, a first objective lens, a second filter, a fourth lens and a multiplication imaging device;
the second light splitting prism is used for sequentially transmitting the first excitation light or the second excitation light to the third dichroic mirror and the third lens;
the third lens is used for coupling the first excitation light or the second excitation light with preset wavelength into the second optical fiber bundle;
the second optical fiber bundle is used for transmitting the first excitation light or the second excitation light to the micro-lens component;
the micro-lens assembly is used for adjusting the beam size of the first excitation light or the second excitation light and transmitting the first excitation light or the second excitation light of a proper beam to the first objective lens;
the first objective lens is used for transmitting the first exciting light or the second exciting light to a fluorescent sample, collecting a second fluorescent signal obtained after the fluorescent sample is excited by the first exciting light or the second exciting light, and sequentially transmitting the collected second fluorescent signal to the micro-lens component, the second optical fiber bundle, the second lens and the third dichroic mirror;
the third dichroic mirror is used for separating the second fluorescent signal from the first exciting light or the second exciting light and transmitting the second fluorescent signal to the second filter plate in a total reflection manner;
the second filter is used for filtering the second fluorescent signal and transmitting the filtered second fluorescent signal to the fourth lens;
and the fourth lens transmits the filtered second fluorescence signal to the multiplication imaging device to obtain a mesoscopic distribution image.
Optionally, the micro-lens assembly comprises: a fifth lens element, a sixth lens element, and a seventh lens element;
the first excitation light or the second excitation light sequentially passes through the fifth lens, the sixth lens and the seventh lens so as to adjust the beam size of the first excitation light or the second excitation light.
Optionally, the microscopic high-resolution imaging unit includes: the system comprises a fourth dichroic mirror, a fifth dichroic mirror, a sixth dichroic mirror, a vibrating mirror, a 4F system, a second objective mirror, a third optical fiber bundle, an eighth lens, a self-focusing lens, a third filter, a ninth lens, a first multimode optical fiber, a first photomultiplier, a fourth filter, a tenth lens, a second multimode optical fiber, a second photomultiplier, a fifth filter, an eleventh lens, a third multimode optical fiber and a third photomultiplier;
the third dichroic prism is used for simultaneously and sequentially transmitting the first excitation light and the second excitation light to the fourth dichroic mirror, the fifth dichroic mirror, the sixth dichroic mirror and the vibrating mirror;
the galvanometer is used for carrying out light beam scanning on the first excitation light and the second excitation light to obtain scanning light beams and transmitting the scanning light beams to the 4F system;
the 4F system is used for adjusting the beam size of the scanning beam and transmitting the adjusted scanning beam to the second objective lens;
the second objective lens is used for coupling the scanning light beam into the third optical fiber bundle;
the third optical fiber bundle is used for transmitting the scanning light beam to the eighth lens;
the eighth lens is used for coupling the scanning light beam into the self-focusing lens;
the self-focusing lens is used for transmitting the scanning light beam to a fluorescent sample, collecting a third fluorescent signal obtained after the scanning light beam excites the fluorescent sample, and transmitting the third fluorescent signal to the eighth lens, the third optical fiber bundle, the second objective lens, the 4F system, the vibrating mirror, the sixth dichroic mirror, the fifth dichroic mirror and the fourth dichroic mirror in sequence in a reverse direction;
the fourth dichroic mirror is used for separating the third fluorescent signal to obtain a fourth fluorescent signal, and totally reflecting the fourth fluorescent signal and sequentially transmitting the fourth fluorescent signal to the third filter, the ninth lens, the first multimode optical fiber and the first photomultiplier tube to obtain a cell membrane distribution map;
the fifth dichroic mirror is used for separating the third fluorescent signal to obtain a fifth fluorescent signal, and totally reflecting the fifth fluorescent signal and sequentially transmitting the fifth fluorescent signal to the fourth filter, the tenth lens, the second multimode optical fiber and the second photomultiplier tube to obtain a nano-drug distribution map;
the sixth dichroic mirror is used for separating the third fluorescent signal to obtain a sixth fluorescent signal, and totally reflecting the sixth fluorescent signal and sequentially transmitting the sixth fluorescent signal to the fifth filter, the eleventh lens, the third multimode optical fiber and the third photomultiplier tube to obtain a nano-drug distribution map.
Optionally, the 4F system includes: a twelfth lens and a thirteenth lens;
the first excitation light and the second excitation light are transmitted from the twelfth lens to the thirteenth lens to adjust the beam size of the first excitation light and the second excitation light.
The invention provides a trans-scale fluorescence endoscopic imaging system, which comprises: an illumination unit and a microscopic imaging unit; wherein the microscopic imaging unit comprises: the device comprises a macroscopic spectrum analysis unit, a mesoscopic imaging unit and a microscopic high-resolution imaging unit; the illumination unit is used for emitting first excitation light and/or second excitation light and transmitting the first excitation light and/or the second excitation light to the macroscopic spectrum analysis unit, the mesoscopic imaging unit and the microscopic high-resolution imaging unit; the macroscopic spectrum analysis unit is used for acquiring a first fluorescence signal generated by exciting the nanoshell and the nano-drug by the incident first excitation light or the incident second excitation light, and generating a fluorescence spectrogram of the nanoshell and the nano-drug according to the first fluorescence signal; the mesoscopic imaging unit is used for acquiring a second fluorescence signal generated by exciting the nanoshell and the nano-drug by the incident first excitation light or the incident second excitation light based on the fluorescence spectrogram and acquiring a mesoscopic distribution image of the nanoshell and the nano-drug according to the second fluorescence signal; the microscopic high-resolution imaging unit is used for acquiring a third fluorescence signal of the incident first excitation light and the incident second excitation light for exciting the nanoshell and the nano-drug based on the mesoscopic distribution image, and performing high-resolution imaging according to the third fluorescence signal to obtain a nano-drug fluorescence image, a nanoshell fluorescence image and a cell membrane distribution map for observing transmembrane, leakage and controlled release of the nano-drug in real time; the system realizes monitoring of drug transport in cells through a nano-drug fluorescence map, a nano-shell fluorescence map and a cell membrane distribution map, and realizes research of cell pharmacokinetic characteristics in deep tissues of living animals.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a cross-scale fluoroscopic endoscopic imaging system architecture diagram provided by the present invention;
fig. 2 is a schematic structural diagram of a lighting unit according to an embodiment of the present invention;
FIG. 3 is a schematic structural diagram of a macro-spectrum analysis unit provided in an embodiment of the present invention;
fig. 4 is a schematic structural diagram of a mesoscopic imaging unit provided in an embodiment of the invention;
FIG. 5 is a schematic structural diagram of a microscopic high-resolution imaging unit provided in an embodiment of the present application;
fig. 6 is an overall structural diagram of a trans-scale fluorescence endoscopic imaging system provided by the present invention.
Detailed Description
In order to make the objects, features and advantages of the present invention more obvious and understandable, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the prior art, various endoscopic systems can identify the tissue damage characteristics of the wall surface, but the resolution cannot meet the requirement of monitoring intracellular drugs; and various optical microscopic techniques have the function of detecting the drug transport in high-resolution cells, but cannot realize the research on the cell pharmacokinetic characteristics in deep tissues of living animals;
in order to solve the defects of the prior art, the invention provides a cross-scale fluorescence endoscopic imaging system, which can monitor the drug transport in cells and solve the technical problem that the optical imaging technology in the prior art cannot realize the research on the cell pharmacokinetic characteristics in the deep tissues of living animals.
Referring to fig. 1 and 6, fig. 1 is an architectural diagram of a cross-scale fluorescence endoscopic imaging system according to the present invention, and fig. 6 is an overall structural diagram of a cross-scale fluorescence endoscopic imaging system according to the present invention;
the system comprises: an illumination unit 100 and a microscopic imaging unit 200;
wherein the microscopic imaging unit 200 includes: a macroscopic spectrum analysis unit 201, a mesoscopic imaging unit 202 and a microscopic high-resolution imaging unit 203;
the illumination unit 100 is used for emitting first excitation light and/or second excitation light, and transmitting the first excitation light and/or the second excitation light to the macro spectrum analysis unit 201, the mesoscopic imaging unit 202 and the microscopic high-resolution imaging unit 203 respectively;
the macro-spectrum analysis unit 201 is configured to obtain a first fluorescence signal generated by exciting the fluorescent sample with the incident first excitation light or the incident second excitation light, and generate a fluorescence spectrum of the fluorescent sample according to the first fluorescence signal;
the mesoscopic imaging unit 202 is configured to obtain a second fluorescence signal generated by exciting the fluorescent sample with the incident first excitation light or the incident second excitation light based on the fluorescence spectrogram, and obtain a mesoscopic distribution image of the fluorescent sample according to the second fluorescence signal;
the microscopic high-resolution imaging unit 203 is configured to obtain a third fluorescence signal of the fluorescence sample excited by the incident first excitation light and the second excitation light based on the mesoscopic distribution image, and perform high-resolution imaging according to the third fluorescence signal to obtain a nano-drug fluorescence image, a nano-shell fluorescence image, and a cell membrane distribution map for observing the fluorescence sample in real time.
In an embodiment of the invention, the imaging system comprises: an illumination unit 100, a macroscopic spectrum analysis unit 201, a mesoscopic imaging unit 202 and a microscopic high-resolution imaging unit 203; the illumination unit 100 is used for realizing functions of light splitting, light intensity adjustment, wavelength selection and the like, a semiconductor continuous laser can be directly adopted as a light source of the illumination unit 100, the wavelength of laser (exciting light) emitted by the laser is 488 nm/nanometer and 650 nm/nanometer, the light intensity of the laser (exciting light) is adjusted at will through an adjusting glass slide in a preset path, and the laser (exciting light) is subjected to light splitting treatment through a light splitting prism in the preset path, so that the laser/exciting light respectively enters the macroscopic spectral analysis unit 201, the mesoscopic imaging unit 202 and the microscopic high-resolution imaging unit 203 according to the preset path;
further, the fluorescent sample comprises: the fluorescent dye composition comprises a nanoshell, a nano-drug, a cell membrane, a first fluorescent dye targeting the nanoshell, a second fluorescent dye targeting the nano-drug and a third fluorescent dye targeting the cell membrane;
in the embodiment, the 488nm wavelength first excitation light is mainly used for exciting the first fluorescent dye FITC targeting the nanoshell, and the first fluorescent dye emits fluorescent light with the wavelength of about 520nm after being excited, so that the distribution of the nanoshell in the tissue can be observed in imaging; the 488nm wavelength first excitation light is also used for exciting a third fluorescent dye DIO targeting cell membranes or a third fluorescent dye self-made by laboratories, and the wavelength of fluorescence emitted by the excited third fluorescent dye is about 500nm, so that effective marking of the cell membranes is realized; the second excitation light with the wavelength of 650nm is mainly used for exciting a Cy5 series second fluorescent dye targeting the nano-drug, and the emitted fluorescent wavelength of the excited second fluorescent dye is about 670nm, so that the aim of tracking the nano-drug is fulfilled; it should be noted that the excitation light needs to enter the macro-spectral analysis unit 201, the meso-imaging unit 202, and the micro high-resolution imaging unit 203 to excite the fluorescent dyes, specifically, the excitation light with a wavelength of 488nm/650nm enters the macro-spectral analysis unit 201, and the corresponding fluorescent dyes are excited to collect the fluorescent spectrogram; selecting a target position to be monitored according to the fluorescence spectrogram, and exciting corresponding fluorescent dye after excitation light with the wavelength of 488nm/650nm enters the mesoscopic imaging unit 202 to obtain a mesoscopic distribution image; exciting light with wavelengths of 488nm and 650nm enters the microscopic high-resolution imaging unit 203 at the same time, and exciting the fluorescent dye according to the mesoscopic distribution map to realize high-resolution imaging so as to obtain a nano-drug fluorescence map, a nano-shell fluorescence map and a cell membrane distribution map for observing transmembrane of the nano-drug, release of the nano-drug and controlled release in real time;
in this embodiment, the basic research of the internal kinetics of the nano-drug clinical precursor can be satisfied according to the fluorescence diagram, the fluorescence diagram of the nanoshell and the cell membrane distribution diagram of the nano-drug, and the animal overall distribution, the intravascular dynamic distribution, the aggregation and damage of the main metabolic organs and the dynamic distribution of the target tissues of the target organs of the nano-drug are revealed by examining the drug transport characteristics in the target cells, so as to observe whether the nano-drug enters the target cells to act or is pumped out by the target cells, and evaluate the damage condition of the main metabolic organs.
Referring to fig. 2, fig. 2 is a schematic view of a lighting unit structure according to an embodiment of the present invention; the lighting unit 100 includes: a light source assembly and a main light path system;
the light source assembly includes: a first semiconductor continuous laser1 and a second semiconductor continuous laser 2;
the main light path system includes: a first reflecting mirror 5, a first dichroic mirror 6, a first adjusting glass sheet 7, a first light splitting prism 8, a second adjusting glass sheet 9, a second light splitting prism 10 and a second reflecting mirror 11;
a first exciting light emitted by a first semiconductor continuous laser1 passes through a first dichroic mirror 6, a first adjusting glass slide 7, a first light splitting prism 8, a second adjusting glass slide 9, a second light splitting prism 10 and a second reflecting mirror 11 in sequence;
the second exciting light emitted by the second semiconductor continuous laser2 passes through a first reflector 5, a first dichroic mirror 6, a first adjusting glass sheet 7, a first light splitting prism 8, a second adjusting glass sheet 9, a second light splitting prism 10 and a second reflector 11 in sequence;
the first light splitting prism 8 is used for transmitting the first excitation light or the second excitation light to the macro-spectrum analysis unit 201 in a light splitting manner, and transmitting the first excitation light or the second excitation light to the second adjusting slide 9 in sequence;
the second beam splitter prism 10 is configured to split and transmit the first excitation light or the second excitation light to the mesoscopic imaging unit 202, and transmit the first excitation light or the second excitation light to the second reflective mirror 11;
wherein the second mirror 11 is used for transmitting the first excitation light and the second excitation light to the microscopic high resolution imaging unit 203.
In the embodiment of the present invention, the illumination unit 100 is used for realizing functions of light splitting, light intensity adjustment, wavelength selection, and the like, and a light source of the illumination unit 100 may directly adopt a semiconductor continuous laser, wherein a wavelength of first laser light (first excitation light) emitted by the first semiconductor continuous laser1 is 488 nm/nm, and a wavelength of second laser light (second excitation light) emitted by the second semiconductor continuous laser2 is 650 nm/nm; the first excitation light and the second excitation light emitted by the first semiconductor continuous laser1 and the second semiconductor continuous laser2 enter the main optical path system according to a preset optical transmission path to realize the light splitting and light intensity adjustment treatment of the emitted first excitation light and the emitted second excitation light, in the embodiment, the light splitting and light intensity adjustment treatment of the emitted laser (excitation light) can be realized by using a combination of a first reflective mirror 5, a first dichroic mirror 6, a first adjusting glass 7, a second adjusting glass 9, a first dichroic prism 8, a second dichroic prism 10, a second reflective mirror 11 and other components, wherein the combination of the reflective mirror and the dichroic mirror can be installed and set according to a preset installation setting scheme, the installation and setting mainly aim at the position and the direction angle of the first reflective mirror 5 and the first dichroic mirror 6 to form a preset path, and the first reflective mirror 5 is used for performing reverse reflection treatment on the 650nm second excitation light emitted by the second semiconductor continuous laser2 Transmitting the second excitation light to the first dichroic mirror 6, wherein the first dichroic mirror 6 is configured to reflect the second excitation light with a wavelength of 650nm, so that the reflected second excitation light enters the main light path system, and meanwhile, the first dichroic mirror 6 is further configured to pass through the first excitation light with a wavelength of 488nm, so as to control the first laser (the first excitation light) to enter the main light path system according to a preset path; the adjusting glass slide and the light splitting prism are installed and set according to a preset installation setting scheme to form a preset path in the main light path system, so that after exciting light enters the main light path system, the light intensity of the laser (exciting light) is randomly adjusted through the adjusting glass slide in the preset path, and the light splitting treatment is performed on the laser (exciting light) through the light splitting prism in the preset path, so that the laser/exciting light respectively enters the macro spectral analysis unit 201, the mesoscopic imaging unit 202 and the microscopic high-resolution imaging unit 203 according to the preset path;
in this embodiment, three types of fluorescence generated by the two excitation lights contained in the system work in real time simultaneously, so that the transmembrane characteristics of the drug, the leakage and controlled release of the drug and other characteristics can be observed in real time through images.
Further, the light source module further comprises: a first electric light barrier 3 and a second electric light barrier 4;
the first electric light barrier 3 is positioned between the first semiconductor continuous laser1 and the first dichroic mirror 6;
a second electrically driven light barrier 4 is located between the second semiconductor continuous laser2 and the first mirror 5.
In the present embodiment, the first electric light barrier 3 and the second electric light barrier 4 are electrically controlled, and include an open/close state, which is mainly used to open or cut off a path for exciting to enter the main optical path system; it should be noted that, at the same time, only one path of excitation light enters the macro-spectral analysis unit 201 and the mesoscopic imaging unit 202, and at this time, the transmission light paths of the excitation light of the first semiconductor continuous laser1 and the second semiconductor continuous laser2 need to be opened or cut off through the first electric light barrier 3 and the second electric light barrier 4 respectively; specifically, when the first electric light barrier 3 is opened, the second electric light barrier 4 is in a closed state, and only 488 nm-wavelength excitation light emitted by the first semiconductor continuous laser1 enters the macro-spectrum analysis unit 201 and the mesoscopic imaging unit 202, and when the second electric light barrier 4 is in an opened state, the first electric light barrier 3 is in a closed state, and only 650 nm-wavelength excitation light emitted by the second semiconductor continuous laser2 enters the macro-spectrum analysis unit 201 and the mesoscopic imaging unit 202; further, for the microscopic high-resolution imaging unit 203, excitation lights with wavelengths of 488nm and 650nm enter simultaneously according to a preset path, and at this time, the first electric light barrier 3 and the second electric light barrier 4 need to be opened simultaneously, so that the two excitation lights enter the main light path system simultaneously.
Referring to fig. 3, fig. 3 is a schematic structural diagram of a macro spectrum analysis unit according to an embodiment of the present invention; the macro-spectrum analysis unit 201 includes: a second dichroic mirror 12, a first lens 13, a first optical fiber bundle 14, a first filter 15, a second lens 16 and a fluorescence spectrometer 17;
the first dichroic prism 8 is used for transmitting the first excitation light or the second excitation light to the second dichroic mirror 12;
the second dichroic mirror 12 is used for transmitting the first excitation light or the second excitation light to the first lens 13;
the first lens 13 is used for coupling the first excitation light or the second excitation light with preset wavelength into the first optical fiber bundle 14;
the first optical fiber bundle 14 is used for transmitting the first excitation light or the second excitation light to the fluorescent sample, collecting a first fluorescent signal obtained by exciting the fluorescent sample with the first excitation light or the second excitation light through a port of the first optical fiber bundle 14, and sequentially transmitting the collected first fluorescent signal to the first optical fiber bundle 14, the first lens 13 and the second dichroic mirror 12;
the second dichroic mirror 12 is configured to separate the fluorescence signal from the first excitation light or the second excitation light, and transmit the first fluorescence signal to the first filter 15 by total reflection;
the first filter 15 is configured to perform filtering processing on the first fluorescence signal, and transmit the filtered first fluorescence signal to the second lens 16;
the second lens 16 is used for transmitting the filtered first fluorescence signal to the fluorescence spectrometer 17 to obtain a fluorescence spectrum.
In the embodiment of the present invention, the macro spectrum analysis unit 201 is mainly used for macro detecting the fluorescence spectrum intensity of the labeled nanoshell and the fluorescence spectrum intensity of the labeled nano-drug, and determining the concentration distribution of the nanoshell and the nano-drug in the blood vessel through the detected fluorescence spectrum intensity. The basic working principle of the channel light path is as follows: electric light baffles (a first electric light baffle 3SH1 and a second electric light baffle 4SH2) are arranged in front of the two lasers, when SH1 is opened, SH2 is in a closed state (or SH1 is in a closed state when SH2 is opened), only one path of excitation light 488nm (or 650nm) exists in the macro spectrum analysis unit 201, the excitation light firstly reaches a second dichroic mirror 12(DM2) after passing through a first light splitting prism 8(SP1), then is coupled to a first optical fiber bundle 14(FB1 containing 3000 optical fibers) through a first lens 13(L1), the excitation light excites a fluorescent sample after passing through the first optical fiber bundle 14, a generated first fluorescent signal is collected through a port of the optical fiber bundle and then returns to the second dichroic mirror 12(DM2) along an original optical path, the second dichroic mirror 12(DM2) is mainly used for separating the excitation light signal from the fluorescent signal, and the separated fluorescent signal is reflected to a detection optical path, the non-signal light is filtered by the first filter 15(F1), transmitted to the second lens 16(L2) and finally enters the fluorescence spectrometer 17(SD), and the effective detection of the fluorescence spectrum intensity is finally realized. The macro spectrum analysis unit 201 needs to pay attention to the fact that there are two excitation lights and two fluorescence lights in the channel, so the requirement on the second dichroic mirror 12(DM2) in the light path is very high, the second dichroic mirror 12(DM2) must meet the requirements of total transmittance for two wavelengths of 488nm and 650nm and total reflection for two wavelengths of 520nm and 670nm, in addition, the first filter 15(F1) in the detection light path is a rotary filter set, a band-pass filter with the wavelength of 520nm should be selected when the fluorescence wavelength is 520nm, and a band-pass filter with the wavelength of 670nm should be selected if the fluorescence wavelength is 670 nm.
Referring to fig. 4, fig. 4 is a schematic structural diagram of a mesoscopic imaging unit according to an embodiment of the invention; the mesoscopic imaging unit 202 comprises: a third dichroic mirror 18, a third lens 19, a second fiber bundle 20, a micro-lens assembly, a first objective lens 24, a second filter 25, a fourth lens 26 and a multiplication imaging device 27;
the second light splitting prism 10 is used for transmitting the first excitation light or the second excitation light to the third dichroic mirror 18 and the third lens 19 in sequence;
the third lens 19 is used for coupling the first excitation light or the second excitation light with the preset wavelength into the second optical fiber bundle 20;
the second optical fiber bundle 20 is used for transmitting the first excitation light or the second excitation light to the micro-lens component;
the micro-lens assembly is used for adjusting the beam size of the first excitation light or the second excitation light and transmitting the first excitation light or the second excitation light of the proper beam to the first objective lens 24;
the first objective lens 24 is used for transmitting the first excitation light or the second excitation light to the fluorescent sample, collecting a second fluorescent signal obtained by exciting the fluorescent sample by the first excitation light or the second excitation light, and sequentially transmitting the collected second fluorescent signal to the microlens assembly, the second optical fiber bundle 20, the second lens 16 and the third dichroic mirror 18;
the third dichroic mirror 18 is configured to separate the second fluorescent signal from the first excitation light or the second excitation light, and transmit the second fluorescent signal to the second filter 25 by total reflection;
the second filter 25 is configured to perform filtering processing on the second fluorescent signal, and transmit the filtered second fluorescent signal to the fourth lens 26;
the fourth lens 26 transmits the filtered second fluorescence signal to the multiplication imaging device 27 to obtain a mesoscopic distribution image.
In this embodiment, the mesoscopic imaging unit 202 is mainly used for mesoscopic microscopic imaging of living cells, after the macroscopic spectral analysis unit 201 finds a target position with a relatively strong fluorescence signal (i.e. a place where nano-drugs or nano-shells are gathered relatively much), the mesoscopic imaging unit 202 is used for real-time wide-field mesoscopic imaging to observe the distribution of the drugs in the blood vessels in real time in the form of images, similarly, the excitation light sources are also derived from two lasers, namely, the first semiconductor continuous Laser 1(Laser1) and the second semiconductor continuous Laser 2(Laser2), when the first electric light barrier 3(SH1) is opened, the second electric light barrier 4SH2 should be in a closed state (or when the second electric light barrier 4(SH2) is opened, the first electric light barrier 3SH1 is in a closed state), at this time, only one excitation light 488nm (or 650nm) exists in the macroscopic spectral analysis unit 201, the laser light passes through the third dichroic mirror 18(DM3) and enters the third lens 19(L3), where the third lens 19L3 is to couple the excitation light into the second fiber bundle 20(FB2, containing 10000 fibers), then the light beam passing through the second fiber bundle 20FB2 is adjusted by the micro lens assemblies (L5, L6 and L7), the size of the light beam is equal to the diameter of the pupil of the first objective lens 24(OBJ1), the diameter of the pupil is 1cm, so that the light beam just fills the pupil of the first objective lens 24(OBJ1, diameter 1cm), the performance of the first objective lens 24 is fully exerted, and finally the fluorescence is excited at the fluorescence sample, and the obtained second fluorescence signal is collected by the first objective lens 24, then returns to the third dichroic mirror 18(DM3) along the original optical path, then is reflected to the detection optical path, filtered by the second filter 25(F2), transmitted to the fourth lens 26(L4), and enters the EMCCD multiplication device (emdm 3627) for mesoscopic imaging, to obtain a macroscopic distribution map. It should be noted that the third dichroic mirror 18(DM3) selected in the optical path of the mesoscopic imaging unit 202 is the same as the second dichroic mirror 12(DM2) in the macroscopic spectral analysis unit 201, and the rotating filter set (F2) is the same as the first filter 15(F1) in the macroscopic spectral analysis unit 201.
Further, the microlens assembly comprises: a fifth lens 21, a sixth lens 22, and a seventh lens 23;
the first excitation light or the second excitation light sequentially passes through the fifth lens 21, the sixth lens 22 and the seventh lens 23 to adjust the light beam size of the first excitation light or the second excitation light.
In the present embodiment, the micro lens assembly in the mesoscopic imaging unit 202 is composed of three lenses, and the micro lens assembly includes: the fifth lens 21, the sixth lens 22 and the seventh lens 23, wherein the sixth microlens is located between the fifth microlens and the seventh microlens, the sizes of the fifth microlens, the sixth microlens and the seventh microlens are the same, and the centers of the fifth microlens, the sixth microlens and the seventh microlens are located on the same straight line, so that the fifth microlens, the sixth microlens and the seventh microlens are sequentially set in parallel, thereby realizing the adjustment of the size of the light beam of the first excitation light or the second excitation light, enabling the light beam of the excitation light to be exactly the same as the pupil size of the first objective lens 24, and enabling the performance of the first objective lens 24 to be fully exerted and utilized.
Referring to fig. 5, fig. 5 is a schematic structural diagram of a microscopic high-resolution imaging unit according to an embodiment of the present disclosure; the microscopic high-resolution imaging unit 203 includes: a fourth dichroic mirror 28, a fifth dichroic mirror 29, a sixth dichroic mirror 30, a vibrating mirror 31, a 4F system, a second objective mirror 34, a third optical fiber bundle 35, an eighth lens 36, a self-focusing lens 37, a third filter 38, a ninth lens 39, a first multimode optical fiber 40, a first photomultiplier 41, a fourth filter 42, a tenth lens 43, a second multimode optical fiber 44, a second photomultiplier 45, a fifth filter 46, an eleventh lens 47, a third multimode optical fiber 48, and a third photomultiplier 49;
the third dichroic prism is used for simultaneously and sequentially transmitting the first excitation light and the second excitation light to a fourth dichroic mirror 28, a fifth dichroic mirror 29, a sixth dichroic mirror 30 and a vibrating mirror 31;
the galvanometer 31 is used for carrying out light beam scanning on the first excitation light and the second excitation light to obtain scanning light beams and transmitting the scanning light beams to the 4F system;
the 4F system is used for adjusting the beam size of the scanning beam and transmitting the adjusted scanning beam to the second objective lens 34;
the second objective lens 34 is used for coupling the scanning beam into a third fiber bundle 35;
the third fiber bundle 35 is used for transmitting the scanning beam to the eighth lens 36;
an eighth lens 36 for coupling the scanning beam into a self-focusing lens 37;
the self-focusing lens 37 is used for transmitting the scanning light beam to the fluorescent sample, collecting a third fluorescent signal obtained by exciting the fluorescent sample by the scanning light beam, and transmitting the third fluorescent signal to the eighth lens 36, the third optical fiber beam 35, the second objective lens 34, the 4F system, the vibrating mirror 31, the sixth dichroic mirror 30, the fifth dichroic mirror 29 and the fourth dichroic mirror 28 in sequence in the reverse direction;
the fourth dichroic mirror 28 is configured to separate the third fluorescent signal to obtain a fourth fluorescent signal, and totally reflect the fourth fluorescent signal and transmit the fourth fluorescent signal to the third filter 38, the ninth lens 39, the first multimode fiber 40, and the first photomultiplier 41 in sequence to obtain a cell membrane distribution map;
the fifth dichroic mirror 29 is configured to separate the third fluorescent signal to obtain a fifth fluorescent signal, and totally reflect the fifth fluorescent signal and sequentially transmit the fifth fluorescent signal to the fourth filter 42, the tenth lens 43, the second multimode optical fiber 44, and the second photomultiplier 45, so as to obtain a nano-drug distribution map;
the sixth dichroic mirror 30 is configured to separate the third fluorescent signal to obtain a sixth fluorescent signal, and totally reflect the sixth fluorescent signal and sequentially transmit the sixth fluorescent signal to the fifth filter 46, the eleventh lens 47, the third multimode fiber 48, and the third photomultiplier 49, so as to obtain a nano-drug distribution map.
In the embodiment of the present invention, the microscopic high-resolution imaging unit 203 is a high-resolution endoscopic imaging channel, and after the mesoscopic microscopic imaging of the mesoscopic imaging unit 202, a macroscopic distribution map of the nano-drugs or the nanoshells can be obtained, since the resolution of the macroscopic distribution map is low, more structural information cannot be seen, a second target position needs to be further selected, and then the microscopic high-resolution imaging unit 203 is used to perform high-resolution imaging on the second target position; the microscopic high-resolution imaging unit 203 comprises two excitation light paths and three detection light paths, wherein the first excitation light with the wavelength of 488nm in the two excitation light paths simultaneously excites two fluorescent dyes: FITC (fluorescent dye FITC) targeting a nanoshell and DIO (fluorescent dye DIO) targeting a cell membrane dye, second excitation light with the wavelength of 650nm is Cy5 series fluorescent dye for exciting a targeted nano-drug, the two paths of light are reflected by a second reflecting mirror 11(M2), then sequentially pass through a fourth dichroic mirror 28, a fifth dichroic mirror 29 and a sixth dichroic mirror 30(DM4, DM5 and DM6) together, then reach a vibrating mirror 31(GM) for beam scanning, enter a 4f system consisting of a twelfth Lens 32(L12) and a thirteenth Lens 33(L13) for adjusting scanning beams, then enter a second objective 34(OBJ2), wherein the second objective 34 mainly couples the scanning beams into a third optical fiber beam 35(FB3, containing 30000), the scanning beams after passing through the third optical fiber beam 35 are coupled into a gradient refractive index changing self-focusing Lens 37 (Lens) through an eighth Lens 36(L8), the scanning excitation of the fluorescent sample is realized through the self-focusing lens 37, and the generated third fluorescent signal is collected through the self-focusing lens 37 and returns along the original optical path;
in this embodiment, the three detection optical paths in the microscopic high-resolution imaging unit 203 are respectively: the first is that fluorescence dye FITC targeting the nanoshell is excited by first excitation light with a wavelength of 488nm, a generated sixth fluorescence signal returns to a sixth dichroic mirror 30(DM6) and is reflected to a detection light path, non-signal light is filtered out by a fifth filter 46(F5), enters an eleventh lens 47(L11), is focused by the eleventh lens 47 and is coupled into a third multimode fiber 48(DMF3), wherein the third multimode fiber 48 not only can transmit the light signal but also can play a role of a pinhole, stray light around a focal spot is filtered to achieve the purpose of improving the resolution of an image, then the sixth fluorescence signal with high resolution information is introduced into a third photomultiplier 49(PMT3) through the third multimode fiber 48 to realize photoelectric signal conversion, and finally image acquisition is realized through imaging software; specifically, the sixth dichroic mirror 30 separates the first excitation light and the second excitation light in the third fluorescence signal to obtain a sixth fluorescence signal containing the first excitation light, and refracts and transmits the sixth fluorescence signal to the fifth filter 46, the fifth filter 46 filters the sixth fluorescence signal, and transmits the filtered sixth fluorescence signal to the eleventh lens 47, the eleventh lens 47 couples the filtered sixth fluorescence signal into the third multimode fiber 48, the third multimode fiber 48 is configured to filter the sixth fluorescence signal to obtain a high-resolution sixth fluorescence signal, and transmits the high-resolution sixth fluorescence signal to the third photomultiplier 49, and the third photomultiplier 49 is configured to perform photoelectric signal conversion on the high-resolution sixth fluorescence signal to obtain a nanosheet distribution map;
meanwhile, the second type is that the fluorescent dye DIO targeting the cell membrane is excited by first excitation light with the wavelength of 488nm, the generated fourth fluorescent signal is collected and returned to the fourth dichroic mirror 28(DM4), is reflected to a detection light path by the fourth dichroic mirror 28(DM4), is filtered by a fourth filter 42(F4), and is transmitted to a tenth lens 43(L10), the tenth lens 43 couples the fourth fluorescent signal into the first multimode optical fiber 40(DMF1), and then is subjected to photoelectric conversion by a first photomultiplier tube 41(PMT1) and then is introduced into imaging software to realize the acquisition of a high-resolution fluorescent signal image; specifically, the fourth dichroic mirror 28 separates the first excitation light from the second excitation light in the third fluorescence signal to obtain a fourth fluorescence signal containing the first excitation light, and refracts and transmits the fourth fluorescence signal to the third filter 38, the third filter 38 filters the fourth fluorescence signal, and transmits the filtered fourth fluorescence signal to the ninth lens 39, the ninth lens 39 couples the filtered fourth fluorescence signal into the first multimode fiber 40, the first multimode fiber 40 is configured to filter the fourth fluorescence signal to obtain a high-resolution fourth fluorescence signal, and transmits the high-resolution fourth fluorescence signal to the first photomultiplier 41, and the first photomultiplier 41 is configured to perform photoelectric signal conversion on the high-resolution fourth fluorescence signal to obtain a cell membrane distribution map;
the third is that the Cy5 series fluorescent dye of the targeted nano-drug is excited by the second excitation light with the wavelength of 650nm, the generated fifth fluorescent signal returns to the fifth dichroic mirror 29(DM5) after being collected, is reflected to the detection light path by the fifth dichroic mirror 29, enters the tenth lens 43(L10) after being filtered by the fourth filter 42(F4) for non-signal light, is coupled into the second multimode optical fiber 44(DMF2) by the tenth lens 43, and then is subjected to photoelectric conversion by the second photomultiplier 45(PMT2) and then is introduced into the imaging software to realize the acquisition of the high-resolution fluorescent signal image; specifically, the fifth dichroic mirror 29 separates the first excitation light from the second excitation light in the third fluorescence signal to obtain a fifth fluorescence signal containing the second excitation light, and refracts and transmits the fifth fluorescence signal to the fourth filter 42, the fourth filter 42 filters the fifth fluorescence signal, and transmits the filtered fifth fluorescence signal to the tenth lens 43, the tenth lens 43 couples the filtered fifth fluorescence signal into the second multimode fiber 44, the second multimode fiber 44 is configured to filter the fifth fluorescence signal to obtain a high-resolution fifth fluorescence signal, and transmits the high-resolution fifth fluorescence signal to the second photomultiplier 45, and the second photomultiplier 45 is configured to perform photoelectric signal conversion on the high-resolution fifth fluorescence signal to obtain a nano-drug distribution map;
in the embodiment of the present invention, the fluorescence distribution image of the nano-drug, the fluorescence distribution map of the nanoshell, and the distribution map of the cell membrane can be obtained through the three detection light paths of the microscopic high-resolution imaging unit 203, and the transmembrane process of the nanoshell, the leakage of the nano-drug, and the controlled release characteristics can be effectively studied by monitoring the changes of the three images in real time.
Further, the 4F system comprises: a twelfth lens 32 and a thirteenth lens 33;
the first excitation light and the second excitation light are transmitted from the twelfth lens 32 to the thirteenth lens 33 to adjust the beam sizes of the first excitation light and the second excitation light.
In the present embodiment, the microscopic high-resolution imaging unit 203 includes a 4F system, which is composed of a twelfth lens 32 and a thirteenth lens 33, wherein the twelfth lens 32 is close to the galvanometer 31(GM) in the microscopic high-resolution imaging unit 203, the thirteenth lens 33 is close to the second objective lens 34 in the microscopic high-resolution imaging unit 203, the first excitation light and the second excitation light enter the microscopic high-resolution imaging unit 203 simultaneously, after being transmitted from the galvanometer 31, the optical lens reaches the twelfth lens 32, and then is transmitted to the thirteenth lens 33 by the twelfth lens 32, and is transmitted to the second objective lens 34 by the thirteenth lens 33, and further, the 4F system can adjust the beam size of the first excitation light and the second excitation light, so that the adjusted light beam is just consistent with the pupil size of the second objective lens 34, and the performance of the second objective lens 34 is fully exerted; furthermore, the 4F system can also be used to translate the imaged image plane to obtain a suitable image plane position, thereby obtaining a high-resolution imaged image plane.
The invention provides a trans-scale fluorescence endoscopic imaging system, which comprises: an illumination unit and a microscopic imaging unit; wherein the microscopic imaging unit includes: the device comprises a macroscopic spectrum analysis unit, a mesoscopic imaging unit and a microscopic high-resolution imaging unit; the illumination unit is used for emitting first excitation light and/or second excitation light and transmitting the first excitation light and/or the second excitation light to the macroscopic spectrum analysis unit, the mesoscopic imaging unit and the microscopic high-resolution imaging unit; the macroscopic spectrum analysis unit is used for acquiring a first fluorescence signal generated by exciting the nanoshell and the nano-drug by the incident first exciting light or the incident second exciting light and generating a fluorescence spectrogram of the nanoshell and the nano-drug according to the first fluorescence signal; the mesoscopic imaging unit is used for acquiring a second fluorescence signal generated by exciting the nanoshell and the nano-drug by the incident first excitation light or the incident second excitation light based on the fluorescence spectrogram and acquiring a mesoscopic distribution image of the nanoshell and the nano-drug according to the second fluorescence signal; the microscopic high-resolution imaging unit is used for acquiring a third fluorescence signal of the incident first excitation light and the incident second excitation light for exciting the nanoshell and the nano-drug based on the mesoscopic distribution image, and performing high-resolution imaging according to the third fluorescence signal to obtain a nano-drug fluorescence image, a nanoshell fluorescence image and a cell membrane distribution map for real-time observation of transmembrane, leakage and controlled release of the nano-drug; the system realizes monitoring of drug transport in cells through a nano-drug fluorescence map, a nano-shell fluorescence map and a cell membrane distribution map, and realizes research of cell pharmacokinetic characteristics in deep tissues of living animals.
In the several embodiments provided in the present application, it should be understood that the disclosed system may be implemented in other ways. For example, the above-described structural embodiments are merely illustrative, and for example, the connection of the "fiber optic device" is merely a logical functional connection, and in fact, there may be additional connections in the implementation, for example, multiple identical devices or components may be combined or integrated into another system, or some features may be omitted, or not implemented. In addition, the shown or discussed couplings or direct couplings or communication connections between each other may be through some interfaces or in other forms.
In addition, the functional "optical fiber devices" in the embodiments of the present invention may be integrated in one system, may exist as functional integrated devices respectively constituting one portion, or may be two or more integrated devices. The integrated device can be realized in a hardware form, and can also be realized in a functional integrated device formed by combining software and hardware.
It should be noted that, for the sake of simplicity, the above-mentioned method embodiments are described as a series of acts or combinations, but those skilled in the art should understand that the present invention is not limited by the described order of acts, as some steps may be performed in other orders or simultaneously according to the present invention. Further, those skilled in the art will appreciate that the embodiments described in the specification are presently preferred and that no acts or modules are necessarily required of the invention.
In the above embodiments, the descriptions of the respective embodiments have respective emphasis, and for parts that are not described in detail in a certain embodiment, reference may be made to related descriptions of other embodiments.
The above description is provided for the generation system of the high energy dissipation soliton resonance square pulse provided by the present invention, and for those skilled in the art, the idea according to the embodiment of the present invention may be changed in the specific implementation manner and the application scope, and in summary, the content of the present specification should not be construed as a limitation to the present invention.
Claims (8)
1. A trans-scale fluoroscopic endoscopic imaging system, the system comprising: an illumination unit and a microscopic imaging unit;
wherein the microscopic imaging unit macro comprises: the device comprises a macroscopic spectrum analysis unit, a mesoscopic imaging unit and a microscopic high-resolution imaging unit;
the illumination unit is used for emitting first excitation light and/or second excitation light and transmitting the first excitation light and/or the second excitation light to the macroscopic spectrum analysis unit, the mesoscopic imaging unit and the microscopic high-resolution imaging unit respectively;
the macroscopic spectrum analysis unit is used for acquiring a first fluorescence signal generated by the incident first excitation light or the second excitation fluorescence sample and generating a fluorescence spectrum diagram of the fluorescence sample according to the first fluorescence signal;
the mesoscopic imaging unit is used for acquiring a second fluorescence signal generated by exciting the fluorescence sample by the incident first excitation light or the second excitation light based on the fluorescence spectrum and acquiring a mesoscopic distribution image of the fluorescence sample according to the second fluorescence signal;
the microscopic high-resolution imaging unit is used for acquiring the incident first excitation light and the incident second excitation light to excite a third fluorescence signal of the fluorescence sample based on the mesoscopic distribution image, and performing high-resolution imaging according to the third fluorescence signal to obtain a nano-drug fluorescence image, a nano-shell fluorescence image and a cell membrane distribution image for observing the fluorescence sample in real time.
2. The trans-scale fluoroscopic endoscopic imaging system according to claim 1, wherein the illumination unit comprises: a light source assembly and a main light path system;
the light source assembly includes: a first semiconductor continuous laser and a second semiconductor continuous laser;
the main light path system includes: the device comprises a first reflective mirror, a first dichroic mirror, a first adjusting glass sheet, a first light splitting prism, a second adjusting glass sheet, a second light splitting prism and a second reflective mirror;
the first exciting light emitted by the first semiconductor continuous laser sequentially passes through the first dichroic mirror, the first adjusting glass sheet, the first light splitting prism, the second adjusting glass sheet, the second light splitting prism and the second reflector;
the second excitation light emitted by the second semiconductor continuous laser sequentially passes through the first reflector, the first dichroic mirror, the first adjusting glass slide, the first light splitting prism, the second adjusting glass slide, the second light splitting prism and the second reflector;
the first light splitting prism is used for transmitting the first exciting light or the second exciting light to the macroscopic spectrum analysis unit in a light splitting mode, and transmitting the first exciting light or the second exciting light to the second adjusting glass slide in sequence;
the second light splitting prism is used for transmitting the first exciting light or the second exciting light to the mesoscopic imaging unit in a split mode and transmitting the first exciting light or the second exciting light to the second reflector;
wherein the second mirror is configured to transmit the first excitation light and the second excitation light to the microscopic high-resolution imaging unit.
3. The system of claim 2, wherein the light source assembly further comprises: a first electric light barrier and a second electric light barrier;
the first electric light barrier is positioned between the first semiconductor continuous laser and the first dichroic mirror;
the second electric light barrier is located between the second semiconductor continuous laser and the first mirror.
4. The system of claim 2, wherein the macro-spectroscopy analysis unit comprises: the device comprises a second dichroic mirror, a first lens, a first optical fiber bundle, a first filter, a second lens and a fluorescence spectrometer;
the first dichroic mirror is used for transmitting the first excitation light or the second excitation light to the second dichroic mirror;
the second dichroic mirror is used for transmitting the first excitation light or the second excitation light to the first lens;
the first lens is used for coupling the first excitation light or the second excitation light with preset wavelength into the first optical fiber bundle;
the first optical fiber bundle is used for transmitting the first excitation light or the second excitation light to a fluorescent sample, collecting a first fluorescent signal obtained after the fluorescent sample is excited by the first excitation light or the second excitation light through a port of the first optical fiber bundle, and sequentially transmitting the collected first fluorescent signal to the first optical fiber bundle, the first lens and the second dichroic mirror;
the second dichroic mirror is used for separating the fluorescent signal from the first exciting light or the second exciting light and transmitting the first fluorescent signal to the first filter sheet in a total reflection manner;
the first filter is used for filtering the first fluorescent signal and transmitting the filtered first fluorescent signal to the second lens;
the second lens is used for transmitting the filtered first fluorescence signal to the fluorescence spectrometer to obtain a fluorescence spectrogram.
5. The trans-scale fluoroscopic endoscopic imaging system according to claim 4, wherein the mesoscopic imaging unit comprises: the third dichroic mirror, a third lens, a second optical fiber bundle, a micro-lens component, a first objective lens, a second filter, a fourth lens and a multiplication imaging device;
the second light splitting prism is used for sequentially transmitting the first excitation light or the second excitation light to the third dichroic mirror and the third lens;
the third lens is used for coupling the first excitation light or the second excitation light with preset wavelength into the second optical fiber bundle;
the second optical fiber bundle is used for transmitting the first excitation light or the second excitation light to the micro-lens component;
the micro-lens assembly is used for adjusting the beam size of the first excitation light or the second excitation light and transmitting the first excitation light or the second excitation light of a proper beam to the first objective lens;
the first objective lens is used for transmitting the first exciting light or the second exciting light to a fluorescent sample, collecting a second fluorescent signal obtained after the fluorescent sample is excited by the first exciting light or the second exciting light, and sequentially transmitting the collected second fluorescent signal to the micro-lens component, the second optical fiber bundle, the second lens and the third dichroic mirror;
the third dichroic mirror is used for separating the second fluorescent signal from the first exciting light or the second exciting light and transmitting the second fluorescent signal to the second filter plate in a total reflection manner;
the second filter is used for filtering the second fluorescent signal and transmitting the filtered second fluorescent signal to the fourth lens;
and the fourth lens transmits the filtered second fluorescence signal to the multiplication imaging device to obtain a mesoscopic distribution image.
6. The trans-scale fluoroscopic endoscopic imaging system according to claim 5, wherein the microlens assembly comprises: a fifth lens element, a sixth lens element, and a seventh lens element;
the first excitation light or the second excitation light sequentially passes through the fifth lens, the sixth lens and the seventh lens so as to adjust the beam size of the first excitation light or the second excitation light.
7. The trans-scale fluoroscopic endoscopic imaging system according to claim 3, wherein the microscopic high resolution imaging unit comprises: the system comprises a fourth dichroic mirror, a fifth dichroic mirror, a sixth dichroic mirror, a vibrating mirror, a 4F system, a second objective mirror, a third optical fiber bundle, an eighth lens, a self-focusing lens, a third filter, a ninth lens, a first multimode optical fiber, a first photomultiplier, a fourth filter, a tenth lens, a second multimode optical fiber, a second photomultiplier, a fifth filter, an eleventh lens, a third multimode optical fiber and a third photomultiplier;
the third light splitting prism is used for simultaneously and sequentially transmitting the first excitation light and the second excitation light to the fourth dichroic mirror, the fifth dichroic mirror, the sixth dichroic mirror and the vibrating mirror;
the galvanometer is used for carrying out light beam scanning on the first excitation light and the second excitation light to obtain scanning light beams and transmitting the scanning light beams to the 4F system;
the 4F system is used for adjusting the beam size of the scanning beam and transmitting the adjusted scanning beam to the second objective lens;
the second objective lens is used for coupling the scanning light beam into the third optical fiber bundle;
the third optical fiber bundle is used for transmitting the scanning light beam to the eighth lens;
the eighth lens is used for coupling the scanning light beam into the self-focusing lens;
the self-focusing lens is used for transmitting the scanning light beam to a fluorescent sample, collecting a third fluorescent signal obtained after the scanning light beam excites the fluorescent sample, and transmitting the third fluorescent signal to the eighth lens, the third optical fiber bundle, the second objective lens, the 4F system, the vibrating mirror, the sixth dichroic mirror, the fifth dichroic mirror and the fourth dichroic mirror in sequence in a reverse direction;
the fourth dichroic mirror is used for separating the third fluorescent signal to obtain a fourth fluorescent signal, and totally reflecting the fourth fluorescent signal and sequentially transmitting the fourth fluorescent signal to the third filter, the ninth lens, the first multimode optical fiber and the first photomultiplier tube to obtain a cell membrane distribution map;
the fifth dichroic mirror is used for separating the third fluorescent signal to obtain a fifth fluorescent signal, and totally reflecting the fifth fluorescent signal and sequentially transmitting the fifth fluorescent signal to the fourth filter, the tenth lens, the second multimode optical fiber and the second photomultiplier tube to obtain a nano-drug distribution map;
the sixth dichroic mirror is used for separating the third fluorescent signal to obtain a sixth fluorescent signal, and totally reflecting the sixth fluorescent signal and sequentially transmitting the sixth fluorescent signal to the fifth filter, the eleventh lens, the third multimode optical fiber and the third photomultiplier tube to obtain a nano-drug distribution map.
8. The trans-scale fluoroscopic endoscopic imaging system according to claim 7, wherein the 4F system comprises: a twelfth lens and a thirteenth lens;
the first excitation light and the second excitation light are transmitted from the twelfth lens to the thirteenth lens to adjust the beam size of the first excitation light and the second excitation light.
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| CN113390844A (en) * | 2021-06-17 | 2021-09-14 | 中国药科大学 | Multi-scale optical fiber fluorescence microscopic imaging system |
| CN114569903A (en) * | 2022-01-20 | 2022-06-03 | 重庆医科大学 | Pulse ultrasound-medicine-cooperated external noninvasive therapeutic apparatus and operation method thereof |
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