CN117731239B - Device and method for noninvasive dynamic monitoring and killing of living circulating tumor cells - Google Patents
Device and method for noninvasive dynamic monitoring and killing of living circulating tumor cells Download PDFInfo
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Abstract
本发明公开了一种活体循环肿瘤细胞无创动态监测及消杀装置，包括循环肿瘤细胞实时动态监测系统和高频脉冲激光消杀系统，所述循环肿瘤细胞实时动态监测系统包括连续波激光器、激光光轴、物镜、载物台、CMOS相机、计算机、光电倍增管，所述物镜上方设有载物台，所述载物台上方设有LED光源，所述CMOS相机、光电倍增管均与计算机连接；所述高频脉冲激光消杀系统包括脉冲激光器、函数发生器。本发明还公开了一种活体循环肿瘤细胞无创动态监测及消杀装置的使用方法。本发明采用上述一种活体循环肿瘤细胞无创动态监测及消杀装置，解决现有的技术仅能实现无创实时动态监测循环肿瘤细胞，但却不能对于监测到的循环肿瘤细胞进行消杀的问题。
The present invention discloses a device for non-invasive dynamic monitoring and disinfecting of circulating tumor cells in living bodies, including a real-time dynamic monitoring system for circulating tumor cells and a high-frequency pulse laser disinfecting system. The real-time dynamic monitoring system for circulating tumor cells includes a continuous wave laser, a laser optical axis, an objective lens, a stage, a CMOS camera, a computer, and a photomultiplier tube. A stage is provided above the objective lens, and an LED light source is provided above the stage. The CMOS camera and the photomultiplier tube are both connected to the computer; the high-frequency pulse laser disinfecting system includes a pulse laser and a function generator. The present invention also discloses a method for using the device for non-invasive dynamic monitoring and disinfecting of circulating tumor cells in living bodies. The present invention adopts the above-mentioned device for non-invasive dynamic monitoring and disinfecting of circulating tumor cells in living bodies to solve the problem that the existing technology can only realize non-invasive real-time dynamic monitoring of circulating tumor cells, but cannot kill the monitored circulating tumor cells.
Description
Technical Field
The invention relates to the technical field of monitoring and killing of circulating tumor cells, in particular to a device and a method for noninvasive dynamic monitoring and killing of living circulating tumor cells.
Background
Cancer is a malignant tumor, which is a disease caused by unrestricted growth and division of abnormal cells in the body. These abnormal cells can invade and destroy normal tissues around the body. Cancer can occur anywhere, with common types including breast, lung, skin, colon, prostate, cervical cancer, and the like. Circulating tumor cells (circulating tumor cells, CTCs) refer to malignant tumor cells that shed from the primary tumor, enter the blood circulation, and are distributed into other organs or tissues. Cancer metastasis refers to the invasion of cancer cells from a primary tumor into surrounding tissues, and further spread to other sites through the blood circulation or lymphatic system, and form new metastatic lesions. Circulating tumor cells play an important role in cancer metastasis. When primary tumors grow to some extent, they can release small numbers of malignant cells into the blood circulation. These circulating tumor cells can be transported through the blood stream to other sites in the body, such as lymph nodes, lungs, liver, bones, etc., where they colonize and grow to form metastatic lesions.
The elimination of the circulating tumor cells has important significance for the treatment of cancers, and the cancer cells usually metastasize early, so that the timely elimination of the circulating tumor cells is beneficial to inhibiting the early metastasis of tumors and reducing the probability of tumor recurrence. The existing living body flow type detection system based on fluorescent markers and living body flow type detection technology based on the photoacoustic effect can only realize noninvasive real-time dynamic monitoring of circulating tumor cells, but cannot kill the monitored circulating tumor cells.
Disclosure of Invention
The invention aims to provide a device and a method for noninvasively and dynamically monitoring and killing living circulating tumor cells, which solve the problem that the existing technology can only realize noninvasive real-time dynamic monitoring of circulating tumor cells, but cannot kill the monitored circulating tumor cells.
The invention provides a living body circulating tumor cell noninvasive dynamic monitoring and killing device, which comprises a circulating tumor cell real-time dynamic monitoring system and a high-frequency pulse laser killing system, wherein the circulating tumor cell real-time dynamic monitoring system comprises a continuous wave laser, a laser optical axis, an objective lens, an objective table, a CMOS camera, a computer and a photomultiplier, the objective table is arranged above the objective lens, an LED light source is arranged above the objective table, and the CMOS camera and the photomultiplier are connected with the computer; the high-frequency pulse laser killing system comprises a pulse laser and a function generator.
Preferably, a cylindrical lens is arranged on one side of the continuous wave laser, a mechanical slit I is arranged at the focal length of the cylindrical lens, a dichroic mirror I is arranged on one side of the mechanical slit I, a reflecting mirror I and a reflecting mirror II are respectively arranged on two sides of the dichroic mirror I, the reflecting mirror I is connected with an objective lens through a convex lens I and a dichroic mirror II, a reflecting mirror III and a spectroscope are sequentially arranged on one side of the dichroic mirror II, a light filter I and a reflecting mirror IV are respectively arranged on two sides of the spectroscope, a reflecting mirror four-way light filter II and a convex lens II are connected with a CMOS camera, and one side of the light filter I is connected with a photomultiplier through the mechanical slit II and the convex lens III.
Preferably, a pulse laser is arranged on one side of the function generator, and the pulse laser is connected with the second reflecting mirror.
Preferably, the distance between the first mechanical slit and the first convex lens is equal to the focal length of the first convex lens, the parameters of the first dichroic mirror are light with reflection wavelengths of 488nm, 532nm and 635nm, the parameters of the second dichroic mirror are light with reflection wavelengths of 488nm, 532nm, 635nm and 1064nm, the parameters of the first dichroic mirror are light with reflection wavelengths of 488nm, 532nm, 635nm and 1064nm, the transmittance of the spectroscope is 90%, the reflectance is 10%, and the parameters of the first filter and the second filter are bandpass filters with wavelengths of 500-520 nm.
Preferably, the included angles between the laser optical axis and the first reflecting mirror, the second reflecting mirror, the third reflecting mirror and the fourth reflecting mirror are 45 degrees respectively.
Preferably, the laser wavelength emitted by the continuous wave laser is 488nm, 532nm and 635nm, the LED light source adopts green light with the wavelength of 510-580nm, the wavelength range of the pulse laser is 780-2000 nm, the repetition frequency range is 1Hz-500KHz, and the pulse width is 1-100ns.
The application method of the living body circulating tumor cell noninvasive dynamic monitoring and killing device comprises the following steps:
(1) The continuous wave laser emits blue laser, one-dimensional compression is carried out through a cylindrical lens, then the blue laser is focused through a convex lens after being reflected by a dichroic mirror I and a reflecting mirror I, and then the blue laser is focused on a blood vessel to be detected of a living animal or human body on an objective table after being reflected by a dichroic mirror II, so as to form a detection and disinfection window, and the circulating tumor cells flowing through the window are detected and disinfected;
(2) The LED light source penetrates through the sample in the step (1), is focused by the objective lens, penetrates through the dichroic mirror II, the reflecting mirror III and the spectroscope, enters the CMOS camera through the reflecting mirror IV, the optical filter II and the convex lens II, and images tumor cells flowing through the window in the computer;
(3) The tumor cells flowing through the window in the step (1) are excited by laser emitted by a continuous wave laser and then generate fluorescent signals, after being focused by an objective lens, the fluorescent signals pass through a dichroic mirror II, a reflecting mirror III and a spectroscope, enter a photomultiplier through a filter I, a mechanical slit II and a convex lens III, and the green fluorescent signals collected by the photomultiplier are input into a computer for counting;
(4) The function generator provides pulse signals to trigger the pulse laser, and the generated pulse laser sequentially passes through the reflecting mirror II, the dichroic mirror I, the reflecting mirror I, the convex lens I and the dichroic mirror II to be focused on tumor cells which are monitored by the object stage and flow through the window, and the cells are vaporized and die due to overhigh local temperature.
The device and the method for noninvasively and dynamically monitoring and killing the living circulation tumor cells have the advantages that:
1. the invention can realize the real-time monitoring of the living circulation tumor cells and kill the circulation tumor cells.
2. The invention can kill for a long time, has important significance for early treatment of tumors in clinic, and has potential for application in the fields of oncology, cytobiology, basic medical research and the like.
The technical scheme of the invention is further described in detail through the drawings and the embodiments.
Drawings
FIG. 1 is a schematic diagram of a device for noninvasive dynamic monitoring and killing of living circulating tumor cells;
FIG. 2 is a graph of fluorescence signal detected by a fluorescence-label-based in vivo flow detection system according to an embodiment of the present invention;
FIG. 3 is a graph showing the relationship between cell death rate and laser flux for cell viability assay of B16F10 cells of the present invention by trypan blue staining, wherein a is the graph of cell viability assay of B16F10 cells on the stage by increasing laser intensity irradiation and B is the graph of cell viability assay by trypan blue staining.
Reference numerals
1. A pulsed laser; 2. a second reflecting mirror; 3. a continuous wave laser; 4. a cylindrical lens; 5. mechanical slit I; 6. a dichroic mirror I; 7. a first reflecting mirror; 8. a first convex lens; 9. a dichroic mirror II; 10. a third reflecting mirror; 11, spectroscope; 12. a first optical filter; 13. mechanical slit II; 14. a convex lens III; 15. a photomultiplier tube; 16. a reflection mirror IV; 17. a second optical filter; 18. a convex lens II; 19. a CMOS camera; 20. an objective lens; 21. an objective table; 22. an LED light source; 23. a computer; 24. a function generator; 25. the laser optical axis.
Detailed Description
The technical scheme of the invention is further described below through the attached drawings and the embodiments.
Unless defined otherwise, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs. The terms "first," "second," and the like, as used herein, do not denote any order, quantity, or importance, but rather are used to distinguish one element from another. The word "comprising" or "comprises", and the like, means that elements or items preceding the word are included in the element or item listed after the word and equivalents thereof, but does not exclude other elements or items. The terms "connected" or "connected," and the like, are not limited to physical or mechanical connections, but may include electrical connections, whether direct or indirect. "upper", "lower", "left", "right", etc. are used merely to indicate relative positional relationships, which may also be changed when the absolute position of the object to be described is changed.
Examples
A non-invasive dynamic monitoring and killing device for living body circulating tumor cells comprises a circulating tumor cell real-time dynamic monitoring system and a high-frequency pulse laser killing system.
As shown in fig. 1, the real-time dynamic monitoring system for circulating tumor cells comprises a continuous wave laser, a laser optical axis, an objective lens, an objective table, a CMOS camera, a computer and a photomultiplier tube. An objective table is arranged above the objective lens, an LED light source is arranged above the objective table, and the CMOS camera and the photomultiplier are connected with a computer.
The continuous wave laser is characterized in that a cylindrical lens is arranged on one side of the continuous wave laser, a mechanical slit I is arranged at the focal length of the cylindrical lens, a dichroic mirror I is arranged on one side of the mechanical slit I, a reflecting mirror I and a reflecting mirror II are respectively arranged on two sides of the dichroic mirror I, the reflecting mirror I is connected with an objective lens through a convex lens I and a dichroic mirror II, a reflecting mirror III and a spectroscope are sequentially arranged on one side of the dichroic mirror II, a light filter I and a reflecting mirror IV are respectively arranged on two sides of the spectroscope, a reflecting mirror four-way light filter II and a convex lens II are connected with a CMOS camera, and one side of the light filter I is connected with a photomultiplier through the mechanical slit II and the convex lens III.
The high-frequency pulse laser killing system comprises a pulse laser and a function generator. One side of the function generator is provided with a pulse laser which is connected with the second reflecting mirror.
The distance between the mechanical slit I and the convex lens I is equal to the focal length of the convex lens I, the parameters of the dichroic mirror I are light with reflection wavelengths of 488nm, 532nm and 635nm, the parameters of the dichroic mirror II are light with reflection wavelengths of 488nm, 532nm, 635nm and 1064nm, the transmittance of the spectroscope is 90%, the reflectivity is 10%, and the parameters of the optical filters I and II are bandpass filters of 500-520 nm.
The included angles of the laser optical axis and the first reflecting mirror, the second reflecting mirror, the third reflecting mirror and the fourth reflecting mirror are 45 degrees respectively.
The laser wavelength emitted by the continuous wave laser is 488nm, 532nm and 635nm, the LED light source adopts green light with wavelength of 510-580nm, the wavelength range of the pulse laser is 780-2000 nm, the repetition frequency range is 1Hz-500KHz, and the pulse width is 1-100ns.
The application method of the non-invasive dynamic monitoring and killing device for the living circulation tumor cells comprises the following steps:
(1) The continuous wave laser emits blue laser, one-dimensional compression is carried out through a cylindrical lens, then the blue laser is focused through a convex lens after being reflected by a dichroic mirror I and a reflecting mirror I, and then the blue laser is focused on B16F10 cells expressing green fluorescent protein on an objective table after being reflected by a dichroic mirror II;
(2) The LED light source penetrates through the sample in the step (1), is focused by the objective lens, penetrates through the dichroic mirror II, the reflecting mirror III and the spectroscope, enters the CMOS camera through the reflecting mirror IV, the optical filter II and the convex lens II, and images B16F10 cells expressing green fluorescent protein in the computer;
(3) The method comprises the following steps that (1) fluorescent signals generated after B16F10 cells expressing green fluorescent proteins are excited by laser emitted by a continuous wave laser are focused by an objective lens, then pass through a dichroic mirror II, a reflecting mirror III and a spectroscope, enter a photomultiplier through a filter I, a mechanical slit II and a convex lens III, and the green fluorescent signals collected by the photomultiplier are input into a computer for counting;
(4) The function generator provides pulse signals to trigger the pulse laser, and the generated pulse laser sequentially passes through the reflecting mirror II, the dichroic mirror I, the reflecting mirror I, the convex lens I and the dichroic mirror II to be focused on the B16F10 cells marked by the green fluorescent protein monitored by the object stage, so that the cells are gasified and dead due to overhigh local temperature.
As shown in FIG. 2, the signal peaks represent the green fluorescent signal generated by B16F10-GFP as monitored by the moving stage, indicating that the real-time dynamic monitoring system of circulating tumor cells of the device can work normally.
As shown in fig. 3, a is a graph of cell viability by increasing laser intensity irradiation of B16F10 cells on the stage, staining with trypan blue, and B is a graph of cell mortality versus laser flux. As can be seen from FIG. 3, the laser flux capable of effectively killing B16F10-GFP cells was 8.8J/cm 2, and subsequent animal experiments as well as clinical experiments were performed based on the laser flux.
The invention realizes the verification of the disinfection effect of 1064nm pulse laser on circulating tumor cells by means of in-vivo fluorescence flow cytometry, and can clinically clear the circulating tumor cells by 1064nm pulse laser and simultaneously treat cancers in a mode of matching with operations, medicines and the like.
Specifically, the in-vitro and in-vivo experiments can verify the sterilizing effect by using the noninvasive dynamic monitoring and sterilizing device for the living circulation tumor cells, wherein in-vitro experiments are to irradiate target tumor cells transfected by fluorescent proteins in different areas on a glass slide by using 1064nm pulse laser, and the optimal energy density of the sterilized target tumor cells is found out by adjusting the laser power; in vivo experiments are to inject target tumor cells transfected by fluorescent protein into the subcutaneous of a mouse, construct a subcutaneous tumor model, periodically irradiate ear blood vessels of the mouse by using the pulse laser optimal energy density determined by in vitro experiments, and periodically monitor the number of the target circulating tumor cells by using in vivo fluorescence flow cytometry. In clinical application, 1064nm pulse lasers with different energy densities can be used for irradiating blood vessels of patients suffering from different types of tumors, so that the treatment effect is achieved.
Therefore, the invention adopts the device and the method for the noninvasive dynamic monitoring and the sterilization of the living circulating tumor cells, and solves the problem that the prior art can only realize the noninvasive real-time dynamic monitoring of the circulating tumor cells, but can not sterilize the monitored circulating tumor cells.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention and not for limiting it, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that: the technical scheme of the invention can be modified or replaced by the same, and the modified technical scheme cannot deviate from the spirit and scope of the technical scheme of the invention.
Claims (4)
1. The utility model provides a living body circulation tumor cell noninvasive dynamic monitoring and kill device which characterized in that: the system comprises a circulating tumor cell real-time dynamic monitoring system and a high-frequency pulse laser sterilizing system, wherein the circulating tumor cell real-time dynamic monitoring system comprises a continuous wave laser, a laser optical axis, an objective lens, an objective table, a CMOS camera, a computer and a photomultiplier, the objective table is arranged above the objective lens, an LED light source is arranged above the objective table, and the CMOS camera and the photomultiplier are connected with the computer; the high-frequency pulse laser killing system comprises a pulse laser and a function generator;
A cylindrical lens is arranged on one side of the continuous wave laser, a mechanical slit I is arranged at the focal length of the cylindrical lens, a dichroic mirror I is arranged on one side of the mechanical slit I, a reflecting mirror I and a reflecting mirror II are respectively arranged on two sides of the dichroic mirror I, the reflecting mirror I is connected with an objective lens through a convex lens I and a dichroic mirror II, a reflecting mirror III and a spectroscope are sequentially arranged on one side of the dichroic mirror II, a light filter I and a reflecting mirror IV are respectively arranged on two sides of the spectroscope, a reflecting mirror four-way filtering sheet II and a convex lens II are connected with a CMOS camera, and one side of the light filter I is connected with a photomultiplier through the mechanical slit II and the convex lens III;
The distance between the first mechanical slit and the first convex lens is equal to the focal length of the first convex lens, the parameters of the first dichroic mirror are light with reflection wavelengths of 488nm, 532nm and 635nm, the parameters of the second dichroic mirror are light with reflection wavelengths of 488nm, 532nm, 635nm and 1064nm, the transmittance of the spectroscope is 90%, the reflectance is 10%, and the parameters of the first optical filter and the second optical filter are bandpass filters with wavelengths of 500-520 nm;
The application method of the living body circulating tumor cell noninvasive dynamic monitoring and killing device comprises the following steps:
(1) The continuous wave laser emits blue laser, one-dimensional compression is carried out through a cylindrical lens, then the blue laser is focused through a convex lens after being reflected by a dichroic mirror I and a reflecting mirror I, and then the blue laser is focused on a blood vessel to be detected of a living animal or human body on an objective table after being reflected by a dichroic mirror II;
(2) The LED light source penetrates through the sample in the step (1), is focused by the objective lens, penetrates through the dichroic mirror II, the reflecting mirror III and the spectroscope, enters the CMOS camera through the reflecting mirror IV, the optical filter II and the convex lens II, and images tumor cells flowing through the window in the computer;
(3) The tumor cells flowing through the window in the step (1) are excited by laser emitted by a continuous wave laser and then generate fluorescent signals, after being focused by an objective lens, the fluorescent signals pass through a dichroic mirror II, a reflecting mirror III and a spectroscope, enter a photomultiplier through a filter I, a mechanical slit II and a convex lens III, and the green fluorescent signals collected by the photomultiplier are input into a computer for counting;
(4) The function generator provides pulse signals to trigger the pulse laser, and the generated pulse laser sequentially passes through the reflecting mirror II, the dichroic mirror I, the reflecting mirror I, the convex lens I and the dichroic mirror II to be focused on tumor cells which are monitored by the object stage and flow through the window.
2. The device for noninvasive dynamic monitoring and killing of living circulating tumor cells according to claim 1, wherein the device comprises: and one side of the function generator is provided with a pulse laser, and the pulse laser is connected with the second reflecting mirror.
3. The device for noninvasive dynamic monitoring and killing of living circulating tumor cells according to claim 1, wherein the device comprises: the included angles of the laser optical axis and the first reflecting mirror, the second reflecting mirror, the third reflecting mirror and the fourth reflecting mirror are 45 degrees respectively.
4. The device for noninvasive dynamic monitoring and killing of living circulating tumor cells according to claim 1, wherein the device comprises: the laser wavelength emitted by the continuous wave laser is 488nm, 532nm and 635nm, the LED light source adopts green light with the wavelength of 510-580nm, the wavelength range of the pulse laser is 780-2000 nm, the repetition frequency range is 1Hz-500KHz, and the pulse width is 1-100ns.
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| US20150335741A1 (en) * | 2008-12-12 | 2015-11-26 | Board Of Trustees Of The University Of Arkansas | In vivo photoacoustic and photothermal nano-theranostics of biofilms |
| WO2011030828A1 (en) * | 2009-09-09 | 2011-03-17 | 学校法人東海大学 | Method for killing tumor cells selectively and apparatus for the method |
| CN105286805A (en) * | 2015-09-28 | 2016-02-03 | 周辉 | Noninvasive circulating tumor cell detection and diagnosis device and detection and diagnosis method thereof |
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| CN116328202A (en) * | 2023-02-16 | 2023-06-27 | 南方医科大学深圳医院 | Tumor laser treatment device |
| CN117204818A (en) * | 2023-09-06 | 2023-12-12 | 北京信息科技大学 | Method to obtain photoacoustic signals of melanoma CTCs with high signal-to-noise ratio in vivo |
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