A photoacoustic system for detecting circulating tumor cells in freely moving mice

Abstract

The present invention discloses a photoacoustic system for detecting circulating tumor cells in freely moving mice. The photoacoustic system includes a light source component, an optical fiber coupling component, a micro-electromechanical scanning galvanometer component, a signal acquisition component, a body surface fixing component and a computer component. The photoacoustic system adopts a wearable design. The micro-electromechanical scanning galvanometer controls the laser to perform reciprocating line scanning. The scanning direction is perpendicular to the blood flow direction. The reciprocating line scanning collects each cross section of a single cell to obtain a high-resolution image of the dynamic flow cell. Based on the difference in absorption of two wavelengths of light by red blood cells and melanoma tumor cells, tumor cells in the blood of freely moving mice are identified, and information such as the morphology, size and concentration of the cells is obtained based on image processing methods. The present invention provides a high-resolution photoacoustic cell detection imaging system that is small in size and can be worn for a long time, which can detect circulating tumor cells in mice for a long time.

Description

Technical Field

[0001] The invention belongs to the technical field of medical imaging equipment, and in particular relates to a photoacoustic system for detecting circulating tumor cells in freely moving mice. Background Art

[0002] At present, the methods used in medicine to detect circulating tumor cells are mainly divided into liquid biopsy, microfluidic chips, optical imaging and other methods. Liquid biopsy uses non-invasive sampling and has the potential to monitor circulating tumor cells for a long time. However, due to the extremely low content of tumor cells, there will be a certain degree of randomness in a small amount of blood samples; in addition, detection methods that rely on external targeted markers will have certain false negatives. The emerging microfluidic chips can distinguish tumor cells based on physical properties such as cell shape, charge, and density. However, the physical properties of most tumor cells are not significantly different from those of white blood cells, resulting in low specificity of this detection method. Optical imaging methods can distinguish and image tumor cells by detecting specific chemical bonds or using fluorescent labeling methods, but blood samples need to be pre-processed, which makes the detection process often cumbersome; the uneven distribution of tumor cells leads to a certain degree of randomness in the test results.

[0003] Both photoacoustic imaging and optical coherence tomography can detect and image in vivo cells, but the imaging contrast of optical coherence tomography is low, making it difficult to automatically detect circulating tumor cells. Photoacoustic imaging based on the photoacoustic effect uses ultrasound waves emitted by endogenous chromophores in biological tissues after absorbing light energy. It has the same high resolution as optical imaging and a similar imaging depth as ultrasound imaging, and can provide high-contrast imaging without labeling.

[0004] Based on the difference in absorption of different wavelengths of illumination light by hemoglobin and melanin, photoacoustic imaging can easily detect circulating tumor cells in the blood using near-infrared wavelength excitation light. Compared with liquid biopsy, microfluidic chips, and traditional optical imaging, photoacoustic technology does not require blood processing and can even detect blood in vivo. However, the optical path of the photoacoustic microscopy system based on mechanical scanning is complex, and the scanning and imaging speeds are slow, making it difficult to detect and image flowing cells. Although the galvanometer-based photoacoustic microscopy system has improved the scanning speed, the device is large and difficult to move, and can only monitor anesthetized or motion-restricted mice. The current photoacoustic imaging technology makes it difficult to achieve long-term monitoring of circulating tumor cells in freely moving mice. Summary of the invention

[0005] 1. Technical issues to be resolved

[0006] The present invention proposes a high-resolution photoacoustic cell detection imaging system that is small in size and can be worn for a long time, in order to solve the problem that the existing photoacoustic imaging technology cannot detect circulating tumor cells in freely moving mice for a long time.

[0007] (II) Technical solution

[0008] In order to solve the technical problem, the present invention provides a photoacoustic system for detecting circulating tumor cells in freely moving mice, and the specific technical solution is as follows.

[0009] 1. A photoacoustic system for detecting circulating tumor cells in freely moving mice, characterized in that the photoacoustic system comprises a light source component, an optical fiber coupling component, a micro-electromechanical scanning galvanometer component, a signal acquisition component, a body surface fixing component and a computer component;

[0010] The light source assembly includes two nanosecond pulse lasers for providing pulse lasers with a first wavelength and a second wavelength, respectively, the two wavelengths of pulse lasers corresponding to the absorption of hemoglobin and the absorption of melanin, respectively; the photoacoustic system identifies tumor cells in the blood according to the difference in absorption of the two wavelengths of light by red blood cells and melanin tumor cells;

[0011] The fiber coupling assembly includes an objective lens, an optical fiber, an optical fiber holder, a three-dimensional translation stage, and a collimating lens, and is connected to a light source assembly;

[0012] The micro-electromechanical scanning galvanometer assembly includes a double cemented lens, a right-angle prism, a dichroic mirror, a micro-electromechanical scanning galvanometer and a light-transmitting anti-acoustic device; a wearable design is adopted, and during the photoacoustic imaging process, the micro-electromechanical scanning galvanometer controls the laser scanning, and the light-transmitting anti-acoustic device and multi-wavelength illumination are used to realize the detection of circulating tumor cells in freely moving mice;

[0013] The signal acquisition component includes an ultrasonic transducer, a signal amplifier and a data acquisition card; the signal acquisition component is connected to the micro-electromechanical scanning galvanometer component, and the ultrasonic transducer is fixed in the system frame;

[0014] The body surface fixation component includes a system frame, a skin fixation sheet and a connection module; the system frame is a 3D printed shell, which contains a scanning component and a collection component; the skin fixation sheet is used to unfold the mouse skin and fix it on the mouse body surface through biological glue, and the skin fixation sheet and the system frame are magnetically connected through an embedded magnet;

[0015] The computer component comprises a computer, which is equipped with acquisition control software and data processing software, and is also equipped with a signal generation card and a data acquisition card for controlling a micro-electromechanical scanning galvanometer.

[0016] Preferably, the connection relationship and working mode between the various components are as follows:

[0017] The pulse laser emitted by the first wavelength nanosecond pulse laser and the laser emitted by the second wavelength nanosecond pulse laser are respectively filtered out of stray light by a spatial filter formed by an objective lens and a pinhole aperture to form a collimated Gaussian beam, which is then focused by the objective lens into a single-mode optical fiber; the optical fiber ends of the two nanosecond pulse lasers are connected to a variable-focus collimating lens and fixed on the system frame;

[0018] The two wavelengths of light are adjusted to be coaxial using a dichroic mirror. After being coaxial, the two beams of illumination light are reflected by a right-angle prism and focused by a double-cemented lens with a high numerical aperture, and then irradiated on the micro-electromechanical scanning galvanometer. The light beam forms a 45-degree angle with the mirror surface of the micro-galvanometer. The reflected light passes through a light-transmitting anti-acoustic device and is vertically focused on the skin surface where the skin fixing sheet is attached. Driven by the micro-galvanometer, the focused pulse laser performs a line scan on the target within the response range of the ultrasound probe.

[0019] The photoacoustic signals generated by the tumor cells in the skin blood vessels are reflected by the light-transmitting anti-acoustic device to the surface of the ultrasound detector, where they are received and converted into electrical signals. After passing through the signal amplifier, the noise is filtered out to obtain a photoacoustic signal with a high signal-to-noise ratio. The signal is then received by the data acquisition card and stored in the computer. The computer processes the collected signals in real time and extracts the information of circulating tumor cells in the blood.

[0020] Preferably, the micro-electromechanical scanning galvanometer of the photoacoustic system controls the laser to perform reciprocating line scanning, and the scanning direction is perpendicular to the blood flow direction; each cross section of a single cell is accurately scanned by reciprocating line scanning with a sub-micron step size, and the collected signal is subjected to Hilbert transform and maximum projection method to obtain a cross-sectional image of the cell; the cross-sectional images are sorted according to the scanning order to obtain a high-resolution image of the dynamic flow cell, and the detailed information of the cell's morphological size and internal structure is obtained based on image processing means.

[0021] Preferably, the photoacoustic system automatically identifies tumor cells in the blood; specifically, based on the obtained high-resolution image of the dynamic flow cells, a cell size threshold is pre-set, and cells larger than the cell size threshold are preliminarily identified as tumor cells; for the result object preliminarily identified as a tumor cell, a photoacoustic signal amplitude threshold is further set, and if its photoacoustic signal amplitude is greater than the photoacoustic signal amplitude threshold, it is finally identified as a tumor cell.

[0022] Preferably, the detected tumor cells are automatically counted using a computer program built into the photoacoustic system; at the same time, the blood is detected using a laser of a first wavelength, the diameter of the blood vessels and three-dimensional information are calculated based on the image information, and the blood flow velocity is calculated based on the photoacoustic Doppler method; based on the calculated blood flow information and the number of tumor cells counted, the concentration of tumor cells in the blood is calculated.

[0023] Preferably, the first wavelength of the light source assembly is 532 nm, and the second wavelength is 770 nm.

[0024] Preferably, the pulse widths of the two pulse lasers provided by the light source assembly are both 1ns-2ns, and the repetition frequencies are both 50KHz.

[0025] Preferably, a delay of 3-8 microseconds is set between the two pulsed nanosecond lasers of the light source assembly.

[0026] (III) Beneficial effects

[0027] Compared with the prior art, the present invention has significantly positive technical effects, and its beneficial effects are at least reflected in the following aspects.

[0028] (1) The device of the present invention adopts a dual-wavelength illumination mechanism, which can not only detect circulating tumor cells in the blood, but also measure blood vessel diameter and blood flow. It does not require blood sampling and can directly monitor mice in vivo for a long time without affecting the normal activities of the mice.

[0029] (2) Compared with the traditional photoacoustic microscope, the device of the present invention has a simple structural design and compact connection, which directly realizes miniaturization. In addition, the system is inexpensive, easy to debug and use, and can be directly fixed on the surface of the mouse to achieve wearable imaging, which is easy to apply and promote.

[0030] (3) The method of the present invention is based on a micro-electromechanical scanning mechanism, which uses a voltage-controlled micro-vibrator for rapid line scanning and uses an ultrasonic detector to detect the photoacoustic signals at each position on the scanning trajectory for image reconstruction, thereby achieving rapid high-resolution imaging and real-time detection feedback.

[0031] (4) The existing technologies have problems such as low resolution, limited detection capability, and reliance on exogenous labels. These technical solutions are often unable to detect tumor cells in in vivo blood vessels. However, the present application, by utilizing the high penetration characteristics of photoacoustic imaging and an independently designed imaging system, can provide high-resolution imaging of tumor cells in in vivo blood vessels, thereby realizing the detection and monitoring of unlabeled tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 A schematic diagram of the system structure of an embodiment of the present invention

[0033] The names of the components shown in the figure are: function generator 1-1, pump pulse laser (532nm) 1-2, pump pulse laser (770nm) 1-3; lens 2-1, aperture diaphragm 2-2, fiber holder 2-3, variable focus collimating lens 2-4; dichroic mirror 3-1, double glued lens 3-2, micro scanning galvanometer 3-3, light-transmitting anti-sound device 3-4, water tank 3-5, ultrasonic transducer 4-1, signal amplifier 4-2, signal filter 4-3, data acquisition card 4-4; small animal surface fixer 5-1, computer 6-1, signal generating card 6-2, surface blood vessels 7-1, tumor cells 7-2. DETAILED DESCRIPTION

[0034] In order to solve the technical problem, the present invention provides a photoacoustic system for detecting circulating tumor cells in freely moving mice. The technical solution of the present invention is further described below by means of specific implementation methods in conjunction with the accompanying drawings of the specification.

[0035] Figure 1 An embodiment of the present invention is shown, and the connection relationship of various subcomponents of the photoacoustic system is shown in the figure.

[0036] The system working principle of this embodiment is as follows.

[0037] The pulsed laser emitted by the 532nm laser and the laser emitted by the 770nm laser are respectively filtered out by a spatial filter formed by an objective lens and a pinhole aperture to form a collimated Gaussian beam, which is then focused into a single-mode optical fiber through an objective lens. The ends of the 532nm and 770nm optical fibers are connected to a variable-focus collimating lens and fixed on the frame of the scanning galvanometer assembly. The 532nm light is coaxial with the 770nm light after being reflected by a right-angle prism and a dichroic mirror. The two coaxial illumination lights are reflected by a right-angle prism and focused by a double-cemented lens with a high numerical aperture to illuminate the mirror surface of the micro-scanning galvanometer, and the light beam is at a 45-degree angle to the mirror surface of the micro-galvanometer. The reflected illumination light passes through a water tank and the light-transmitting and anti-sound device therein, and is vertically focused on the imaging target surface plane. At this time, the focused pulsed laser is driven by the micro-galvanometer to perform a line scan of the target within the response range of the ultrasonic probe.

[0038] Since hemoglobin has a strong absorption characteristic for 532nm excitation light, it produces a thermal expansion effect after absorbing light energy, generating ultrasonic waves, i.e. photoacoustic signals. Melanin in melanoma cells has a strong absorption of 770nm laser, while hemoglobin has a very low absorption, and the ultrasonic waves it produces are very weak and can be ignored. The photoacoustic signal generated by each excitation point within the scanning range is reflected by the light-transmitting anti-sound device to the surface of the ultrasonic detector, where it is received and converted into an electrical signal. After the signal is amplified by the amplifier, the noise is filtered out, and the photoacoustic signal with a good signal-to-noise ratio is obtained, which is then received and stored by the data acquisition card for image reconstruction.

[0039] Due to the difference in light absorption between hemoglobin and circulating tumor cells, the photoacoustic system can use 532nm laser to find the location of blood vessels and obtain the diameter and flow of blood vessels, and use 770nm laser to monitor circulating tumor cells. In order to avoid the superposition of ultrasonic signals under 532nm and 770nm excitation light, there will be a delay of 3-8μs between the two lasers.

[0040] Since the focused laser beam is scanned correspondingly within the focal area of ​​the ultrasonic detector, the ultrasonic detector can detect the photoacoustic signals generated at each scanning position without moving. On the other hand, since the difference in the optical refractive index between the glass sheet and the coupling liquid in the light-transmitting anti-sound device is small, the loss of light is small, and the difference in the acoustic impedance between the glass sheet and the coupling liquid is large, the ultrasonic reflection efficiency is high, so the detected photoacoustic signal has a good signal-to-noise ratio.

[0041] The specific implementation cases described in this application are merely examples of the main ideas of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in similar ways, but they will not deviate from the spirit of the present invention or exceed the scope defined by the attached claims.

Claims

1. A photoacoustic system for detecting circulating tumor cells in freely moving mice, characterized in that: The photoacoustic system includes a light source component, an optical fiber coupling component, a micro-electromechanical scanning galvanometer component, a signal acquisition component, a body surface fixing component and a computer component; The light source assembly includes two nanosecond pulse lasers for providing pulse lasers with a first wavelength and a second wavelength, respectively, the two wavelengths of pulse lasers corresponding to the absorption of hemoglobin and the absorption of melanin, respectively; the photoacoustic system identifies tumor cells in the blood according to the difference in absorption of the two wavelengths of light by red blood cells and melanin tumor cells; The fiber coupling assembly includes an objective lens, an optical fiber, an optical fiber holder, a three-dimensional translation stage, and a collimating lens, and is connected to a light source assembly; The micro-electromechanical scanning galvanometer assembly includes a double cemented lens, a right-angle prism, a dichroic mirror, a micro-electromechanical scanning galvanometer and a light-transmitting anti-acoustic device; a wearable design is adopted, and during the photoacoustic imaging process, the micro-electromechanical scanning galvanometer controls the laser scanning, and the light-transmitting anti-acoustic device and multi-wavelength illumination are used to realize the detection of circulating tumor cells in freely moving mice; The signal acquisition component includes an ultrasonic transducer, a signal amplifier and a data acquisition card; the signal acquisition component is connected to the micro-electromechanical scanning galvanometer component, and the ultrasonic transducer is fixed in the system frame; The body surface fixation component includes a system frame, a skin fixation sheet and a connection module; the system frame is a 3D printed shell, which contains a scanning component and a collection component; the skin fixation sheet is used to unfold the mouse skin and fix it on the mouse body surface through biological glue, and the skin fixation sheet and the system frame are magnetically connected through an embedded magnet; The computer component comprises a computer, which is equipped with acquisition control software and data processing software, and is also equipped with a signal generation card and a data acquisition card for controlling a micro-electromechanical scanning galvanometer.

2. The photoacoustic system for detecting circulating tumor cells in freely moving mice according to claim 1, characterized in that: The connection relationship and working mode between each component are as follows: The pulse laser emitted by the first wavelength nanosecond pulse laser and the laser emitted by the second wavelength nanosecond pulse laser are respectively filtered out of stray light by a spatial filter formed by an objective lens and a pinhole aperture to form a collimated Gaussian beam, which is then focused by the objective lens into a single-mode optical fiber; the optical fiber ends of the two nanosecond pulse lasers are connected to a variable-focus collimating lens and fixed on the system frame; The two wavelengths of light are adjusted to be coaxial using a dichroic mirror. After being coaxial, the two beams of illumination light are reflected by a right-angle prism and focused by a high numerical aperture doublet lens with an aperture value of 0.4-0.

6. They are then irradiated on the micro-electromechanical scanning galvanometer, and the light beam forms a 45-degree angle with the mirror surface of the micro-galvanometer. The reflected light passes through a light-transmitting anti-acoustic device and is vertically focused on the skin surface where the skin fixing sheet is attached. Driven by the micro-galvanometer, the focused pulse laser performs a line scan on the target within the response range of the ultrasound probe. The photoacoustic signals generated by the tumor cells in the skin blood vessels are reflected by the light-transmitting anti-acoustic device to the surface of the ultrasound detector, where they are received and converted into electrical signals. After passing through the signal amplifier, the noise is filtered out to obtain a photoacoustic signal with a high signal-to-noise ratio. The signal is then received by the data acquisition card and stored in the computer. The computer processes the collected signals in real time and extracts the information of circulating tumor cells in the blood.

3. The photoacoustic system for detecting circulating tumor cells in freely moving mice according to claim 2, characterized in that: The photoacoustic system's microelectromechanical scanning galvanometer controls the laser to perform reciprocating line scanning, with the scanning direction perpendicular to the direction of blood flow. It uses reciprocating line scanning with submicron steps to accurately scan each cross-section of a single cell, and obtains a cross-sectional image of the cell by subjecting the collected signal to Hilbert transform and maximum projection method. The cross-sectional images are sorted in the scanning order to obtain high-resolution images of the dynamic flowing cells, and detailed information on the cell's morphological size and internal structure is obtained based on image processing methods.

4. The photoacoustic system for detecting circulating tumor cells in freely moving mice according to claim 3, characterized in that: The photoacoustic system automatically identifies tumor cells in the blood; based on the high-resolution images of the dynamic flow cells obtained, a cell size threshold is pre-set, and cells larger than the cell size threshold are preliminarily identified as tumor cells; for the result objects preliminarily identified as tumor cells, a photoacoustic signal amplitude threshold is set, and if the photoacoustic signal amplitude is larger than the photoacoustic signal amplitude threshold, it is finally identified as a tumor cell.

5. A photoacoustic system for detecting circulating tumor cells in freely moving mice according to claim 4, characterized in that: The computer program built into the photoacoustic system is used to automatically count the detected tumor cells. At the same time, the blood is detected using a laser of the first wavelength, and the diameter and three-dimensional information of the blood vessels are calculated based on the image information, and the blood flow velocity is calculated based on the photoacoustic Doppler method. The concentration of tumor cells in the blood is calculated based on the calculated blood flow information and the number of tumor cells counted.

6. The photoacoustic system for detecting circulating tumor cells in freely moving mice according to any one of claims 1 to 5, characterized in that: The first wavelength of the light source assembly is 532nm, and the second wavelength is 770nm.

7. The photoacoustic system for detecting circulating tumor cells in freely moving mice according to any one of claims 1 to 5, characterized in that: The pulse widths of the two pulse lasers provided by the light source assembly are both 1 ns-2 ns, and the repetition frequencies are both 50 kHz.

8. The photoacoustic system for detecting circulating tumor cells in freely moving mice according to any one of claims 1 to 5, characterized in that: A delay of 3-8 μm is set between the two pulse nanosecond lasers of the light source assembly.

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