A real-time cerebral vascular morphology detection system based on second harmonic imaging

Abstract

The present application belongs to the technical field of biological detection, and particularly discloses a cerebral vascular shape real-time detection system based on second harmonic imaging. The present application comprises two parts of a second harmonic imaging device and a software system. A femtosecond two-photon laser scanning microscope is used to perform in-vivo imaging detection on cerebral vessels, and the collagen molecules in the cerebral vessel wall are the detection objects. Experiments prove that the present application uses collagen in the vessel wall as the target molecules, and the blood vessels show a structure of a hollow in the middle and two thin lines on both sides in the imaging, wherein the hollow part is the blood vessel cavity, and the two thin lines are the blood vessel walls on both sides. The present application system is suitable for the detection of living brain, has the advantages of high spatiotemporal resolution, no need for exogenous molecular labeling, and the like, and can detect the cerebral vascular state in real time, quickly and accurately, and has a wide application prospect in the pathological examination of brain in clinical medicine.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a label-free real-time detection system for cerebral vascular morphology. Background Technology

[0002] The vascular structure in animals (including humans) is mainly divided into two parts: the vessel wall and the vessel lumen. The vessel lumen is the channel for serum and blood cells, while the vessel wall, located on the outer side of the lumen, protects the vessel and restricts the flow of components between the inside and outside of the vessel. Compared with other blood vessels in the body, blood vessels in the brain also play a role in connecting the circulatory system and the nervous system. The vessel walls of cerebral blood vessels not only protect the blood vessels and isolate blood from nerves, but are also an important component of the blood-brain barrier.

[0003] The walls of cerebral blood vessels consist of a cellular layer and an extracellular matrix layer. The cellular layer is mainly composed of endothelial cells and smooth muscle cells, while the extracellular matrix layer is mainly composed of collagen fibers and elastic fibers. Existing research has shown that the collagen constituting the blood vessel wall is mainly type I and type III. Both of these collagen subtypes have a special non-centrosymmetric structure, which exhibits second-harmonic emission properties. This is an inherent endogenous luminescent property of collagen molecules, thus allowing for imaging without the need for exogenous fluorescent markers.

[0004] Collagen with second harmonic characteristics mainly includes two features: first, the wavelength of the emitted light signal is exactly equal to 1 / 2 of the wavelength of the incident light; second, the emitted light signal has polarized light characteristics, and the optimal polarization angle has a period of 180°.

[0005] Current cerebral vascular imaging research primarily focuses on brain slices, falling under the category of in vitro studies. The limitations of in vitro studies include the inability to investigate vascular function under real physiological or pathological conditions, the significant damage caused by the high energy of the light source to the slice samples, and poor reproducibility of operations on the same sample. In vivo imaging using live animals as samples can overcome these shortcomings. Live animal imaging mainly relies on femtosecond two-photon laser scanning microscopes, which offer advantages such as high spatial resolution, high sensitivity, high specificity, and minimal sample damage. By modifying the hardware and designing the software of the two-photon scanning microscope to enable second harmonic imaging, the goal of observing and analyzing the fine structure of cerebral blood vessel walls in vivo can be achieved.

[0006] The core of this invention is to design a system with unique hardware and software based on a two-photon laser imaging device, which enables the system to acquire label-free second harmonic imaging images of animal cerebral blood vessel walls in real time and to perform quantitative analysis on various parameters related to cerebral blood vessel morphology. Summary of the Invention

[0007] The purpose of this invention is to provide a system for real-time detection of cerebral vascular morphology based on second harmonic imaging with high spatial resolution.

[0008] The real-time cerebral vascular morphology detection system based on second harmonic imaging provided by this invention comprises two parts: a second harmonic imaging device and a software system; wherein:

[0009] The second harmonic imaging device, as a hardware component, has the following structure: Figure 2 As shown, the system includes a femtosecond laser 1, a polarizer 2, a scanning galvanometer 3, a microscope system 4, a dichroic mirror 5, a microscope objective 6, a second harmonic filter 7, a fluorescence filter 8, an image acquisition / display computer 9, a laser control computer 10, a main program control computer 11, and an analysis and calculation computer 12. Its optical path is as follows: the femtosecond laser 1 emits laser light, which passes through the polarizer 2, the scanning galvanometer 3, and is reflected at a 90-degree angle into the microscope system 4. Finally, the laser light passes through the microscope objective 6 and illuminates the object under test. The reflected light is split into two paths after passing through the dichroic mirror 5, passing through the second harmonic filter 7 and the fluorescence filter 8 respectively. The imaging signal enters the image acquisition / display computer 9 for real-time monitoring, and finally the acquired signal is transmitted to the analysis and calculation computer 12 for analysis and calculation. The laser excitation and power adjustment of the femtosecond laser 1 are controlled by the laser control computer 10; the operation of the scanning galvanometer 3 and the microscope objective 6 is controlled by the main program control computer 11.

[0010] The femtosecond laser 1 provides a stable pulsed laser source with a laser pulse emission interval of 100 femtoseconds, and the emitted laser spot remains highly convergent throughout the optical path. The laser emitted by the femtosecond laser 1 can excite second-harmonic emission of collagen molecules in the extracellular matrix of the inner and outer membranes of cerebral blood vessels. This second-harmonic phenomenon can be used to detect collagen molecules in cerebral blood vessels. The excitation wavelength range of the femtosecond laser 1 is 710-910 nanometers, which covers the entire excitation spectrum of type I / III collagen subtypes.

[0011] The polarizing mirror 2 is used to filter the laser emitted by the femtosecond laser 1, allowing only laser light with a specific polarization angle to pass through. The polarizing mirror is continuously adjustable within the range of 0-359°, and the polarization angle can be set as needed. The polarizing mirror 2 includes a rotatable disk with graduations accurate to 1°. A short line on the top of the polarizing mirror serves as an indicator graduation. When a graduation on the disk is aligned with the polarizing mirror's indicator graduation, the polarizing mirror only allows transmission of laser light at the angle indicated by that graduation (i.e., allows laser light with a specific polarization angle to pass through). Adjusting the polarizing mirror 2 allows laser light with a specific polarization angle to irradiate the brain blood vessel being tested. When the polarization angle of the laser is exactly equal to the polarization angle of the collagen molecules in the brain blood vessel wall, the detected optical signal is the second harmonic signal.

[0012] The scanning galvanometer 3 consists of two vertically placed mirrors. One mirror vibrates at high speed in the horizontal direction (called the X-mirror), and the other mirror vibrates at high speed in the vertical direction (called the Y-mirror), enabling the small spot of the laser source to achieve a large-area surface scanning. Specifically, the small spot first achieves a linear laser trajectory through the high-speed vibration of the X-mirror, and its trajectory is reflected onto the Y-mirror, where it then achieves a planar light trajectory through high-speed vibration. In this way, a rectangular plane can be scanned with a single spot.

[0013] The microscope system 4, as needed, incorporates lenses with different magnifications to magnify the laser beam to a specific magnification after passing through the microscope system. Furthermore, by adjusting the position and angle of the lens group, the laser beam propagates strictly along a predetermined optical path. The microscope system 4 allows the laser beam to pass through the optical path with minimal deflection and maximum magnification.

[0014] The microscope objective 6 can converge the divergent light spot from the microscope system 4 into an extremely precise light spot, focus the imaging plane on a specific position of the object under test, and observe different sizes of imaging fields through objective lenses with different magnifications.

[0015] The dichroic mirror 5 receives the reflected light from the microscope objective 6 illuminating the object under test. It allows unidirectional transmission of the laser light source, ensuring that the reflected light signal from the light source is reflected only by the dichroic mirror and cannot pass through. After reaching the dichroic mirror 5, the reflected light is split into two independent optical paths. The reflected light in one path passes through the second harmonic filter 7, while the reflected light in the other path passes through the fluorescence filter 8.

[0016] The second harmonic filter 7 selectively transmits the second harmonic signal of a specific wavelength reflected by the dichroic mirror 5. This signal is the source of light emission from collagen molecules in the extracellular matrix of the inner and outer membranes of cerebral blood vessels. Its central wavelength range is 355-455 nanometers, which covers the entire emission spectrum of type I / III collagen subtypes.

[0017] The fluorescent filter 8 selectively transmits fluorescence signals of a specific wavelength reflected by the dichroic mirror 5. In the absence of exogenous fluorescent molecular labeling, it is used only to detect molecules with autofluorescence in the cerebral cortex. After intravenous injection of a fluorescent indicator into an animal, it can detect exogenous fluorescence signals from blood cells within the blood vessel lumen. The purpose of detecting the autofluorescence signal is to correct data during analysis and calculation. Specifically, because the autofluorescence signal in the brain is relatively stable, there is almost no overexposure or attenuation of signal intensity or positional shift within the imaging time window. This makes it suitable for calibrating the stability of the light source and various components of the hardware system, and monitoring the normal physiological state of experimental animals. Only when all of the above conditions are normal does the analysis and calculation of the second harmonic imaging data have scientific significance. The purpose of detecting the fluorescence signal of the fluorescent indicator is to mark the blood flow in cerebral blood vessels. By observing the fluorescence signal, information such as the diameter of the blood vessel lumen and the direction of blood flow can be determined. The fluorescence signal representing the diameter of the blood vessel lumen and the second harmonic signal representing the diameter of the blood vessel wall together constitute the complete diameter information of a cerebral blood vessel.

[0018] The imaging signal acquisition / display computer 9 receives reflected light through the second harmonic filter 7 and the fluorescence filter 8. Its function is to acquire, display and record the imaging signal in real time. Its significance is to collect the location and morphological information of the target cerebral blood vessels to prepare for subsequent analysis and calculation. Then, the image is transmitted to the analysis and calculation computer 12.

[0019] The analysis and calculation computer 12 is connected to the imaging signal acquisition / display computer 9 via a local area network. It is used to receive imaging image data from the imaging signal acquisition / display computer 9 and further perform quantitative analysis on the second harmonic imaging image data through the calculation and analysis software module.

[0020] The software system mainly includes an image display module and an analysis and calculation module. Specifically:

[0021] The image display module, deployed in the imaging signal acquisition / display computer 9, includes: a real-time display program and an image post-viewing program, wherein:

[0022] The real-time display program is used to monitor the image acquisition process in real time. The main program control computer 11 controls the scanning galvanometer 3 to start working, and the image obtained from scanning the sample is displayed in real-time in the image display window of the imaging signal acquisition / display computer 9. This allows for real-time adjustment of the imaging field of view, including the horizontal and depth positions of the imaging focal plane, ultimately locating the desired imaging target area. The real-time display program only allows for real-time viewing of the image; it becomes ineffective after image acquisition is complete and cannot be used to view or modify the acquired image. To view or adjust image parameters of the acquired image, a post-image viewing program can be used.

[0023] The image post-viewing program is used to view the acquired 3D images and make simple adjustments. The acquired 3D images are single files; the software allows for frame-by-frame viewing by dragging the scroll bar. The image adjustment function allows for real-time adjustment of parameters such as brightness and contrast of the entire 3D image or individual frames. Note that the parameter adjustments made in the image post-viewing program are for better display of the image and do not enable quantitative analysis operations such as image analysis and calculation. Quantitative image analysis is performed by the analysis and calculation module.

[0024] The analysis and calculation module is deployed in the analysis computer 12, which is connected to the signal presentation / acquisition computer 9 via a local area network. It enables precise display, detail adjustment, and accurate analysis of 3D images. Using a programming language, the module can split the acquired 3D image into single-frame 2D images for display. It can also superimpose specific frames from a 3D image into a single 2D image, or superimpose all frames into a single 2D image. The analysis and calculation program allows for frame-by-frame correction between different 3D images, enabling equal calculations between two sets of images. Specific calculations include (but are not limited to): calculating the average diameter of cerebral blood vessels, calculating the imaging signal intensity of the cerebral blood vessel walls, and calculating the thickness of the cerebral blood vessel walls.

[0025] (1) Calculation of the average diameter of cerebral blood vessels: The image of the blood vessel wall is obtained by second harmonic imaging. The distance between the two blood vessel wall imaging signals is the diameter of the blood vessel. First, rotate the blood vessel to the vertical direction and draw a horizontal straight line that is perpendicular to the two blood vessel wall imaging signals. The absolute value of the length of the line segment between the two intersection points of the horizontal straight line and the imaging signal is the diameter of the blood vessel at that position. According to the imaging image, it can be found that there are undulations at the edge of the blood vessel lumen, so the line segments are also of different lengths. The blood vessel wall imaging signal is composed of several pixels in the vertical direction. The total number of pixels can be calculated by the analysis and calculation module. The length of the line segment between each pair of pixels (two corresponding pixels on the left and right blood vessel walls constitute a pair) can be calculated. It is necessary to calculate the length between all pairs of pixels in the vertical direction. Finally, the average of all lengths is calculated to obtain the average diameter of the blood vessel.

[0026] (2) Calculation of the signal intensity of cerebral blood vessel wall imaging; First, a second harmonic imaging image is obtained through an image acquisition / viewing program. Each pixel in the image is displayed as a 16-bit grayscale image. The signal intensity of the imaging image ranges from 0 to 255, and the unified unit of intensity value is written as au. Then, the average value of all intensity values ​​of the blood vessel wall is calculated as the average intensity value of the blood vessel wall. Since the optical lens is perpendicular to the horizontal section of the brain, the acquired imaging image is distributed from the surface to the inside along the vertical axis. Each set of data usually consists of several images forming a three-dimensional data set, with the spacing between each image being 0.5-10 micrometers. If two three-dimensional data sets are to be compared, three-dimensional correction is required: In the vertical direction, the data correction adopts a "layer-to-layer" strategy, with each layer representing a two-dimensional image. The maximum cross-correlation coefficient between the two data sets is calculated to determine the range to be superimposed for each set of data. In the horizontal direction, the data correction adopts a "pixel-to-pixel" cross-correlation analysis strategy. After calculating the displacement difference between the two data sets, the image is translated to finally achieve alignment correction between the data sets. Only after the imaging image has been corrected can the signal strength be calculated, compared, and analyzed.

[0027] (3) Calculation of the thickness of the cerebral blood vessel wall; Second harmonic imaging mainly acquires horizontally oriented blood vessels in the brain. Each blood vessel has two second harmonic signals of the blood vessel wall. First, the blood vessel is rotated to the vertical direction. The signal of each blood vessel wall has a peak in the row where each pixel point is located (i.e., "pixel row"). Then, Gaussian function fitting is performed on the row where each "pixel row" is located. The half width and height of each "pixel row" position can be calculated by Gaussian function fitting. Finally, the average value of all the half width and height values ​​of a blood vessel wall is calculated to obtain the thickness value of the blood vessel wall on one side; the same calculation is performed on the other side.

[0028] Furthermore, in this invention, the laser excitation and power adjustment of the femtosecond laser 1 are controlled by a laser control computer 10. Specifically, the laser control computer 10 is directly connected to the femtosecond laser 1, and this computer has a built-in control program to control the femtosecond laser 1, including controlling the opening and closing of the femtosecond laser 1 and adjusting the emitted laser wavelength. Here, adjusting the emitted laser wavelength uses a tunable scroll bar. By dragging the scroll bar (or entering a specific value in the input box), the femtosecond laser 1 emits laser light of the specified wavelength.

[0029] Furthermore, in this invention, the operation of the scanning galvanometer 3 is controlled by the main program control computer 11. The main program control computer 11 is connected to the scanning galvanometer 3 and controls the opening and closing of the scanning galvanometer 3, adjusting parameters such as the scanning frequency. Specifically, this includes controlling scanning without data archiving for dynamic observation of the selected imaging window; and controlling data archiving while scanning to save a three-dimensional image file for subsequent analysis. The scanning frequency of the scanning galvanometer 3 can be adjusted by inputting parameters into the input box of the built-in program interface of the corresponding computer 11. By setting different sampling rates, the scanning galvanometer 3 can vibrate at different frequencies. The lower the vibration frequency, the longer the laser stays at each pixel, and the more detailed information in the image.

[0030] Furthermore, in this invention, the operation of the microscope objective 6 is controlled by the main program control computer 11, which is connected to the microscope objective 6 and controls the spatial position of the microscope objective 6. Specifically, three input boxes can be set in the built-in program interface of the computer 11, corresponding to the X-axis, Y-axis and Z-axis positions of the objective 6 in space, respectively. By inputting specific values, the objective is moved to a specified position. Among them, the "Horizontal Zero" button is used to move the microscope objective 6 to a preset initial position in the XY-axis direction, and the "Vertical Zero" button is used to move the microscope objective 6 to a preset initial position in the Z-axis direction. The "Start" input box and the "End" input box can be used to input specific values, so that the microscope objective 6 moves continuously in the Z-axis direction within a specified value range.

[0031] This invention provides a system for real-time detection of cerebral vascular morphology. Through in vivo second harmonic imaging, it delivers cerebral vascular imaging signals with high spatiotemporal resolution, thereby obtaining cerebral vascular morphology-related information in a living state. This overcomes the limitation of real-time detection in ex vivo cerebral vascular imaging and also provides a label-free imaging system for clinical brain imaging. In this invention, the detection of cerebral vascular morphology uses the second harmonic signal generated by collagen in the vessel wall as a structural intrinsic parameter to observe information related to vascular structure, such as vessel diameter, vessel wall thickness, and collagen content in the vessel wall.

[0032] The system for real-time detection of cerebral vascular morphology provided by this invention has the following specific operating procedure:

[0033] (1) Preparation of imaging window: First, the skull is thinned by grinding, then the window is sealed with a coverslip, and agarose is used to fill the gap. Finally, the coverslip is fixed with adhesive.

[0034] (2) Place the microscope objective 6 on the window described in step (1) and collect data on the cerebral blood vessels through the hardware module and software module. The target of the signal acquisition is the second harmonic imaging signal of the blood vessels, specifically the second harmonic signal emitted by the collagen in the extracellular matrix of the inner and outer membranes of the blood vessel wall.

[0035] (3) In the hardware system described in step (2), the excitation wavelength of the femtosecond laser 1 is set to any wavelength in the range of 710-910nm. The excitation light is focused onto the blood vessels in the cerebral cortex by the microscope objective 6, and the generated reflected light signal is collected by the same microscope objective 6. The collected reflected light is further filtered by the second harmonic filter 7, and its effective wavelength range is 355-455 nm. The allowed wavelength value fluctuation range is any width of 10-40 nm. The cerebral blood vessels are scanned frame by frame through the full layer depth by the microscope objective 6. The interval between each frame of the frame scan is determined according to the specific blood vessel diameter, the depth of the blood vessel in the brain, and other factors. Usually, the minimum scanning interval is set to 0.5 micrometers and the maximum interval is set to 10 micrometers. The main program control computer 11 in the control module realizes the control of the spatial position of the microscope objective 6.

[0036] (4) The method described in step (3) is implemented by the graphics display module. First, the region with clear cerebral blood vessels is accurately located through the real-time display program. After acquiring a set of three-dimensional images, the selected region is analyzed by the image post-viewing program to determine if it is optimal. If the selected imaging region still needs to be adjusted, the above steps are repeated until the selected imaging region includes complete vascular wall brightness information and depth information. Second harmonic imaging is performed on the horizontally oriented blood vessels in the cerebral cortex. The obtained image is characterized by two thin, elongated line images. Between the two lines is a hollow background imaging signal region. The two lines are the two sides of the blood vessel wall, and the hollow region is the vascular cavity.

[0037] (5) Further analyze the blood vessel wall imaging image described in step (4), determine the effective range of the imaging signal through the image analysis calculation module, perform a fitting analysis on the half-width at half-maximum (WHM), and select the effective part of the fitting curve to obtain the effective range of 2-100 micrometers on both sides of the blood vessel wall signal; when selecting the blood vessel wall signal for quantitative analysis, it is necessary to accurately select the imaging data within the effective range, and the calculated specific WHM value can represent the thickness of the blood vessel wall.

[0038] (6) The second harmonic imaging signal of the blood vessel wall needs to be tested for polarization characteristics. A continuously adjustable polarizer 2 with a range of 0-359° is set in the hardware module. The control module of the polarizer 2 is adjusted to allow lasers to pass through at different angles. The polarization angle at which the second harmonic signal is strongest (i.e., the optimal polarization angle) and the polarization angle at which the signal is weakest (i.e., the background polarization angle) are found. There are two polarization angles that can obtain the strongest imaging signal. The difference between these two angles must be 180° (this difference is valid within a range of 20°). Similarly, there are two polarization angles that can obtain the weakest imaging signal. The difference between these two angles must also be 180° (this difference is valid within a range of 20°). The angle difference between the optimal polarization angle and the background polarization angle must be 90° (this difference is valid within a range of 20°).

[0039] (7) The signal intensity of the second harmonic imaging of the blood vessel wall described in step (4) is quantitatively analyzed by the analysis and calculation module. The calculated value represents the relative number of collagen molecules in the blood vessel wall. The higher the signal intensity, the more collagen molecules there are.

[0040] (8) The average distance between the two blood vessel wall imaging signals mentioned in step (4) can be calculated by the analysis and calculation module to obtain the diameter information of the blood vessel;

[0041] (9) The thickness of the blood vessel wall mentioned in step (5) is calculated by analyzing and calculating the half-width of each “pixel row” of the blood vessel wall on both sides. The average half-width of the blood vessel wall on each side is then calculated to obtain the average thickness of the two blood vessel walls.

[0042] The key feature of this invention is the modification of an existing, mature two-photon laser microscopy hardware system, along with the development of customized data analysis and computation software. This enables the acquisition of second harmonic distortion (HSD) images of mouse cerebral blood vessels and the quantitative analysis of various parameters related to cerebral vascular morphology. The advantages of this invention primarily include: the ability to study cerebral blood vessels in live animals; the ability to acquire high-resolution imaging signals of cerebral blood vessels in real time; the ability to detect intrinsic optical signals of blood vessels (i.e., without the need for external fluorescent markers); and the ability to rapidly and quantitatively assess various important parameters related to cerebral blood vessels, such as diameter, wall thickness, and collagen content. Attached Figure Description

[0043] Figure 1 This is the experimental flowchart of the present invention.

[0044] Figure 2 This is a diagram of a live second harmonic imaging device.

[0045] Figure 3It is a schematic diagram of cerebrovascular imaging image acquisition.

[0046] Figure 4 It is an in-vivo second harmonic / fluorescence imaging map of cerebral blood vessels. Among them, the left figure is the second harmonic imaging map; the middle figure is the fluorescence imaging map of the same area of the same blood vessel; the right figure is the superimposed map of the left figure and the middle figure. The arrows in the figure indicate the blood vessel walls on both sides, and the fluorescence signal indicates the position of the blood vessel lumen.

[0047] Figure 5 It is the polarization characteristic analysis of the second harmonic signal of the blood vessel wall, and the data is displayed in the form of a radar statistical chart.

[0048] Numbers in the figure: 1. Femtosecond laser, 2. Polarizer, 3. Scanning galvanometer, 4. Microscope system, 5. Dichroic mirror, 6. Microscopic objective lens, 7. Second harmonic filter, 8. Fluorescence filter, 9. Image display / acquisition computer, 10. Laser control computer, 11. Main program control computer, 12. Image analysis computer; 13. Blood vessel lumen, 14. Blood vessel wall, 15. Continuously acquired images, 16. Brain horizontal section. Specific implementation mode

[0049] Unless otherwise specified, the experimental methods used in the examples are all conventional methods. The materials, reagents, etc. used in the examples can be obtained through commercial channels unless otherwise specified. The protection scope of the present invention is not limited to the following examples.

[0050] The experimental animals were C57BL / 6 mice, 4 - 6 weeks old, weighing 20 - 25 g, provided by Shanghai Slake Experimental Animal Co., Ltd. (license number SCXK (Shanghai) 2012 - 0002). Hardware system: Femtosecond laser 1 (model MaiTai DeepSee, parameters 100 fs, 80 MHz), polarizer 2, Spectra-Physics. Scanning galvanometer 3, Cambridge. Microscope system 4, dichroic mirror 5, Sutter (MOM). Microscopic objective lens 6, second harmonic filter 7, Olympus. Fluorescence filter 8, Chroma. Image acquisition / display computer 9, laser control computer 10, main program control computer 11, analysis calculation computer 12, DELL. Software system: The programs in the control module, image display module, and calculation analysis module are all written in the programming language Matlab (Mathworks).

[0051] Example 1: An imaging system for real-time in-vivo second harmonic detection of cerebral blood vessels

[0052] The implementation flowchart is as Figure 1This includes the design and assembly of the in-body second harmonic imaging system hardware, the display of second harmonic imaging images, and the analysis and calculation of second harmonic imaging images. Specifically:

[0053] I. Design and Assembly of In Vivo Second Harmonic Imaging Hardware System

[0054] The hardware and software modules of the imaging system, such as Figure 2 As shown. The hardware modules include: the excitation wavelength of the femtosecond laser 1 is set to 870 nm; the polarizer 2 is switched to a non-working state and temporarily not set in the optical path; the scanning galvanometer 3 is in its initial position; the reflecting mirror group inside the microscope system 4 is adjusted to the optimal angle, i.e., the laser optical path is parallel to the internal path of the microscope; the dichroic mirror 5 is in its optimal position, i.e., the mirror exactly covers the entire range of the laser scan; the microscope objective 6 is positioned in the center of the field of view, and the position of the foremost part of the lens is exactly the optimal position for imaging the sample; the wavelength of the second harmonic filter is set to 435 nm; and the wavelength of the fluorescence filter is set to 610 nm. The software modules are deployed in four computers, including: the laser control module (control module) in the laser control computer 10; the scanning galvanometer 3 control module and the microscope objective 6 control module (control module) in the main program control computer 11; the real-time image display program and the image post-viewing program (image display module) in the image display / acquisition computer 9; and the image analysis and calculation program (analysis and calculation module) in the analysis and calculation computer 12.

[0055] The laser is emitted by a femtosecond laser 1 and focused onto the animal's cerebral blood vessels via a microscope objective 6. The resulting reflected light signal is collected by a microscope objective 6 with a magnification of 20x. The collected reflected light is filtered using a bandpass filter, and the wavelength of the second harmonic filter 7 is set to 435nm. The second harmonic imaging method for cerebral blood vessels involves scanning the target blood vessels frame by frame from superficial to deep, as illustrated in the diagram. Figure 3 As shown, the vibration frequency of the scanning galvanometer 3 is set to 0.49 Hz, the step between frames is 1 micrometer, and the image acquisition size is 7.78 micrometers / pixel.

[0056] II. Display of Second Harmonic Imaging Images of Cerebral Vascular System

[0057] Using the image acquisition method described above, the obtained cerebral vascular second harmonic image is as follows: Figure 4As shown in the left image, the resulting image consists of two thin, elongated lines, representing the two sides of the blood vessel wall. Between these lines lies a hollow region without imaging signal, which is the blood vessel lumen. The blood vessel wall contains collagen, elastin, endothelial cells, and smooth muscle cells, while the blood vessel lumen contains blood cells and various blood components. However, none of these cells and molecules possess second-harmonic emission characteristics; therefore, in second-harmonic imaging of cerebral blood vessels, only the imaging signal of the blood vessel wall collagen is obtained.

[0058] Whether the imaging image obtained by the image display module is indeed an imaging signal of the blood vessel wall needs further verification. The method involves using the same hardware module and image display module to fluorescently label the blood flow within the blood vessel lumen. The steps are as follows: Bengal red is selected as the fluorescent indicator for labeling the blood vessel lumen; a 0.5% concentration of the fluorescent indicator is prepared and injected via the tail vein of a mouse; the wavelength of the fluorescent filter 8 is set to 610 nm; the fluorescent signal of the blood vessel lumen can be observed through the image display module. The fluorescent indicator molecules are uniformly distributed in the blood flow, and this fluorescent signal indicates the location of the blood vessel lumen, such as... Figure 4 As shown in the middle figure. The second harmonic imaging signal (…) is analyzed by the image analysis program in the analysis and calculation module. Figure 4 (left image) and fluorescence imaging signal ( Figure 4 By overlaying the images (in Chinese and English), we obtain... Figure 4 (Right image) The former is located exactly outside the latter, forming a structure of blood vessel wall-blood vessel lumen, which confirms that the second harmonic imaging image is the blood vessel wall of the cerebral blood vessel.

[0059] III. Quantitative analysis of second harmonic imaging images of cerebral blood vessel walls.

[0060] Quantitative analysis of the images is primarily achieved through the computation module in the analysis and computing computer 12, with the image display module in the image display / analysis computer 9 serving as an auxiliary tool. The objectives of the quantitative analysis mainly include the signal polarization characteristics of the imaging image, the signal intensity of the imaging image, the diameter of cerebral blood vessels, and the thickness of the cerebral blood vessel walls. Specifically:

[0061] 1. Polarization Characteristic Identification. Polarizing mirror 2 is set to working mode, its control module is activated, and the 0° scale in the control module is aligned with the indicator scale on the polarizing mirror. At this point, a set of three-dimensional images of cerebral blood vessels is acquired. The control module disk is rotated in 10° increments, sequentially acquiring three-dimensional images of the same cerebral blood vessel when polarizing mirror 2 is pointed at 10°, 20°, 30°…350°. Then, the average intensity of the second harmonic imaging signal in each set of three-dimensional images is calculated using the analysis and calculation module. All the above average values ​​are plotted as a radar statistical chart. Figure 5 Therefore, we can conclude that for Figure 4For the blood vessels shown, the optimal polarization angle is 150 / 330°, while the polarization angle of the background signal intensity is 60 / 240°. When the polarization angle is between 150-240° / 330-60°, the signal gradually weakens, and when the polarization angle is between 60-150° / 240-330°, the signal gradually strengthens, exhibiting a periodic change pattern with a period of 90°, which conforms to the polarization characteristics of the second harmonic signal.

[0062] 2. Calculation of signal intensity in cerebral blood vessel wall imaging. This is performed using the analysis and calculation module. Figure 4 The second harmonic imaging signal intensity of the cerebral blood vessel wall shown in the figure was calculated by taking the signal intensity value of each pixel on the walls of the two blood vessels, and then averaging all the intensity values ​​to obtain an average second harmonic signal intensity of 168 (au) for the blood vessel wall. The average intensity of the second harmonic imaging signal of the cerebral blood vessel wall reflects the relative number of collagen molecules in the blood vessel wall. A higher imaging signal intensity value indicates a relatively high collagen molecule content, and vice versa.

[0063] 3. Calculation of cerebral blood vessel diameter. This is achieved through the analysis and calculation module. Figure 4 To calculate the diameter of the cerebral blood vessels shown, it is necessary to calculate the distance between the second harmonic imaging signals of the two blood vessel walls at each location, and then average all the above distances to finally calculate the average diameter of the blood vessel as 22.5 micrometers.

[0064] 4. Calculation of vessel wall thickness. This is performed using the analysis and calculation module. Figure 4 The thickness of the cerebral blood vessel wall shown is calculated by taking the average half-width of each vessel wall. The average thickness of the left vessel wall is 2.65 micrometers, and the average thickness of the right vessel wall is 2.12 micrometers.

[0065] This invention provides a system for real-time detection of cerebral vascular morphology based on in vivo second harmonic imaging. This invention is applicable to the detection of cerebral blood vessels in vivo. The detection method has advantages such as high spatiotemporal resolution and no need for exogenous molecular markers. It can quantitatively analyze multiple parameters of cerebral blood vessels in real time, rapidly, and accurately, and has great application prospects in clinical medical brain pathological examination. Many ideas and methods exist for implementing this solution. It should be noted that those skilled in the art can make many improvements without departing from the principles of this solution, and these improvements are considered within the scope of protection of this invention.

Claims

1. A real-time cerebral vascular morphology detection system based on second harmonic imaging, characterized in that, It comprises two parts of a second harmonic imaging device and a software system, wherein: The second harmonic imaging device comprises a femtosecond laser 1, a polarizer 2, a scanning galvanometer 3, a microscope system 4, a dichroic mirror 5, a microscope objective 6, a second harmonic filter 7, a fluorescence filter 8, an image acquisition / display computer 9, a laser control computer 10, a main program control computer 11, and an analysis calculation computer 12; the optical path connection is as follows: the femtosecond laser 1 emits laser light, which passes through the polarizer 2, the scanning galvanometer 3, and a 90-degree angle reflection into the microscope system 4, and finally the laser light is irradiated to the object to be measured through the microscope objective 6; the reflected light is divided into two paths after passing through the dichroic mirror 5, and then passes through the second harmonic filter 7 and the fluorescence filter 8, respectively, and the imaging signals enter the image acquisition / display computer 9 for real-time monitoring, and finally the collected signals are transmitted to the analysis calculation computer 12 for analysis and calculation; wherein the laser excitation and power adjustment of the femtosecond laser 1 are controlled by the laser control computer 10; the scanning galvanometer 3 and the microscope objective 6 are controlled by the main program control computer 11; wherein: The femtosecond laser 1 provides a stable pulsed laser light source; the laser light emitted by the femtosecond laser 1 is used to excite the second harmonic luminescence of collagen molecules in the adventitial and intimal extracellular matrix of the cerebral vascular wall, and the second harmonic phenomenon can be used for detection of collagen molecules in the cerebral blood vessels; the excitation light wavelength range of the femtosecond laser 1 is 710-910 nanometers; The polarizer 2 is used to filter the laser light emitted by the femtosecond laser 1, and only allows laser light of a specific polarization angle to pass through; the polarizer is continuously adjustable within the range of 0-359°, and the polarization angle is set according to the needs; specifically, a rotatable disc with a scale is arranged in the polarizer 2, and the disc scale is accurate to 1°; there is a short line on the top of the polarizer piece as an indication scale; when a certain scale on the disc is aligned with the polarizer indication scale, the polarizer only has transmittance to laser light of the angle indicated by the scale, that is, it allows laser light of a specific polarization angle to pass through; adjust the polarizer 2 so that laser light of a specific polarization angle irradiates the cerebral blood vessel to be measured; when the polarization angle of the laser light is exactly equal to the polarization angle of the collagen molecules in the cerebral vascular wall, the detected optical signal is the second harmonic signal; The scanning galvanometer 3 is composed of two perpendicularly placed mirrors, one of which vibrates at high speed in the horizontal direction and is called X mirror, and the other vibrates at high speed in the vertical direction and is called Y mirror, so that the small spot of the laser light source realizes large-range plane scanning; specifically, the small spot first passes through the X mirror to realize linear laser track, and then passes through the Y mirror to realize planar light track, so that a small spot realizes scanning of a rectangular plane; The microscope system 4 sets different magnification lenses in the system according to the needs, so that the laser light is magnified to a certain multiple after passing through the microscope system, and the propagation direction of the laser light is strictly along the set optical path by adjusting the position and angle of the lens group. The microscope objective 6 converges the divergent light spot from the microscope system 4 to a very precise light spot, focuses the imaging plane on a specific position of the object to be measured, and observes different size imaging fields of view through different magnification objective lenses; The dichroic mirror 5 receives the reflected light from the measured object irradiated by the microscope objective 6, and allows the laser light source to be transmitted in one direction, so that the reflected light signal after the light source irradiates the measured object can only be reflected by the dichroic mirror and cannot be transmitted; the reflected light reaching the dichroic mirror 5 is divided into two independent light paths, one of which passes through the second harmonic filter 7, and the other of which passes through the fluorescent filter 8; The second harmonic filter 7 selectively transmits the second harmonic signal of a specific wavelength reflected by the dichroic mirror 5, and the signal is the light source of the collagen molecules in the extracellular matrix of the intima and adventitia of the cerebral vascular wall, with a central wavelength range of 355-455 nm; The fluorescent filter 8 selectively transmits the fluorescent signal of a specific wavelength reflected by the dichroic mirror 5; when there is no exogenous fluorescent molecule labeling, it is only used for detecting molecules with spontaneous fluorescence in the cerebral cortex; after injecting a fluorescent indicator into the animal vein, it can detect the exogenous fluorescent signal of blood cells in the vascular lumen; The imaging signal acquisition / display computer 9 receives the reflected light passing through the second harmonic filter 7 and the fluorescent filter 8, and collects, displays and records the imaging signal in real time; then the image is transmitted to the analysis computer 12; The analysis computer 12 is connected to the imaging signal acquisition / display computer 9 through a local area network, and is used to receive the imaging image data from the imaging signal acquisition / display computer 9, and further quantitatively analyze the second harmonic imaging image data through a calculation software module; The software system includes an image display module and an analysis calculation module; The image display module is deployed in the imaging signal acquisition / display computer 9, and includes a real-time display program and an image post-viewing program, wherein: The real-time display program is used to monitor the image acquisition process in real time; the main program control computer 11 controls the scanning galvanometer 3 to start working, and the image obtained by scanning the sample is displayed in the image display window of the imaging signal acquisition / display computer 9 in real time, so as to realize real-time adjustment of the imaging field of view, including the horizontal position and depth position of the imaging focal plane, and finally locate the required imaging target area; The image post-viewing program is used to view the three-dimensional image collected above and to simply adjust the image; the collected three-dimensional image is a single file, and the three-dimensional image can be viewed frame by frame by dragging the scroll bar of the software; the image adjustment function can realize real-time adjustment of brightness and contrast of the whole or single frame of the three-dimensional image; The analysis calculation module is deployed in the analysis computer 12, and the specific calculation content includes the average diameter of the cerebral blood vessel, the imaging signal intensity of the cerebral blood vessel wall, and the thickness of the cerebral blood vessel wall; wherein: (1) Calculation of the average diameter of the cerebral blood vessels; the distance between two blood vessel wall imaging signals is the diameter of the blood vessel; first, the blood vessel is rotated to the vertical direction, and a horizontal straight line is drawn perpendicular to the two blood vessel wall imaging signals; the absolute value of the length of the line segment between the two intersection points of the horizontal straight line and the imaging signals is the diameter of the blood vessel at that position; according to the imaging image, it can be found that the edges of the blood vessel lumen have ups and downs, so the length of the line segment is not the same; the blood vessel wall imaging signal is composed of a plurality of pixel points in the vertical direction, and the number of all pixel points is calculated by the analysis calculation module; the length of the line segment between each pair of pixel points is calculated; the length between all pairs of pixel points in the vertical direction is calculated, and finally the average value of all lengths is calculated to obtain the average diameter of the blood vessel; the pair of pixel points refers to the two corresponding pixel points on the left and right blood vessel walls; (2) Calculation of the intensity of the cerebral blood vessel wall imaging signal; first, the second harmonic imaging image is obtained through the image acquisition / viewing program, and each pixel point in the image is displayed in 16-bit grayscale; the imaging signal intensity has a value range of 0-255, and the unit of the intensity value is written as a.u.; then the average value of all intensity values of the blood vessel wall is calculated as the average intensity value of the blood vessel wall; (3) Calculation of the thickness of the cerebral blood vessel wall; second harmonic imaging mainly collects blood vessels with a horizontal orientation in the brain, and each blood vessel has two blood vessel wall second harmonic signals; first, the blood vessel is rotated to the vertical direction, and the signal of each blood vessel wall has a peak value at each pixel row; then, a Gaussian function is fitted to each pixel row, and the half-height width of each pixel row is calculated; finally, the average value of all half-height width values of a blood vessel wall is calculated to obtain the thickness value of one side of the blood vessel wall; the other side is also calculated.

2. The cerebral vascular shape real-time detection system according to claim 1, characterized by, The laser excitation and power adjustment of the femtosecond laser 1 are controlled by a laser control computer 10, specifically, the laser control computer 10 is directly connected with the femtosecond laser 1, the computer has a built-in control program to control the femtosecond laser 1, including controlling the opening and closing of the femtosecond laser 1, and adjusting the wavelength of the emitted laser; here, an adjustable scroll bar is used to adjust the wavelength value, by dragging the scroll bar or inputting specific values in the input box, the femtosecond laser 1 emits laser of specified wavelength.

3. The cerebral vascular morphology real-time detection system according to claim 1, characterized in that, The operation of the scanning galvanometer 3 is controlled by the main program control computer 11, including controlling the opening and closing of the scanning galvanometer 3, adjusting the scanning frequency parameters, and specifically including: only controlling scanning without data archiving, for dynamic observation of the selected imaging window; controlling scanning while archiving data to save three-dimensional image files for subsequent analysis; adjusting the scanning frequency of the scanning galvanometer 3, which can be adjusted by inputting parameters in the input box of the built-in program interface of the corresponding computer 11, and different sampling rates are set to realize the vibration of the scanning galvanometer 3 at different frequencies. The lower the vibration frequency, the longer the laser stays at each pixel, and the more detailed information the imaging image has.

4. The cerebral vascular shape real-time detection system according to claim 1, characterized by, The operation of the microscope objective 6 is controlled by the main program control computer 11, that is, the spatial position of the microscope objective 6 is controlled, and specifically, three input boxes are set in the built-in program interface of the computer 11, corresponding to the X-axis, Y-axis and Z-axis positions of the objective 6 in space, and the objective is moved to the specified position by inputting specific values; wherein the "horizontal direction zero" button is used to move the microscope objective 6 in the X-Y axis direction to the preset initial position, and the "vertical direction zero" button is used to move the microscope objective 6 in the Z-axis direction to the preset initial position; the "start" input box and the "end" input box can input specific values to make the microscope objective 6 move continuously in the Z-axis direction within the specified value interval.

5. The cerebral vascular morphology real-time detection system according to any one of claims 1 to 4, characterized by, The specific operation process is as follows: (1) Prepare the imaging window: first grind the skull to thin it, then package the window with a cover glass, fill it with agarose in between, and finally fix the cover glass with adhesive; (2) Place the microscope objective 6 above the window described in step (1), collect the brain blood vessels through the second harmonic imaging device and software system, and the target of signal collection is the second harmonic imaging signal of the blood vessel, specifically the second harmonic signal emitted by the collagen in the adventitial and intimal extracellular matrix of the blood vessel wall; (3) In the second harmonic imaging device described in step (2), the excitation light wavelength of the femtosecond laser 1 is set to any wavelength in the range of 710-910 nm, the excitation light is focused on the cerebral cortex blood vessel by the microscope objective 6, and the reflected light signal is collected by the same microscope objective 6; the collected reflected light is further filtered using a second harmonic filter 7, and the effective wavelength range is 355-455 nanometers, and the allowed wavelength value floating range is any width of 10-40 nanometers; the brain blood vessel is scanned frame by frame in the whole layer depth by the microscope objective 6, and the interval distance between each frame of the frame-by-frame scanning is determined according to factors such as the specific blood vessel diameter and the depth of the blood vessel in the brain. The smallest scanning interval is set to 0.5 microns, and the largest interval is set to 10 microns. The spatial position of the microscope objective 6 is controlled by the main program control computer 11 in the control module. (4) The process of step (3) is displayed by a graphic display module. Firstly, the real-time display program is accurately positioned to the area with clear cerebral blood vessels. After a set of three-dimensional images are collected, the selected area is analyzed by the image post-processing program to determine whether it is optimal. If the selected imaging area still needs to be adjusted, the above steps are repeated until the selected imaging area includes complete blood vessel wall brightness information and depth information. Secondary harmonic imaging is performed on the blood vessels in the cerebral cortex that run horizontally. The obtained image has two thin linear images, and a hollow background imaging signal region is contained between the two lines. The two lines are the two sides of the blood vessel wall, and the hollow region is the blood vessel lumen; (5) The blood vessel wall imaging image of step (4) is further analyzed by an image analysis and calculation module to determine the effective range of the imaging signal. The half-height width is fitted and analyzed, and the effective range of 2-100 microns on both sides of the blood vessel wall signal is obtained by selecting the effective part of the fitting curve. When the blood vessel wall signal is selected for quantitative analysis, the imaging data in the effective range needs to be accurately selected, and the calculated specific half-height value can represent the thickness of the blood vessel wall; (6) The secondary harmonic imaging signal of the blood vessel wall needs to be tested for polarization characteristics. In the secondary harmonic imaging device, the polarizer 2 is continuously adjustable and the range is 0-359°. Adjusting the control part of the polarizer 2 allows the laser to pass through at different angles, and the polarization angle at which the strongest secondary harmonic signal is obtained is found, which is the optimal polarization angle, and the polarization angle at which the weakest signal is obtained is the background polarization angle. There are two polarization angles that can obtain the strongest imaging signal, and the difference between the two angles must be 180°, and the difference within the range of 20° up and down is effective. Similarly, there are also two polarization angles that can obtain the weakest imaging signal, and the difference between the two angles must also be 180°, and the difference within the range of 20° up and down is effective. The angle difference between the optimal polarization angle and the background polarization angle must be 90°, and the difference within the range of 20° up and down is effective; (7) The blood vessel wall secondary harmonic imaging signal intensity in step (4) is quantitatively analyzed by an analysis and calculation module. The calculated value represents the relative number of collagen molecules in the blood vessel wall. The higher the signal intensity, the more the number of collagen molecules; (8) The average distance between the two blood vessel wall imaging signals in step (4) is calculated by an analysis and calculation module to obtain the diameter information of the blood vessel; (9) The blood vessel wall thickness in step (5) is calculated by an analysis and calculation module. The half-height width of each "pixel row" of the two blood vessel walls is calculated by Gaussian fitting, and the average half-height width of each side of the blood vessel wall is calculated to obtain the average thickness of each blood vessel wall.

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