A blood flow velocity measurement system and method coupling laser speckle and fluorescence imaging

Abstract

The present invention discloses a blood flow velocity measurement system and method coupling laser speckle and fluorescence imaging. Scattered laser and excitation light fluorescence signals are obtained through a beam splitting module. The scattered laser is transmitted through a stereomicroscope to irradiate the surface of the skin tissue, and then reflected back to the stereomicroscope and input into the first beam splitting module to obtain the laser backscattered light. The fluorescence particle excitation light is transmitted through the stereomicroscope to irradiate the surface of the skin tissue, and then reflected back to the stereomicroscope and input into the second beam splitting module to obtain the emitted light fluorescence signal. A light processing module is used to process the laser backscattered light and the emitted light fluorescence signal, and accurate blood information can be obtained. An image acquisition module acquires the focused laser speckle signal and the fluorescence particle emitted light, and can realize real-time acquisition of information. The laser speckle blood flow relative velocity field image and the blood absolute velocity field image are obtained through the flow field information processing module, and the two images are input into the flow velocity analysis module, and accurate blood flow velocity can be obtained.

Description

Technical Field

[0001] The present invention belongs to the technical field of bio - optical imaging, and relates to a blood flow velocity measurement system and method that couples laser speckle and fluorescence imaging. Background Art

[0002] The existing laser speckle contrast imaging for blood flow velocity measurement is a very promising non - invasive surface imaging technology. It has the advantages of non - contact, non - invasive, large imaging field of view, no need to use contrast agents, and simple equipment with low cost. However, it currently only measures the relative velocity of blood flow and has disadvantages such as inaccurate blood flow velocity measurement, and it is a semi - quantitative velocity measurement method. Fluorescent particle image velocimetry is a micro - scale flow velocity measurement technology that combines optical microscopy technology and traditional particle velocimetry technology, with advantages such as full - field, transient, and high - precision quantitative velocity measurement. However, it requires the use of fluorescent particle tracers and is an invasive velocity measurement method. Summary of the Invention

[0003] The purpose of the present invention is to solve the problem in the prior art that it is difficult to accurately and real - time measure blood flow velocity information, and to provide a blood flow velocity measurement system and method that couples laser speckle and fluorescence imaging.

[0004] To achieve the above - mentioned purpose, the present invention adopts the following technical solutions:

[0005] A blood flow velocity measurement system that couples laser speckle and fluorescence imaging proposed by the present invention includes a flow field information acquisition module, a flow field information processing module, and a flow velocity analysis module. The flow field information acquisition module includes a laser speckle light source module, a fluorescence light source module, a first beam splitting module, a second beam splitting module, a stereomicroscope, a first light processing module, a second light processing module, and an image acquisition module;

[0006] The laser speckle light source module outputs a laser. After the laser is input into the first beam splitting module, scattered laser is output. The scattered laser is transmitted through the stereomicroscope and irradiated onto the surface of the skin tissue, and then reflected back to the stereomicroscope and input into the first beam splitting module. The first beam splitting module outputs the laser and then sends the scattered light to the first light processing module. The first light processing module outputs a focused laser speckle signal, and the image acquisition module acquires the focused laser speckle signal;

[0007] The fluorescence light source module outputs fluorescence particle excitation light. After the fluorescence particle excitation light is input into the second beam splitting module, fluorescence particle excitation light is output. The fluorescence particle excitation light is transmitted through the stereomicroscope and irradiated onto the surface of the skin tissue, and then reflected back to the stereomicroscope and input into the second beam splitting module. The second beam splitting module outputs an emitted fluorescence signal to the second light processing module. The second light processing module outputs a focused fluorescence particle emission light, and the image acquisition module acquires the focused fluorescence particle emission light;

[0008] The collected and focused laser speckle signal is input into the flow field information processing module, and then the laser speckle blood flow relative velocity field image is output; the collected and focused fluorescence particle emission light is input into the flow field information processing module, and then the blood absolute velocity field image is output. The laser speckle relative blood flow image and the blood absolute velocity field image are input into the flow velocity analysis module, and then the blood flow velocity is output.

[0009] Preferably, the flow field information processing module includes a speckle contrast analysis module and a fluorescence particle image velocimetry analysis module;

[0010] The focused laser speckle signal is input into the speckle contrast analysis module to output the laser speckle blood flow relative velocity field image; the focused fluorescence particle emission light is input into the fluorescence particle image velocimetry analysis module to output the blood absolute velocity field image.

[0011] Preferably, the first beam splitting module is a polarization beam splitting unit; the second beam splitting module is a dichroic mirror unit.

[0012] Preferably, the laser speckle light source module includes a 905nm semiconductor laser unit and a laser collimation and beam expansion unit;

[0013] The 905nm semiconductor laser unit outputs laser light, and inputs the laser light into the laser collimation and beam expansion unit to output amplified laser light, and then inputs the amplified laser light into the polarization beam splitting unit.

[0014] Preferably, the fluorescence light source module includes a xenon lamp unit and an excitation light filtering unit;

[0015] The xenon lamp unit outputs broadband white light, and inputs the broadband white light into the excitation light filtering unit to output fluorescence particle excitation light, and then inputs the fluorescence particle excitation light into the dichroic mirror unit.

[0016] Preferably, the first light processing module includes an interference band-pass filtering unit and a first focusing lens unit; the second light processing module includes an emission light filtering unit and a second focusing lens unit;

[0017] The laser backscattered light is input into the interference band-pass filtering unit for filtering and then input into the first focusing lens unit to output the focused laser speckle signal;

[0018] The emission light fluorescence signal is input into the emission light filtering unit for filtering and then input into the second focusing lens unit to output the focused fluorescence particle emission light.

[0019] Preferably, the image acquisition module includes a first camera and a second camera;

[0020] The first input port of the first camera is connected to a synchronizer, and the second input port of the first camera is connected to a trigger. The first input port of the second camera is connected to the synchronizer, and the second input port of the second camera is connected to the trigger;

[0021] The first camera collects the focused laser speckle signal, and the second camera collects the emitted light of the fluorescent particles after focusing.

[0022] A method for measuring blood flow velocity by coupling laser speckle and fluorescence imaging proposed by the present invention includes the following steps:

[0023] The laser speckle light source module outputs laser to the first beam splitting module and then outputs scattered laser. The scattered laser is transmitted through the stereomicroscope to the surface of the skin tissue, and then reflected back to the stereomicroscope and transmitted to the first beam splitting module. The first beam splitting module outputs laser and then sends the scattered light to the first light processing module, and the first light processing module outputs the focused laser speckle signal;

[0024] The fluorescence light source module outputs the excitation light of the fluorescent particles to the second beam splitting module and then outputs the excitation light of the fluorescent particles. The excitation light of the fluorescent particles is transmitted through the stereomicroscope to the surface of the skin tissue, and then reflected back to the stereomicroscope and transmitted to the second beam splitting module. The second beam splitting module outputs the emitted fluorescence signal to the second light processing module, and the second light processing module outputs the focused emitted light of the fluorescent particles;

[0025] The image acquisition module is used to collect the focused laser speckle signal and the focused emitted light of the fluorescent particles; the collected focused laser speckle signal is input into the flow field information processing module to output the laser speckle blood relative velocity field image; the collected focused emitted light of the fluorescent particles is input into the flow field information processing module to output the blood absolute velocity field image;

[0026] Both the laser speckle blood relative velocity field image and the blood absolute velocity field image are input into the flow velocity analysis module to obtain the blood flow velocity and realize the real-time measurement of the blood flow velocity.

[0027] Preferably, the method for obtaining the blood flow velocity is as follows:

[0028] The pyramid Lucas–Kanade optical flow method is used for the blood absolute velocity field image to obtain the absolute velocity field of the fluorescent particle image;

[0029] Spatial contrast analysis is performed on the laser speckle blood relative velocity field image to obtain the laser speckle imaging blood relative velocity field;

[0030] The absolute velocity field of the fluorescent particle image is locally averaged according to the M×M spatial grid to obtain the local average velocity vector field and conversion coefficient of the M×M spatial grid of the fluorescent particle image;

[0031] Multiply the speckle blood flow index image by the conversion coefficient to obtain the blood flow velocity.

[0032] Preferably, the method for obtaining the relative blood flow velocity field of laser speckle imaging is as follows:

[0033] The method for calculating the speckle blood flow index SFI of the pixel points of the laser speckle image is as follows:

[0034]

[0035] where K is the contrast value within the M×M spatial window pane; the mean image of two consecutively acquired laser speckle blood flow index images is used as the final relative blood flow velocity field of laser speckle imaging;

[0036] The expression of the contrast K within the M×M spatial window pane is as follows:

[0037]

[0038] where σ s represents the standard deviation of the pixel gray levels within the M×M spatial window pane, It represents the average grayscale value of M×M pixel points.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] A blood flow velocity measurement system that couples laser speckle and fluorescence imaging proposed by the present invention obtains scattered laser light through a first beam splitting module and obtains emitted light fluorescence signals through a second beam splitting module; the scattered laser light is irradiated onto the surface of the skin tissue through a stereomicroscope and then reflected back to the stereomicroscope and input into the first beam splitting module, aiming to obtain the laser backscattered light; the excitation light fluorescence signal is irradiated onto the surface of the skin tissue through a stereomicroscope and then reflected back to the stereomicroscope and input into the second beam splitting module, aiming to obtain the emitted light fluorescence signal; a first light processing module is used to process the laser backscattered light, and a second light processing module is used to process the emitted light fluorescence signal, aiming to reduce interference information and obtain accurate blood information. Moreover, the stereomicroscope has a long working distance, a large depth of field, and can simultaneously image light of different wavelengths, and is suitable for simultaneous imaging of laser speckles and fluorescent particles; an image acquisition module is used to collect the focused laser speckle signal and the focused fluorescent particle emitted light, enabling real-time acquisition of information; a flow field information processing module is used to obtain the relative blood flow image of the laser speckle and the absolute blood velocity field image, and input the relative blood flow image of the laser speckle and the absolute blood velocity field image into the flow velocity analysis module, enabling accurate blood flow velocity to be obtained. Therefore, the measurement system proposed by the present invention can solve the problem in the prior art that it is difficult to accurately and real-time measure blood flow velocity information.

[0041] Furthermore, the xenon lamp unit can output a strong continuous spectrum from ultraviolet to near-infrared, the light color in the visible region is extremely similar to sunlight, the energy density is high, the output is stable, and the excitation light of the fluorescent particle band is obtained through a narrowband filter.

[0042] Furthermore, a synchronizer is connected to the first camera and the second camera, aiming to control the first camera and the second camera to be able to synchronously collect images, and a trigger is connected to the first camera and the second camera, aiming to control the first camera and the second camera to simultaneously collect images.

[0043] A blood flow velocity measurement method that couples laser speckle and fluorescence imaging proposed by the present invention can real-time collect the focused laser speckle signal and the focused fluorescent particle excitation light through an image acquisition module, and can analyze and process the relative blood flow image of the laser speckle and the absolute blood velocity field image through a flow velocity analysis module to obtain the blood flow velocity. Brief Description of the Drawings

[0044] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for the embodiments will be briefly introduced below. It should be understood that the following accompanying drawings only show some embodiments of the present invention, and therefore should not be regarded as a limitation of the scope. For those of ordinary skill in the art, without creative efforts, other related accompanying drawings can also be obtained based on these drawings.

[0045] Figure 1 Structural diagram of the blood flow velocity measurement system for coupling laser speckle and fluorescence imaging of the present invention.

[0046] Figure 2 Flowchart of the blood flow velocity measurement method for coupling laser speckle and fluorescence imaging of the present invention.

[0047] Figure 3 Schematic diagram of the fluorescence particle image velocimetry of the present invention.

[0048] Figure 4 Schematic diagram of the conversion coefficient between laser speckle velocimetry and fluorescence particle image velocimetry of the present invention ((a) is the absolute velocity field of the fluorescence particle image, (b) is the local average velocity vector field of the M×M grid of the fluorescence particle image, (c) is the relative velocity field of the speckle blood flow index). Detailed implementation manners

[0049] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Usually, the components of the embodiments of the present invention described and shown in the accompanying drawings here can be arranged and designed in various different configurations.

[0050] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed present invention, but merely represents the selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0051] It should be noted that: similar reference numerals and letters indicate similar items in the following accompanying drawings. Therefore, once an item is defined in one accompanying drawing, it does not need to be further defined and explained in subsequent accompanying drawings.

[0052] In the description of the embodiments of the present invention, it should be noted that if terms such as "upper", "lower", "horizontal", "inner", etc. are used to indicate the orientation or positional relationship, it is based on the orientation or positional relationship shown in the drawings, or the orientation or positional relationship in which the invention product is usually placed during use. This is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation. Therefore, it should not be construed as a limitation to the present invention. In addition, terms such as "first", "second", etc. are only used for distinguishing descriptions and cannot be understood as indicating or implying relative importance.

[0053] In addition, if the term "horizontal" appears, it does not mean that the component is required to be absolutely horizontal, but it can be slightly inclined. For example, "horizontal" only means that its direction is more horizontal relative to "vertical", and does not mean that the structure must be completely horizontal, but it can be slightly inclined.

[0054] In the description of the embodiments of the present invention, it should also be noted that unless otherwise clearly specified and limited, if terms such as "set", "installed", "connected", "coupled" are used, they should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and it can be the communication inside two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific situations.

[0055] The following further describes the present invention in detail with reference to the drawings:

[0056] A blood flow velocity measurement system that couples laser speckle and fluorescence imaging proposed by the present invention, as Figure 1 shown, includes a flow field information acquisition module, a flow field information processing module, and a flow velocity analysis module. Among them, the flow field information acquisition system includes a laser speckle light source module, a first beam splitting module, a fluorescence light source module, a second beam splitting module, a stereomicroscope, a first light processing module, a second light processing module, an image acquisition module, and a control module. The flow field information processing module includes a speckle contrast analysis module and a fluorescence particle image velocimetry analysis module.

[0057] The laser speckle light source module outputs laser light, which is input into the first beam splitting module and then scattered laser light is output; the scattered laser light is transmitted through the stereomicroscope and irradiated onto the surface of the skin tissue, and then reflected back to the stereomicroscope and input into the first beam splitting module. The first beam splitting module outputs laser light and directs the scattered light to the first light processing module, and the first light processing module outputs the focused laser speckle signal. The fluorescence light source module outputs fluorescence particle excitation light, which is then input into the second beam splitting module and then fluorescence particle excitation light is output. The fluorescence particle excitation light is transmitted through the stereomicroscope and irradiated onto the surface of the skin tissue, and then reflected back to the stereomicroscope and input into the second beam splitting module. The second beam splitting module outputs the emitted fluorescence signal to the second light processing module, and the second light processing module outputs the focused fluorescence particle emission light. The stereomicroscope has a long working distance, a large depth of field, can simultaneously image light of different wavelengths, and is suitable for simultaneous imaging of laser speckles and fluorescence particles.

[0058] The focused laser speckle signal is collected by the image acquisition module, and the focused fluorescence particle emission light is collected by the image acquisition module; the focused laser speckle signal is input into the flow field information processing module and then the laser speckle blood flow relative velocity field image is output, and the focused fluorescence particle emission light is input into the flow field information processing module and then the blood absolute velocity field image is output; the laser speckle blood flow relative velocity field image and the blood absolute velocity field image are input into the flow velocity analysis module to obtain the blood flow velocity, realizing accurate and real-time measurement of the blood flow velocity.

[0059] The beam splitting module includes a first beam splitting module and a second beam splitting module, and the light processing module includes a first light processing module and a second light processing module.

[0060] The first beam splitting module is a polarization beam splitting unit, and the laser speckle light source module includes a 905nm semiconductor laser unit and a laser collimation and beam expansion unit; the 905nm semiconductor laser unit outputs laser light, inputs the laser light into the laser collimation and beam expansion unit, outputs the amplified laser light, and inputs the amplified laser light into the polarization beam splitting unit. The first light processing module includes an interference band-pass filter unit and a first focusing lens unit; the laser backward scattered light is input into the interference band-pass filter unit for filtering and then input into the first focusing lens unit, and the focused laser speckle signal is output.

[0061] The second beam splitting module is a dichroic mirror unit, and the fluorescence light source module includes a xenon lamp unit and an excitation light filter unit; the xenon lamp unit outputs broadband white light, inputs the broadband white light to the excitation light filter unit, outputs fluorescence particle excitation light, and inputs the fluorescence particle excitation light to the dichroic mirror unit. The second light processing module includes an emission light filter unit and a second focusing lens unit; the emitted light fluorescence signal is input to the emission light filter unit for filtering and then input to the second focusing lens unit to output the focused fluorescence particle emission light. The xenon lamp unit can output a strong continuous spectrum from ultraviolet to near-infrared, the light color in the visible region is very similar to sunlight, the energy density is high, the output is stable, and the excitation light in the fluorescence particle band is obtained through a narrowband filter.

[0062] The image acquisition module includes a first camera and a second camera. A synchronizer is connected to the first input port of the first camera, and a trigger is connected to the second input port of the first camera. The first input port of the second camera is connected to the synchronizer, and the second input port of the second camera is connected to the trigger; the synchronizer is used to control the first camera and the second camera to synchronously acquire images, and the trigger is used to control the first camera and the second camera to acquire images simultaneously. The first camera is a CCD camera, and the second camera is an sCMOS camera. The first camera acquires the focused laser speckle signal, and the second camera acquires the focused fluorescence particle emission light signal.

[0063] A blood flow velocity measurement method for coupling laser speckle and fluorescence imaging proposed by the present invention, as Figure 2 shown, includes the following steps:

[0064] S1. The laser speckle light source module outputs laser to the first beam splitting module and then outputs scattered laser. The scattered laser is transmitted through a stereomicroscope and irradiated onto the surface of the skin tissue, and then reflected back to the stereomicroscope and transmitted to the first beam splitting module. The first beam splitting module outputs the laser and then outputs the scattered light to the first light processing module, and the first light processing module outputs the focused laser speckle signal;

[0065] S2. The fluorescence light source module outputs fluorescence particle excitation light to the second beam splitting module and then outputs fluorescence particle excitation light. The fluorescence particle excitation light is transmitted through a stereomicroscope and irradiated onto the surface of the skin tissue, and then reflected back to the stereomicroscope and transmitted to the second beam splitting module. The second beam splitting module outputs the emitted light fluorescence signal to the second light processing module, and the second light processing module outputs the focused fluorescence particle emission light;

[0066] S3. The image acquisition module is used to acquire the focused laser speckle signal and the focused fluorescence particle emission light; the acquired focused laser speckle signal is input to the flow field information processing module to output the laser speckle blood flow relative velocity field image; the acquired focused fluorescence particle emission light is input to the flow field information processing module to output the blood absolute velocity field image;

[0067] S4. Input both the laser speckle blood flow relative velocity field image and the blood absolute velocity field image into the flow velocity analysis module to obtain the blood flow velocity, achieving real-time measurement of the blood flow velocity.

[0068] The method for obtaining the blood flow velocity is as follows:

[0069] 1). Use the pyramidal Lucas–Kanade optical flow method for the blood absolute velocity field image to obtain the absolute velocity field of the fluorescent particle image;

[0070] 2). Conduct spatial contrast analysis on the laser speckle blood flow relative velocity field image to obtain the relative blood flow velocity field of laser speckle imaging;

[0071] The method for obtaining the relative blood flow velocity field of laser speckle imaging is as follows:

[0072] The method for calculating the speckle blood flow index SFI of the pixel points of the laser speckle image is as follows:

[0073]

[0074] where K is the contrast value within the M×M spatial window pane; the mean image of two continuously acquired laser speckle blood flow index images is used as the final relative blood flow velocity field of laser speckle imaging;

[0075] The expression of the contrast K within the M×M spatial window pane is as follows:

[0076]

[0077] where σ s represents the standard deviation of the pixel gray levels within the M×M spatial window pane, represents the average gray value of M×M pixel points;

[0078] 3), perform local averaging on the absolute velocity field of the fluorescent particle image according to the M×M spatial grid to obtain the local average velocity vector field and conversion coefficient of the M×M spatial grid of the fluorescent particle image;

[0079] 4), multiply the speckle blood flow index image by the conversion coefficient to obtain the blood flow velocity.

[0080] The following describes a blood flow velocity measurement system and method that couples laser speckle and fluorescence imaging proposed by the present invention with a specific example. The in-vivo sample of the present invention uses SD rats.

[0081] Step 1, production of the rat spinal window:

[0082] The experimental animals used are KM female mice at 3 - 4 weeks old with a body weight controlled at 30±2 g. Put the head of the experimental mouse into the anesthesia mask. In the small animal anesthesia machine, isoflurane is volatilized by the oxygen gas flow, and the volatilized isoflurane (4% concentration) is transported to the anesthesia mask through the air pump of the anesthesia machine to perform short-term anesthesia on the mouse for 2 - 3 min to avoid the mouse struggling during intraperitoneal injection and causing the needle hole to pierce the internal organs and death. During the onset of short-term anesthesia of the mouse, anesthetize the mouse by intraperitoneal injection of 10% chloral hydrate solution (4 mL / kg). When the mouse lies on its back with uniform heartbeat and breathing, relaxed muscles, no movement of the limbs, no reaction when the whiskers are touched, and the pedal reflex disappears, it is considered to have reached the fully anesthetized state. If the forelimbs of the mouse start to shake, it is judged that the mouse starts to recover. The anesthesia mask needs to be placed at the mouth and nose of the mouse, and 10% chloral hydrate solution is injected intraperitoneally to restore the anesthetized state. Generally, the additional dose does not exceed 1 / 10 - 1 / 5 of the total injection amount. After confirming that the anesthetic state is stable, place the mouse on a disposable surgical sheet on the heating pad, use a clipper to roughly shave off the hair on the back of the mouse, then apply depilatory cream to the back of the mouse for about 8 - 9 min, and then clean and wipe off the depilatory cream. Use fixed forceps to pick up the skin on the back of the mouse, turn on the light source to irradiate the skin from behind the mouse spine to find a blood vessel of appropriate thickness, use scissors and forceps to dig out a circular area with a diameter of about 10 mm on one side of the skin, and then cut off and remove the fascia on the surface of the blood vessel as much as possible to avoid reflection under the microscope affecting the image quality. Finally, tightly clamp the stretched double-layer skin of the spine with two symmetric titanium frames, and fix the titanium frames with small clips.

[0083] Select the fluorescent particle model FluoSpheres Red, with a diameter of 1 μm, and the excitation light wavelength λ ex = 580±15 nm, and the emission light wavelength λ em = 580 ± 15 nm, prepare a fluorescent microsphere solution (fluorescent microspheres: 0.9% NaCl = 1:4), and evenly and steadily inject the fluorescent microsphere solution (1 mL / Kg) into the tail vein of the mouse through the tail vein injection, and then place it under a stereomicroscope for imaging.

[0084] Step 2, register the laser speckle image and the fluorescence image:

[0085] The images obtained by the sCMOS camera and the CCD camera in their respective independent optical paths are often not in exactly the same spatial position, and image misalignment will occur. Registration is required to overlap the two image fields obtained by the cameras. In the present invention, the method of spatial feature points is used for registration. From the absorption spectrum of hemoglobin, it can be seen that the contrast of the laser speckle pattern at a wavelength of 905 nm is relatively poor, and it is impossible to directly obtain sufficiently clear feature points. It is necessary to first calculate the laser speckle contrast velocity map from the laser speckle pattern at a wavelength of 905 nm, and then register the fluorescence image according to 10 feature points selected from this map to obtain a geometric transformation matrix. Applying this matrix to the fluorescence image can obtain the image sequence after the fluorescence image is registered.

[0086] Step 3, perform pyramid Lucas–Kanade optical flow method calculation on the blood absolute velocity field image to obtain the fluorescence particle image absolute velocity field:

[0087] If the number of pyramid layers is set to 3 and three-layer pyramid decomposition is performed, as Figure 3 shown, because when each layer of the image is decomposed, the size of the image in this layer is half of the size of the image in the previous layer, in order to ensure that the size of the image obtained by each decomposition is an integer, it is necessary to adjust the size of the original fluorescence image (including the front and back two frames of images) through image scaling operations.

[0088] The image pyramid is a set of images with gradually decreasing resolution and size towards the upper layer. The bottom of the pyramid is the original image with high resolution, and the top is the image with the lowest resolution. Image pyramidization mainly includes two steps: smoothing the image using a low-pass filter (usually a Gaussian filter), and then sampling the smoothed image to obtain a series of image pyramids with reduced size and resolution.

[0089] The principle of smoothing the image using a low-pass filter: The color of a pixel is not only determined by itself, but also weighted by the pixels around it. Objectively reducing the difference from the surrounding pixels can play a role in smoothing the image. The weights of the surrounding pixels satisfy the rule that the closer to the center, the greater the weight, and theoretically satisfy the Gaussian distribution.

[0090] Before pyramid tracking, it is necessary to add additional pixel circles to the fluorescence images of two consecutive frames. This is because when the pixel points to be calculated are close to the image edge, the points in its neighborhood may exceed the boundary. To solve the problem that the neighborhoods of these edge - close points exceed the image boundary and the optical flow of them cannot be calculated normally, it is necessary to add additional pixel circles around the image before calculating the optical flow in each layer of the image, and fill the pixel values within the pixel circles with the true boundary values of the image. In this way, when calculating the neighborhood of the edge points, it is equivalent to only calculating the valid part of the neighborhood of this point.

[0091] Pyramid tracking starts from the top layer to calculate the optical flow, and initializes the optical flow estimate value in the top - layer image to 0. At this time, the least - squares method is used to find the derivative of the sum of the matching errors of all pixel points in the neighborhood. The derivative is 0 at the most - suitable point, and at this time, the similarity of the corresponding points in the two consecutive frames of images is the highest, and the residual optical flow vector of the current layer of pyramid image is obtained. Then, according to the calculation result of the optical flow of the previous layer as the initial value of the optical flow of the next layer, because the size of the previous - layer pyramid is half of the size of the next - layer pyramid image, the initial value of the optical flow of the next - layer image is 2 times that of the previous - layer optical flow vector. Then, according to this optical flow estimate value, the neighborhood window is translated to make the calculated residual displacement vector smaller, meeting the requirement of moving less in the Lucas–Kanade optical flow method, and the exact value of the residual optical flow vector on the next - layer image is calculated by the same principle as the previous layer. Finally, through cyclic accumulation, the exact values of the residual optical flow vectors of each layer of the pyramid are calculated, and the sum is the final optical flow value of this pixel point. Select the fluorescence tracking particles in the absolute velocity field image of blood, and perform the pyramid - type Lucas–Kanade optical flow algorithm calculation to obtain the absolute velocity field of the fluorescence particle image, that is, the actual blood flow velocity field.

[0092] Step 4: Conduct spatial contrast analysis on the laser - speckle blood flow relative velocity field image to obtain the laser - speckle imaging blood relative flow velocity field.

[0093] Specifically, as Figure 4 shown, (a) is the absolute velocity field of the fluorescence particle image, (b) is the local average velocity vector field of the M×M pane of the fluorescence particle image, and (c) is the relative velocity field of the speckle blood flow index. Select a spatial pane with a size of M×M, calculate the ratio of the standard deviation to the mean of the gray - scale values of M×M pixel points within this spatial pane according to formula (1), and assign it to the central pixel point as its spatial contrast value. For each calculation of the contrast of a central pixel point, translate this unit pane by one pixel and calculate the contrast of the next central pixel point, and so on until all pixels have completed the above operations, and finally obtain a complete contrast image.

[0094]

[0095] In the formula, σ s Represents the standard deviation of pixel grayscale within the M×M spatial pane, It represents the average grayscale value of M×M pixel points. The spatial contrast image calculated with a spatial window size of 5×5 or 7×7 has a better effect.

[0096] Calculate the speckle blood flow index of pixel points for the speckle image according to formula (2).

[0097]

[0098] In the formula, SFI represents the speckle blood flow index, and K represents the spatial contrast value.

[0099] Calculate the average image of two consecutive acquired speckle blood flow index images as the final laser speckle imaging blood relative flow velocity field.

[0100] Step 5: Calculate the laser speckle imaging blood relative flow velocity field and the absolute velocity field of the fluorescent particle image to obtain the conversion coefficient between the two.

[0101] Specifically: Perform local averaging on the absolute velocity field of the fluorescent particle image according to the M×M spatial window to obtain the local average velocity vector field of the M×M spatial window of the fluorescent particle image. Ignoring the velocity direction, calculate the conversion coefficient between the laser speckle imaging blood relative flow velocity field and the absolute velocity field of the fluorescent particle image.

[0102] Continuously collect speckle images, perform contrast analysis and calculation on them to obtain speckle blood flow index images, multiply the conversion coefficient by the speckle blood flow index images, and obtain the blood flow velocity, thereby realizing the rapid, accurate and real-time measurement of blood flow velocity.

[0103] A blood flow velocity measurement system and method coupling laser speckle and fluorescence imaging proposed by the present invention. The laser and the excitation light of fluorescent particles are coaxially coupled through a beam splitting module, and the coaxially coupled light is incident on a stereomicroscope. The stereomicroscope irradiates the coaxially coupled light onto the surface of the skin tissue. The light processing module filters and focuses the received laser backscattered light and emitted fluorescence signal returned from the skin tissue, and projects them onto an image acquisition module. The image acquisition module collects the focused laser speckle signal and the emitted light of fluorescent particles, and transmits them to the flow field information processing module. The flow field information processing module includes a laser speckle imaging analysis module and a fluorescent particle image velocity measurement analysis module. The speckle contrast analysis module generates a laser speckle blood relative velocity field image from the collected laser speckle signal, and the fluorescent particle image velocity measurement analysis module generates a blood absolute velocity field image from the collected fluorescent particle image. Inputting the laser speckle blood relative velocity field image and the blood absolute velocity field image into the flow velocity analysis module can obtain accurate blood flow velocity, and can solve the problem of difficult accurate and real-time measurement of blood flow velocity information in the prior art.

[0104] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A blood flow velocity measurement system that couples laser speckle and fluorescence imaging, characterized in that, it includes a flow field information acquisition module, a flow field information processing module, and a flow velocity analysis module. The flow field information acquisition module includes a laser speckle light source module, a fluorescence light source module, a first beam splitting module, a second beam splitting module, a stereomicroscope, a first light processing module, a second light processing module, and an image acquisition module; The laser speckle light source module outputs laser light. After the laser light is input into the first beam splitting module, scattered laser light is output. The scattered laser light is irradiated onto the surface of the skin tissue through the stereomicroscope and then reflected back to the stereomicroscope and input into the first beam splitting module. The first beam splitting module outputs laser light and directs the scattered light to the first light processing module. The first light processing module outputs a focused laser speckle signal, and the image acquisition module acquires the focused laser speckle signal; The fluorescence light source module outputs fluorescence particle excitation light. After the fluorescence particle excitation light is input into the second beam splitting module, fluorescence particle excitation light is output. The fluorescence particle excitation light is irradiated onto the surface of the skin tissue through the stereomicroscope and then reflected back to the stereomicroscope and input into the second beam splitting module. The second beam splitting module outputs an emitted light fluorescence signal to the second light processing module. The second light processing module outputs a focused fluorescence particle emission light, and the image acquisition module acquires the focused fluorescence particle emission light; The acquired focused laser speckle signal is input into the flow field information processing module, and then a laser speckle blood flow relative velocity field image is output. The acquired focused fluorescence particle emission light is input into the flow field information processing module, and then a blood absolute velocity field image is output. The laser speckle relative blood flow image and the blood absolute velocity field image are input into the flow velocity analysis module, and then the blood flow velocity is output.

2. The blood flow velocity measurement system that couples laser speckle and fluorescence imaging according to claim 1, characterized in that, the flow field information processing module includes a speckle contrast analysis module and a fluorescence particle image velocimetry analysis module; The focused laser speckle signal is input into the speckle contrast analysis module, and then a laser speckle blood flow relative velocity field image is output. The focused fluorescence particle emission light is input into the fluorescence particle image velocimetry analysis module, and then a blood absolute velocity field image is output.

3. The blood flow velocity measurement system that couples laser speckle and fluorescence imaging according to claim 1, characterized in that, the first beam splitting module is a polarization beam splitting unit; the second beam splitting module is a dichroic mirror unit.

4. The blood flow velocity measurement system that couples laser speckle and fluorescence imaging according to claim 3, characterized in that, the laser speckle light source module includes a 905nm semiconductor laser unit and a laser collimation and beam expansion unit; The 905nm semiconductor laser unit outputs laser light, inputs the laser light into the laser collimation and beam expansion unit, outputs amplified laser light, and inputs the amplified laser light into the polarization beam splitting unit.

5. The blood flow velocity measurement system that couples laser speckle and fluorescence imaging according to claim 3, characterized in that, the fluorescence light source module includes a xenon lamp unit and an excitation light filter unit; The xenon lamp unit outputs broadband white light, inputs the broadband white light into the excitation light filtering unit, outputs fluorescence particle excitation light, and inputs the fluorescence particle excitation light into the dichroic mirror unit.

6. The blood flow velocity measurement system for coupling laser speckle and fluorescence imaging according to claim 1, characterized in that the first light processing module includes an interference band-pass filtering unit and a first focusing lens unit; the second light processing module includes an emission light filtering unit and a second focusing lens unit; The laser backscattered light is input into the interference band-pass filtering unit for filtering and then input into the first focusing lens unit to output the focused laser speckle signal; The emission light fluorescence signal is input into the emission light filtering unit for filtering and then input into the second focusing lens unit to output the focused fluorescence particle emission light.

7. The blood flow velocity measurement system for coupling laser speckle and fluorescence imaging according to claim 1, characterized in that the image acquisition module includes a first camera and a second camera; A synchronizer is connected to the first input port of the first camera, a trigger is connected to the second input port of the first camera, the first input port of the second camera is connected to the synchronizer, and the second input port of the second camera is connected to the trigger; The first camera acquires the focused laser speckle signal, and the second camera acquires the focused fluorescence particle emission light.

8. A blood flow velocity measurement method for coupling laser speckle and fluorescence imaging, characterized in that it includes the following steps: The laser speckle light source module outputs laser to the first beam splitting module and then outputs scattered laser, the scattered laser is transmitted through the stereomicroscope and irradiated onto the surface of the skin tissue, and then reflected back to the stereomicroscope and transmitted to the first beam splitting module. The first beam splitting module outputs the laser backscattered light to the first light processing module, and the first light processing module outputs the focused laser speckle signal; The fluorescence light source module outputs fluorescence particle excitation light to the second beam splitting module and then outputs fluorescence particle excitation light. The fluorescence particle excitation light is transmitted through the stereomicroscope and irradiated onto the surface of the skin tissue, and then reflected back to the stereomicroscope and transmitted to the second beam splitting module. The second beam splitting module outputs the emission light fluorescence signal to the second light processing module, and the second light processing module outputs the focused fluorescence particle emission light; The image acquisition module is used to acquire the focused laser speckle signal and the focused fluorescence particle emission light; the acquired focused laser speckle signal is input into the flow field information processing module to output the laser speckle blood flow relative velocity field image; the acquired focused fluorescence particle emission light is input into the flow field information processing module to output the blood absolute velocity field image; Both the laser speckle blood flow relative velocity field image and the blood absolute velocity field image are input into the flow velocity analysis module to obtain the blood flow velocity and realize the real-time measurement of the blood flow velocity.

9. The blood flow velocity measurement method for coupling laser speckle and fluorescence imaging according to claim 8, characterized in that The method for obtaining the blood flow velocity is as follows: The pyramid Lucas–Kanade optical flow method is used for the blood absolute velocity field image to obtain the fluorescence particle image absolute velocity field; Perform spatial contrast analysis on the laser speckle blood flow relative velocity field image to obtain the laser speckle imaging blood relative flow velocity field; Perform local averaging on the fluorescence particle image absolute velocity field according to the M×M spatial grid to obtain the M×M spatial grid local average velocity vector field and conversion coefficient of the fluorescence particle image; Multiply the speckle blood flow index image by the conversion coefficient to obtain the blood flow velocity.

10. The method for measuring blood flow velocity by coupling laser speckle and fluorescence imaging according to claim 9, characterized in that, The method for obtaining the laser speckle imaging blood relative flow velocity field is as follows: The method for calculating the speckle blood flow index SFI of the pixel points of the laser speckle image is as follows: where K is the contrast value within the M×M spatial grid; the mean image of two consecutive laser speckle blood flow index images obtained is used as the final laser speckle imaging blood relative flow velocity field; The expression of the contrast K within the M×M spatial grid is as follows: Among them, σ s represents the standard deviation of pixel grayscale within the M×M spatial window pane, represents the mean of the gray values of M×M pixel points.

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