High-resolution laser speckle vascular blood flow imaging method and system

Abstract

This invention discloses a high-resolution laser speckle vascular blood flow imaging method and system. The method includes irradiating the tissue under test with laser transmission illumination, capturing a set of laser speckle images of the tissue under test with short exposure time and a set of laser speckle images of the tissue under test with long exposure time, and then fusing the two sets of images to obtain a blood perfusion index image and angiography image. Two sets of laser speckle images were acquired, one with short exposure time and the other with long exposure time. The laser speckle images with short exposure time can take into account the degree of scattering and the velocity of scattering particles in the tissue under test. After fusing with the laser speckle images with long exposure time, the contributions of scattered and non-scattered photons in the transmitted light are separated, which can significantly reduce the estimation error. It can not only resolve the flow velocity information of microvessels, but also achieve high-resolution imaging of fine microvascular structures.

Description

Technical Field

[0001] This invention belongs to the field of laser speckle imaging, and more specifically, relates to a method and system for high-resolution laser speckle vascular blood flow imaging. Background Technology

[0002] Improving the detection depth and spatial resolution of laser speckle contrast imaging has always been a focus. Classical laser speckle contrast imaging systems typically employ a reflective, wide-field imaging structure, where most of the reflected signal originates from shallow biological tissue. While utilizing the low absorption and scattering coefficients of biological tissue in the near-infrared band and employing a 785nm wavelength as the illumination source can improve the detection depth of reflective laser speckle contrast imaging, it still struggles to detect blood flow signals deeper than 700µm. Furthermore, the scattering signals from microvessels within tissue are often masked by the scattering signals from surrounding tissues. Without introducing depth resolution mechanisms, the proportion of signals from microvessels in the speckle images obtained by reflective systems is very low. This results in a lack of resolution capability for microvessels in reflective laser speckle contrast imaging systems.

[0003] When a transmissive imaging structure is used, where the illumination source and image sensor are positioned opposite the tissue being measured, the incident light penetrates the tissue, carrying all blood flow information along its propagation path. Therefore, transmissive imaging structures offer the possibility of detecting deep tissue blood flow signals and achieving high-resolution blood flow velocity imaging. However, according to the principles of tissue optics, if the thickness of biological tissue is on the order of hundreds of micrometers or even millimeters, the forward scattered light obtained using transmissive illumination includes not only diffused photons from multiple scatterings but also a large number of directly transmitted ballistic photons and serpentine photons with fewer scatterings. For such a mixture of scattered and non-scattered forward scattering, directly using the traditional laser speckle contrast theory model corresponding to a reflective detection structure to estimate the decorrelation time of the optical field related to flow velocity will lead to serious estimation errors, and may even fail to resolve blood flow information in microvessels. Summary of the Invention

[0004] In view of the above-mentioned defects or improvement needs of the prior art, the present invention provides a high-resolution laser speckle vascular blood flow imaging method and system, the purpose of which is to solve the technical problem that the complex composition of scattered light and the large difference in the degree of scattering under transmitted illumination in the prior art leads to large errors in blood flow information estimation.

[0005] To achieve the above objectives, according to one aspect of the present invention, a high-resolution laser speckle vascular blood flow imaging method is provided, comprising the following steps:

[0006] Using laser transmission illumination, a set of laser speckle images was captured with a short exposure time and a set of laser speckle images was captured with a long exposure time. The two sets of laser speckle images were then fused to obtain blood perfusion index images and angiography images.

[0007] Using the above technical solution, two sets of laser speckle images were obtained under short-time and long-time exposure. The laser speckle image under short-time exposure can take into account both the degree of scattering in the tissue under test and the velocity of the scattering particles in the tissue. After being fused with the laser speckle image under long-time exposure, the contributions of scattered and non-scattered photons in the transmitted light are separated, which can significantly reduce the estimation error. It can not only resolve the flow velocity information of microvessels, but also realize high-resolution imaging of fine microvascular structures at the same time.

[0008] The present invention also proposes a high-resolution laser speckle vascular blood flow imaging system, comprising a laser, a laser modulator, a sample stage, and an imaging device. The sample stage is used to place the tissue to be tested, the laser is used to provide incident light to the tissue to be tested and generate transmitted light, the imaging device is used to collect the transmitted light, and the laser modulator is used to adjust the intensity of the incident light. The incident light and the transmitted light are located on opposite sides of the sample stage.

[0009] This imaging system can be used to implement the optical path in the above imaging method. By adjusting the intensity of the incident light through a laser modulator, the intensity of the two sets of speckle images under short and long exposure times in the above imaging method is similar. Attached Figure Description

[0010] Figure 1 This is a schematic diagram illustrating the principle of the method of the present invention;

[0011] Figure 2 This is a schematic diagram of the system structure of the present invention;

[0012] Figure 3 Image (a) is the blood perfusion index obtained in Example 1;

[0013] Figure 3 Image (b) is an angiography image obtained in Example 1;

[0014] Figure 3 (c) is a vascular fluorescence angiography image obtained in Comparative Example 1;

[0015] Figure 3 (d) is a conventional time-contrast image obtained under transmitted illumination in Scale 2;

[0016] Figure 3 (e) is a spatial contrast image of transmitted illumination obtained in Scale 3;

[0017] Figure 3 (f) is a conventional time-contrast image obtained under reflective illumination as shown in Scale 4;

[0018] Figure 3 (g) is the normalized intensity distribution of LOI for Example 1 and Comparative Examples 1 to 4;

[0019] Figure 4 These are images of blood perfusion index, angiography, conventional time-contrast images under transmitted illumination, spatial contrast images under transmitted illumination, and conventional time-contrast images under reflected illumination, as shown in scales 5 to 8.

[0020] In the diagram, 1 is the laser; 2 is the collimating lens; 3 is the neutral density filter; 4 is the mirror; 5 is the sample stage; 6 is the objective lens; 7 is the lens barrel; and 8 is the camera. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0022] like Figure 1 As shown, this invention proposes a high-resolution laser speckle vascular blood flow imaging method, comprising the following steps:

[0023] The tissue under test was irradiated with laser transmission illumination. A set of laser speckle images of the tissue under test was captured with a short exposure time and a set of laser speckle images of the tissue under test were captured with a long exposure time. The two sets of laser speckle images were then fused to obtain blood perfusion index images and angiography images.

[0024] Existing transmission speckle imaging techniques acquire only one set of laser speckle images for processing, employing traditional time-contrast calculations, which can lead to incorrect estimations of blood flow velocity in tissues. The specific reasons for this have been explained in the background section. Our method, however, builds upon transmission speckle imaging by acquiring two sets of laser speckle images under both short and long exposures. It considers both the degree of scattering in the tissue and the velocity of scattering particles within it. Therefore, by fusing the two sets of images, the contributions of scattered and non-scattered photons in the transmitted light are separated. This allows us to not only resolve the flow velocity information of microvessels (i.e., the blood flow index) but also simultaneously achieve high-resolution imaging of fine microvascular structures, i.e., angiographic images.

[0025] Specifically, both short exposure time and long exposure time refer to the exposure time for capturing one frame of a laser speckle image, i.e., one exposure time. Therefore, to acquire two sets of laser speckle images with different exposure times, one can first perform N exposures with a short exposure time to capture N frames of laser speckle images, forming one set; then perform N exposures with a long exposure time to capture N frames of laser speckle images, forming another set. Alternatively, one can alternate between short and long exposure times N times, acquiring one frame of laser speckle image with a short exposure time and one frame of laser speckle image with a long exposure time each time, and repeating this cycle N times to obtain N frames of laser speckle images with short exposure times as one set and N frames of laser speckle images with long exposure times as another set. Furthermore, the order of short and long exposures is not important; each set of images must have at least one frame captured, and the number of frames can be adjusted according to the tissue being tested or the application scenario.

[0026] Furthermore, the specific process of fusion processing is as follows: The temporal contrast K is obtained from a set of laser speckle images captured with a short exposure time. t，short The temporal contrast K was obtained from a set of laser speckle images taken with long exposure time. t，log Then according to Calculate the electric field decorrelation time τ c , among which, T long Indicates long exposure time. The resulting blood perfusion index image is shown.

[0027] More specifically, temporal contrast is defined as the ratio of the standard deviation to the mean of an image sequence, i.e. Based on this well-known definition, the short exposure time contrast K under this method can be obtained. t，short Contrast with long exposure time K t，long ,Right now: Where, σ t，short represents the pixel standard deviation in the temporal neighborhood of laser speckle images acquired with short exposure times. t，short The pixel mean in the temporal neighborhood of a laser speckle image obtained by short exposure time; Where, σ t，long The pixel standard deviation in the temporal neighborhood of laser speckle images obtained by long exposure time photography. t，long This represents the average pixel value in the temporal neighborhood of a laser speckle image obtained through long exposure. Therefore, after capturing a set of laser speckle images, the temporal contrast K can be calculated from this set of speckle images.

[0028] The expression for laser speckle under transmission speckle imaging is: Therefore, under short exposure time, T is very small. According to L'Hôpital's rule, we know that... so Where β represents ..., ξ represents the tissue scattering coefficient, βξ 2 The resulting angiographic image is represented, and K is calculated using the well-known definition of time-contrast. t，short With K t，long After obtaining two values, the time contrast ratio of the two different exposure times is substituted into the laser speckle expression for transmission speckle imaging described above, thereby obtaining... In this formula, the time contrast ratio of the two different exposure times is a known parameter, and the long exposure time is also a known parameter. Finally, the electric field decorrelation time τ can be solved using Newton's iteration method. c .

[0029] βξ 2 This reflects the proportion of scattered light to the total light intensity, i.e., the scattering intensity of the tissue being tested. Therefore, this method utilizes the time contrast under short exposure time, which is approximately equal to βξ. 2 Replace βξ with time contrast under short exposure. 2 Substituting this into the laser speckle expression for transmission speckle imaging can reduce estimation errors and obtain... It can simultaneously display blood flow velocity information from both large and small blood vessels, correcting estimation errors caused by directly using time-contrast calculations.

[0030] Furthermore, the short exposure time is T short T short ≤100us, T long ≥1ms. The thickness of the tissue under test is on the order of hundreds of micrometers. Therefore, when laser speckle imaging is performed using transmitted illumination, the transmitted light contains not only diffused photons but also ballistic and serpentine photons. If the traditional time-contrast algorithm is used directly, the resulting estimation error will be 1 / K of the error in the large blood vessel region. 2 Higher than that of tissues without blood flow, while the 1 / K ratio corresponding to small blood vessels is higher. 2 Significantly lower than that of tissues without blood flow, exhibiting the "reverse color of blood vessels" phenomenon in blood flow maps, this phenomenon not only misinterprets the blood flow distribution of biological tissues, but also makes it difficult to segment and extract vascular morphology parameters.

[0031] Furthermore, when capturing two sets of laser speckle images, the laser power irradiated onto the tissue under test is adjusted to regulate the light intensity of the laser speckle images obtained at different exposure times. The purpose of this step is to make the intensity of the two sets of speckle images at short and long exposure times similar.

[0032] like Figure 2 As shown, the present invention also proposes a high-resolution laser speckle vascular blood flow imaging system, including a laser 1, a laser modulator 3, a sample stage 5, and an imaging device. The sample stage 5 is used to place the tissue to be tested, the laser 1 is used to provide incident light to the tissue to be tested and generate transmitted light, the imaging device is used to collect the transmitted light, and the laser modulator 3 is used to adjust the light intensity of the incident light. The incident light and the transmitted light are located on opposite sides of the sample stage 5.

[0033] By adjusting the intensity of the incident light using laser modulator 3, it can be ensured that the light intensity of images acquired at two different exposure times is comparable.

[0034] Furthermore, it also includes an image acquisition control module: This module controls the imaging device to capture a set of laser speckle images of the tissue under test using both short and long exposure times, based on the light intensity adjusted by the laser modulator 3. This module controls the exposure time according to the light intensity adjusted by the laser modulator 3.

[0035] Furthermore, it also includes: an angiography image generation module, used to generate a tissue angiography image by squared time contrast from a set of laser speckle images acquired with a short exposure time.

[0036] Furthermore, it also includes a blood perfusion index image generation module, which is used to generate a blood perfusion index image by fusing a set of laser speckle images acquired with a short exposure time and a set of laser speckle images acquired with a long exposure time.

[0037] In some embodiments, the laser modulator 3 is a current driver or acousto-optic modulator that adjusts the amplitude, pulse width, and frequency of the laser output optical pulse.

[0038] In other embodiments, the laser modulator is a neutral density filter (NDB) positioned between the laser and the sample stage, movable into / out of the incident light path. During short exposures, the NDB is not located in the incident light path; during long exposures, it is located in the incident light path. Moving the NDB 3 out of the incident light path during short exposures and moving it into the incident light path during long exposures attenuates the intensity of the incident light, reducing the light intensity irradiating the tissue to be tested within the sample stage 5, thereby ensuring that the light intensity of the images acquired at two different exposure times is comparable.

[0039] In some embodiments, a rotating device is installed between the laser 1 and the sample stage 5, and a neutral density filter is installed inside the rotating device. The rotating device drives the neutral density filter to rotate, thereby moving it into / out of the incident light path. Of course, the neutral density filter can also be moved into / out of the incident light path by means of a detachable configuration, etc., as long as the structure can adjust the position of the neutral density filter.

[0040] In some embodiments, a beam expander and a collimating lens 2 are also provided in the optical path between the laser 1 and the laser modulator 3. The collimating lens 2 may have a focal length of 30mm.

[0041] Furthermore, the imaging device includes an objective lens 6, a lens barrel 7, and a camera 8. Transmitted light passes sequentially through the objective lens 6 and lens barrel 7 before being acquired by the camera 8. The overall magnification of the imaging device is adjustable within the range of 0.4x to 4.0x. In some embodiments, the laser 1 is a semiconductor laser with a wavelength of 785nm and an optical power of 90mW, and the camera 8 is a CMOS camera with a frame rate of 50fps. Depending on the placement between the sample stage 5 and the laser 1, optical lenses such as reflectors 4 can be further placed between them. This optimizes the positional structure of each component and adjusts the direction of the incident light, ensuring that the incident light accurately strikes the tissue to be tested on the sample stage 5.

[0042] To verify and illustrate the technical effects of the present invention, the following embodiments and comparative examples are provided:

[0043] C57 mice were selected and anesthetized by intraperitoneal injection of 2% chloral hydrate and 10% urethane (0.01 ml / g) solution.

[0044] Example 1 and Comparative Examples 1 to 4: The test tissues were mouse ears, with a thickness of hundreds of micrometers.

[0045] Example 1

[0046] T short =21us,T long =20ms, by alternating between short and long exposure times 50 times, 50 frames of laser speckle images are obtained under short exposure time and 50 frames of laser speckle images under long exposure time respectively. In the system of this invention, during the shooting and acquisition process, during each short exposure time, the rotating device moves the neutral density filter out of the incident light optical path, and then during the long exposure time, it moves the neutral density filter back into the incident light optical path. This alternation is repeated 50 times to obtain two sets of images.

[0047] Then the two sets of images are fused together. Obtain the blood flow index, also known as the blood perfusion index image, such as... Figure 3 As shown in (a); with βξ 2 Obtain angiographic images, such as Figure 3 As shown in (b).

[0048] Comparative Example 1

[0049] Further, mice were injected via tail vein with a 1% tetramethylrhodamine isothiocyanate-dextran solution (TRITC-Dextran 70, injection dose 7.5 uL / g), followed by fluorescence angiography imaging using an independent fluorescence microscope (excitation wavelength 540 nm, emission fluorescence wavelength 605 nm) to obtain vascular fluorescence angiography images of the tested tissues, such as... Figure 3 As shown in (c).

[0050] Comparative Example 2

[0051] Under the same transmissive illumination conditions as in Example 1, 50 frames of laser speckle images were captured with a uniform exposure time of 20 ms. The temporal contrast K of this set of images was then obtained directly using the definition of temporal contrast. t1 ,by This represents a conventional time-contrast image under transmitted illumination, such as Figure 3 As shown in (d).

[0052] Comparative Example 3

[0053] The difference from Comparative Example 2 is that it uses the definition of spatial contrast, which is the ratio of the standard deviation of pixels to the mean of pixels in the spatial neighborhood of an image within a sliding window. This ratio is calculated within a 5×5 sliding window to obtain the spatial contrast K of the image set. s ,by This represents a spatial contrast image under transmitted illumination, such as Figure 3 As shown in (e).

[0054] Comparative Example 4

[0055] The difference from Comparative Example 2 is that reflected illumination is used, meaning that after the incident light shines on the tissue to be tested, the reflected light is collected on the same side as the incident light. The time contrast K is obtained in the same manner as in Comparative Example 2. t2 ,by This represents a conventional time-contrast image under reflected illumination, such as Figure 3 As shown in (f).

[0056] By comparing the images of Example 1 with those of Comparative Examples 1 to 4, it can be seen that... Figure 3 In the study (f), it was found that when using reflected illumination, vascular areas would be present in areas with small blood vessels. The phenomenon of being below the background tissue region; from Figure 3 In (e), it was found that under transmitted illumination, traditional spatial contrast algorithms can only obtain flow velocity information for a few large blood vessels, and cannot obtain the blood flow velocity of microvessels; from Figure 3 As can be seen in (d), under transmitted illumination, the traditional time-contrast algorithm exhibits the "vascular color reversal" phenomenon mentioned earlier. From Figure 3 As found in (a)-(c), under transmissive illumination, the blood flow index obtained using this invention can simultaneously display blood flow velocity information in both large and small blood vessels, i.e., it is corrected for... Figure 3 The estimation error in (d) indicates that the spatial resolution of this application is higher than that of comparative examples two, three, and four. Furthermore, under transmissive illumination, the angiography images obtained by this invention show the difference in scattering between vascular and non-vascular regions in the tissue, achieving label-free imaging of microvascular structural information, and its spatial resolution is comparable to that of fluorescence angiography images.

[0057] To further verify the technical effect of the present invention, a linear region of interest (LOI) was selected from the image results obtained in Example 1 and Comparative Examples 1-4, and the normalized intensity distribution was analyzed. The analysis results are as follows: Figure 3 As shown in (g), it can be observed that the two images of Example 1 can distinguish tiny blood vessels with a diameter of up to 20 μm. However, in Comparative Examples 2 and 4, which also use time-contrast, the intensity distribution curves show a "depression" at the blood vessels, indicating that there are problems with the traditional laser speckle time-contrast analytical transmission illumination light field.

[0058] Comparative Example 5

[0059] The tissue to be tested was mouse skin. Then, following the methods in Example 1, Comparative Examples 2, 3, and 4, images of the blood perfusion index, angiography, conventional time-contrast images under transmitted illumination, spatial contrast images under transmitted illumination, and conventional time-contrast images under reflected illumination were obtained sequentially from the mouse skin.

[0060] Comparative Example 6

[0061] The difference from Comparative Example 5 is that the test tissue was the hind paw of a mouse, and the blood perfusion index image, angiography image, conventional time-contrast image under transmitted illumination, spatial contrast image under transmitted illumination, and conventional time-contrast image under reflected illumination of the mouse hind paw were acquired sequentially.

[0062] Comparative Example 7

[0063] The difference from Comparative Example 5 is that the test tissue was the hind limb of a mouse, and the blood perfusion index image, angiography image, conventional time-contrast image under transmitted illumination, spatial contrast image under transmitted illumination, and conventional time-contrast image under reflected illumination of the mouse hind limb were acquired sequentially.

[0064] Comparative Example 8

[0065] The difference from Comparative Example 5 is that the test tissue was a mouse forepaw, and the blood perfusion index image, angiography image, conventional time-contrast image under transmitted illumination, spatial contrast image under transmitted illumination, and conventional time-contrast image under reflected illumination were acquired sequentially.

[0066] like Figure 4 As shown, five images from each of Comparative Examples 5, 6, 7, and 8 are presented. It can be observed that in Comparative Examples 6, 7, and 8, the conventional time-contrast images and spatial-contrast images under transmitted illumination have the same resolution as the blood perfusion index images obtained using the content of this invention. The verification results shown in Comparative Example 5 are consistent with the verification results after comparing with Examples 1, 2, 3, and 4. This is because the thickness of the tissue tested in Comparative Example 5 is the same as that in Example 1, both being on the order of hundreds of micrometers. In contrast, the thickness of the tissues tested in Comparative Examples 6, 7, and 8 is approximately on the order of millimeters. For such thick tissues, when using transmitted illumination, the incident light is almost completely scattered. Therefore, both traditional time-contrast images and spatial-contrast images under transmitted illumination have the same resolution as the perfusion index images obtained using this invention. Furthermore, since the incident light is completely scattered, there is no difference in the degree of scattering within the imaging field of view. Thus, the angiography images in Comparative Examples 6, 7, and 8 cannot distinguish vascular structures. Only thinner tissues under transmitted illumination show differences in the degree of scattering. Therefore, this invention is more suitable for samples on the order of hundreds of micrometers. In addition, it can be observed that regardless of the thickness of the tissue being tested, the perfusion index images obtained using transmitted illumination still have better resolution of vascular details than those obtained using reflected illumination.

[0067] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A high-resolution laser speckle angiography method, characterized by, The method comprises the following steps: A set of laser speckle images is collected by using laser transmission illumination and short exposure time, and another set of laser speckle images is collected by using laser transmission illumination and long exposure time, and then the two sets of laser speckle images are fused to obtain a blood perfusion index image and an angiography image; The specific process of the image fusion processing is: obtaining time contrast of a group of laser speckle images shot under a short exposure time according to the group of laser speckle images ; obtaining time contrast of a group of laser speckle images shot under a long exposure time according to the group of laser speckle images ; Then according to Computing the electric field decorrelation time , representing the resulting angiogram image, representing a long exposure time, representing the resulting blood perfusion index image.

2. The method of claim 1, wherein, Short exposure times are , , .

3. The method of claim 1, wherein, During the collection of the two sets of laser speckle images, the laser power under laser transmission illumination is adjusted to adjust the light intensity of the laser speckle images obtained under different exposure times.

4. A high-resolution laser speckle angiography system for implementing the method according to any one of claims 1 to 3, characterized in that The method comprises a laser, a laser modulator, a sample stage and an imaging device, the sample stage is used to place a tissue to be measured, the laser is used to provide incident light to the tissue to be measured and generate transmitted light, the imaging device is used to collect the transmitted light, and the laser modulator is used to adjust the light intensity of the incident light, the incident light and the transmitted light being located on opposite sides of the sample stage.

5. The system of claim 4, wherein, The method further comprises an image collection control module: according to the light intensity adjusted by the laser modulator, the imaging device is controlled to collect a set of laser speckle images of the tissue to be measured with short exposure time and to collect another set of laser speckle images of the tissue to be measured with long exposure time.

6. The system of claim 5, wherein, The method further comprises: An angiography image generation module is configured to generate an angiography image of the tissue by squaring the time contrast of the set of laser speckle images collected with short exposure time.

7. The system of claim 5, wherein, The method further comprises: A blood perfusion index image generation module is configured to generate a blood perfusion index image by fusing the set of laser speckle images collected with short exposure time and the set of laser speckle images collected with long exposure time.

8. The system according to any of claims 5-7, characterized in that, The laser modulator is a current driver or an acousto-optic modulator that adjusts the amplitude, pulse width and frequency of the light pulses output by the laser.

9. The system according to any of claims 5-7, characterized in that, The laser modulator is a neutral density filter arranged between the laser and the sample stage and capable of being moved into or out of the light path of the incident light, when the exposure time is short, the neutral density filter is not located in the light path of the incident light, and when the exposure time is long, the neutral density filter is located in the light path of the incident light.

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