CN114041771A

CN114041771A - System and method for quantitative evaluation of skin blood vessels and blood flow based on optical imaging

Info

Publication number: CN114041771A
Application number: CN202111460113.6A
Authority: CN
Inventors: 孙娅楠; 李俐霜; 王毅
Original assignee: EXPERIMENTAL RESEARCH CENTER CHINA ACADEMY OF CHINESE MEDICAL SCIENCES
Current assignee: EXPERIMENTAL RESEARCH CENTER CHINA ACADEMY OF CHINESE MEDICAL SCIENCES
Priority date: 2021-12-02
Filing date: 2021-12-02
Publication date: 2022-02-15

Abstract

The invention discloses a quantitative evaluation system and method for skin blood vessels and blood flow based on optical imaging, and belongs to the technical field of biological information processing. The skin blood vessel and blood flow quantitative evaluation system based on optical imaging includes a laser speckle blood flow imaging unit, a two-photon imaging unit, a capillary detection unit and an immunofluorescence staining unit. The laser speckle blood flow imaging unit, capillary detection unit, and immunofluorescence staining unit are used for macroscopic observation, and the two-photon imaging unit is used for microscopic morphological observation. The observation results of each unit are integrated to realize the establishment of a quantitative evaluation system for dermal blood vessels and blood flow. , which provides a strong guarantee for the research of vascular blood microcirculation system.

Description

Skin blood vessel and blood flow quantitative evaluation system and method based on optical imaging

Technical Field

The invention belongs to the technical field of biological information processing, and particularly relates to a skin blood vessel and blood flow quantitative evaluation system and method based on optical imaging.

Background

The skin microcirculation blood vessel network has three-dimensional construction characteristics, the capillary loop in hairpin shape on the outermost surface layer is positioned in the dermal papilla, each dermal papilla has one or more loops which are different, and the capillary artery and vein transition part is arranged below the dermal papilla and is in a hedgehog skin-like appearance as a whole; a layer of polygonal capillary network characterized by polygonal meshes is arranged below the papilla at the junction of the dermal papilla layer and the reticular layer, and the abundant vascular network provides sufficient nutrition for peripheral nerves, hair follicles, glands and the like, thereby having very important significance.

At present, the observation of the form of the blood vessel of the dermis and the blood flow perfusion amount still has limitations, on one hand, a single method cannot evaluate a result in a one-sided way, the accuracy is poor, and a comprehensive blood vessel and blood flow evaluation system is not constructed; on the other hand, most of the capillary observation means stay at the XY axis level, that is, the level of the cross section, and are limited to the papillary ring level depth of the dermis, and the three-dimensional evaluation of the blood vessel is incomplete, so that a more comprehensive and complete quantitative evaluation system for the blood vessel flow of the dermis is required to be constructed.

Disclosure of Invention

The invention discloses a system and a method for quantitatively evaluating skin blood vessels and blood flow based on optical imaging, which are used for observing and evaluating a plurality of angles from an XY axis and a Z axis so as to enable the blood flow indexes of blood vessels in a dermis layer to be more comprehensive and detailed.

In order to achieve the purpose, the invention adopts the following technical scheme:

the skin blood vessel and blood flow quantitative evaluation system based on optical imaging comprises a laser speckle blood flow imaging unit, a two-photon imaging unit, a capillary vessel detection unit and an immunofluorescence staining unit;

the laser speckle blood flow imaging unit is used for macroscopic observation of blood vessels and blood flow perfusion;

the two-photon imaging unit is used for microscopic observation of the shape and the form of the blood vessel;

the capillary vessel detection unit is used for observing a capillary vessel network;

the immunofluorescence staining unit is used for counting the blood vessels in the transverse row and the longitudinal row at different detection depths.

The laser speckle blood flow imaging has the advantages that the blood flow perfusion amount of different parts can be counted macroscopically, the diameter and the angle of a blood vessel can be roughly counted, but the requirement on the light transmittance of the skin is higher, after a transparent agent is smeared, only the approximate running of the blood vessel can be seen, the detection depth is limited, the blood vessel is limited to the superficial dermis, and the blood vessel is not suitable for accurate measurement, particularly the measurement of a micro blood vessel smaller than 2 mu m; the two-photon imaging unit is arranged, so that the running and the shape of a microscopic blood vessel can be obtained, particularly, the measurement of a micro blood vessel with the diameter less than 2 mu m and the accurate angle measurement of a blood vessel branch can be obtained, and the hair follicle surrounded by the blood vessel can be seen; the method makes up the deficiency of laser speckle blood flow imaging, and enables the index quantification of the blood vessel blood flow to be more comprehensive and accurate. The capillary vessel detection unit has the advantages that the polygonal capillary vessel network under the papilla at the junction of the dermal papilla layer and the reticular layer and the surrounding hair follicles are observed macroscopically, and the number of the capillary vessel networks and the number of nodes can be calculated through software so as to represent the density of the capillary vessels. On the longitudinal section, because the blood vessels in the microcirculation network have variable running, the immunofluorescence staining unit can respectively distinguish the transverse blood vessels from the longitudinal blood vessels in the superficial layer and the deep layer of the dermis for counting, and is used for counting the number of the blood vessels in different forms and categories of each layer. And a more comprehensive and complete blood vessel blood flow evaluation system can be formed by integrating the observation results of all the units.

Preferably, the system further comprises an information processing unit;

the information processing unit is used for comprehensively analyzing the image information obtained by the laser speckle blood flow imaging unit, the two-photon imaging unit, the capillary detection unit and the immunofluorescence staining unit.

Further, the diameter of the blood vessel observed by the laser speckle blood flow imaging unit is larger than 2 μm.

The method for quantitatively evaluating the blood vessels and the blood flow of the skin based on optical imaging uses any system to quantitatively evaluate the blood vessels and the blood flow of the skin.

The method for quantitatively evaluating the blood vessels and the blood flow of the skin based on optical imaging comprises the following steps:

macroscopic observation of blood vessels and blood perfusion amount is carried out through the laser speckle blood flow imaging unit;

microscopic observation of the shape and the form of the blood vessel is carried out through the two-photon imaging unit;

observing a capillary network through a capillary detection unit;

observing the transverse and longitudinal blood vessels at different detection depths by an immunofluorescence staining unit;

and integrating the image information of each unit to quantify the indexes of the skin blood vessels and the blood flow.

In summary, the invention utilizes various optical imaging units to respectively observe and count from XY-axis and Z-axis levels, wherein the laser speckle blood flow imaging unit, the capillary detection unit and the immunofluorescence staining unit are used for macroscopic observation, the two-photon imaging unit is used for microscopic observation, observation results of all units are integrated, establishment of a quantitative evaluation system of blood vessels and blood flow in a dermis layer is realized, and powerful guarantee is provided for research of a blood vessel blood flow microcirculation system.

Drawings

FIG. 1 shows the speckle pattern observation results during the observation position selection process;

wherein: a. a live-action picture after rat back depilation, wherein a black line is divided into six areas;

b. rat dorsal blood perfusion map (boxed area is late selection observation area);

C. and (4) a blood perfusion quantity statistical chart of six regions on the back.

FIG. 2 shows the observation result of the capillary probe and the observation result of the OCT in the process of selecting the observation position;

wherein: a-f are observation images of the rat back by a capillary vessel detector:

a. an upper left image; b. an upper right image; c. a middle left image; d. the middle right image; e. a lower left image; f. lower right image;

g-l is the observation image of the rat back by OCT:

g. an upper left image; h. an upper right image; i. a middle left image; j. the middle right image; k. a lower left image; lower right image.

FIG. 3 shows the results of the XY-axis hierarchical observation;

imaging the blood vessel at the back under the speckle instrument (the line segment shows the diameter of the blood vessel);

b. imaging the blood vessels at the back under the speckle apparatus (the blood flow perfusion statistics in the area is shown by a circle);

c. imaging the blood vessel at the back under the speckle instrument (the line segment angle is the branch angle of the blood vessel);

d. FIG. 3a is a statistical map of vessel diameters;

e. FIG. 3b is a blood perfusion statistics.

f. Two-photon blood vessel imaging (the angle of a line segment is the branch angle of a blood vessel; the line segment shows the diameter of the blood vessel);

g. capillary imager back vessel image (hair follicle indicated by white arrow);

h. manually tracing the running of the capillary vessel;

automatically extracting a blood vessel network image by imagej;

automatically analyzing the nodes of the blood vessel network by imagej;

imagej automatically extracts the vessel network nodes.

FIG. 4 shows a Z-axis hierarchy observation;

wherein, a transverse blood vessel immunofluorescence image of a dermal-epidermal junction;

c. FIG. 4a is a vessel count map (dots indicate locations of transverse vessels; dashed lines indicated by yellow arrows indicate typical transverse vessel cross-sections;

d. a longitudinal blood vessel immunofluorescence image at the junction of the dermis and the epidermis;

f. FIG. 4d is a graph of a vessel count (dark circles indicate the locations of longitudinal vessels; dashed lines indicated by dark arrows indicate typical longitudinal vessel cross-sections; white circles indicate the locations of transverse vessels; dashed lines indicated by white arrows indicate typical transverse vessel cross-sections;

g. a transverse blood vessel immunofluorescence image of deeper layers of dermis;

i. FIG. 4g is a graph of the blood vessel count (dots indicating the locations of transverse blood vessels; dashed lines indicated by dark arrows indicate typical transverse blood vessel cross-sections; dashed lines indicated by white arrows indicate deeper layers of large blood vessel cross-sections; dashed lines indicated by arrows within deeper layers of large blood vessel cross-sections indicate large intravascular red blood cells;

j. longitudinal blood vessel immunofluorescence image of deeper layer of dermis;

the vessel count plot of FIG. 4j (dots indicate where the longitudinal vessel is located; dashed lines indicated by white arrows indicate the travel of the branch vessel;

b. e, h and k are superimposed graphs of blood vessel immunofluorescence, cell nucleus blue staining and tissue autofluorescence, and the labeling meanings in the graphs are respectively the same as those in the graphs c, f, i and l.

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

the laser speckle blood flow imaging unit selects a laser speckle blood flow imaging instrument (model: SIM BFI HR Pro) for macroscopic observation of blood vessels and blood flow perfusion.

The two-photon imaging unit comprises a two-photon confocal scanning microscope (Olympus FV1000) and a Mai Tai deep See laser (120fs, 80MHz) and is used for microscopic observation of the blood vessel deformation and morphology;

the capillary vessel detection unit selects a capillary vessel detector (model: CAM1CVF) for observing a capillary vessel network;

the immunofluorescence staining unit comprises a section preparation material, an immunofluorescence reaction material and fluorescence observation equipment and is used for counting the transverse and longitudinal blood vessels at different detection depths.

Further, the system can also comprise an information processing unit, wherein image processing software is selected and used for comprehensively analyzing the image information obtained by the laser speckle blood flow imaging unit, the two-photon imaging unit, the capillary detection unit and the immunofluorescence staining unit, and the image information comprises indexes such as capillary density, diameter, mesh node number and blood flow perfusion quantity.

Example 2

Quantitative evaluation of skin vessels and blood flow was performed using the system of example 1.

1. Observation position selection

(1) Speckle appearance observation

3 male Wistar rats with a body weight of 190 + -20 g were selected.

Rats are anesthetized with isoflurane, most of the hair on the back is shaved off with a shaver, the remaining hair is cleaned with depilatory cream, and finally the rat is wiped with warm physiological saline. The rat is in a continuous anesthesia state, is placed under a microscope with a laser speckle blood flow imager, observes the back skin of the rat, is connected with a computer image, carries out real-time positioning and calibration, simultaneously collects a real-time image and a blood flow image, carries out image collection when the two images are relatively clear, and the collection time is 100 s.

The back of the human body is divided into six areas, namely an upper area, a middle area and a lower area, each area is uniformly divided into a left side and a right side, the difference of blood perfusion amount of each area is observed and compared, statistics is carried out, and the optimal observation part is screened.

As shown in fig. 1, it can be seen from fig. 1c that the blood perfusion in the upper, middle and lower parts of the back of the rat is mostly characterized by the right blood perfusion significantly better than the left blood perfusion in the data analysis statistics, and the blood perfusion in the middle part is significantly higher than that in the upper and lower parts and the blood perfusion in the lower part is the lowest in consideration of individual differences caused by the exercise habits of the rat. And as can be observed from fig. 1b, the red area in the middle part accounts for the most, and is distributed uniformly, and the high blood flow perfusion area in the middle area of the back can also be visually observed. In summary, the blood flow perfusion in the middle part is the most intensive and is most suitable for the site of blood flow observation.

(2) Observation by capillary vessel detector

Anesthetizing a rat with isoflurane, cleaning back hairs, fully exposing back skin, collecting image information on the back of the rat by using a capillary vessel detector, smearing white light oil on the surface of the skin, aligning to a measurement area, starting laser, adjusting focal length until the visual field is clear, collecting an image, and recording an image file on a Capiscope processing unit. Image recording is performed for each of the six divided regions.

As shown in fig. 2, it can be observed from fig. 2a, 2b, 2c, 2d, 2e, and 2f that the capillaries on the back of the rat are in a net-like distribution, the upper and lower part of the rat have fewer vascular networks, the middle part of the rat has more vascular networks, especially the middle part (fig. 2d) has a significantly richer vascular network than other parts, the results observed by the speckle pattern apparatus are consistent, the middle part and the right part have rich vascular networks, and the corresponding blood perfusion is dense.

(3) OCT (optical coherence tomography) Observation

Rats were anesthetized with isoflurane, the back hair was cleaned, the back skin was fully exposed, OCT (optical coherence tomography) was used, the line scan was chosen, the length of the line was 10mm, the lens height and direction were adjusted so that the line scan area was aligned with the back viewing area until the collected signal remained at a high level and the image was substantially horizontal, and the image was collected. Image recording is performed for each of the six divided regions.

The results are shown in fig. 2, which is observed from the general observations of fig. 2g, 2h, 2i, 2j, 2k, 2l, the upper skin layer is thin and uneven, the light penetration of the tissue is poor, resulting in a weak signal, while the middle and lower skin layers are thicker, homogeneous, smooth surface, good light penetration and strong signal.

In summary, the observation result of the speckle meter is that the blood perfusion volume of the middle area of the back is the highest, and corresponding to the result of the capillary vessel detector, the capillary vessel network corresponding to the area with high blood perfusion volume is also abundant, and the observation result of the OCT is that the light transmission of the middle and lower parts is better and the signal reflection is stronger; therefore, based on the consideration of light transmittance and blood perfusion, the middle area of the back of the rat is finally selected as the observation area.

2, observing the blood vessel in the XY-axis layer

(1) Speckle appearance observation and statistics

The rats are anesthetized by isoflurane, back hairs are cleaned, back skin is fully exposed, image signal acquisition is carried out on the middle area of the back, and the specific acquisition mode is the same as that of the back. And the diameter of the capillary vessel and the perfusion volume of the blood flow are counted by using self-contained analysis software of the speckle apparatus.

As shown in fig. 3a and 3d, under the observation of the speckle pattern, the blood vessel running of normal skin shows a higher blood perfusion color labeling area (yellow-green area), and branches from the main blood vessel to the small blood vessel, so that the blood vessel running is clear and the blood vessel diameters are counted, the main blood vessel diameter is 4.328 μm (left four line segment), the blood vessel near branch diameter is 3.213 μm (left three line segment), the branch blood vessel diameter is 2.245 μm (left two line segment), and the blood vessel diameter is presumed to be too thin at the left line segment label, so that the measurement value is extremely unstable, and the diameter cannot be measured. And in fig. 3d it can be observed that the diameter of the main vessel increases twice due to the increased perfusion of the blood flow, and the blood flow at the secondary vessel also increases, with the same increase in diameter, but the diameter of the secondary small vessel does not change significantly. It is shown that the blood flow of the main and secondary vessels are mutually affected, and the closer to the main vessel, the more obvious the effect is.

As shown in fig. 3b and 3e, the perfusion rates of the thicker main vessel branches are 41.8 (upper right circle) and 43.6 (lower right circle), the perfusion rate of the secondary vessel branches is 40.8 (lower middle circle), the perfusion rate of the terminal vessel branches is 39.6 (upper left circle) and 37.5 (lower left circle), respectively; it can be observed from fig. 3e that the perfusion amount fluctuates with the respiratory movement of the rat, and the perfusion amounts between the primary and secondary blood vessels affect each other with the same change rule. Fig. 3c can count the angles of the blood vessel branches, which are the included angles formed by black line segments: 147 °, 104 °, 112 °.

Therefore, on the one hand, the blood perfusion of the available primary and secondary blood vessels fluctuates along with the respiration of the rat, and the diameter of the blood vessels is influenced, and the change trend among the primary and secondary blood vessels is the same. On the other hand, the perfusion of blood flow from the main vessel to the secondary vessel to the terminal vessel decreases in diameter.

(2) Two-photon observation and statistics

Rats were anesthetized with isoflurane, the back hair was cleaned, the back skin was fully exposed, and the tail vein was injected with rhodamine dextran solution (8ml/ml) at an injection dose of 0.4ml/100 g. The signal was collected from the back using a two-photon fluorescence imaging unit. Signal collection is started, the two-photon fluorescence excitation wavelength is 860nm, excitation light is focused to an observation part through an objective lens, a generated fluorescence signal is collected through a water immersion microscope objective lens with the same objective lens 25 multiple value and the aperture of 1.05, a high-pass filter is used for a two-photon fluorescence channel, and the wavelength is 860 nm; and (3) carrying out full-layer Z-axis scanning on the tissues, stepping by 1 mu m, carrying out image acquisition at the speed of 10 mu m/pixel, and carrying out Z-axis superposition on the obtained images.

As a result, as shown in FIG. 3f, the diameter of the blood vessel can be calculated by a ruler, the diameter of the thicker main blood vessel is 6.8 μm, the diameter of the branch blood vessel is 4.83 μm, the diameter of the thinner blood vessel can reach 1.63 μm, so clear blood vessel running can not be observed under a speckle instrument, and the diameter of the micro blood vessel smaller than 2 μm can not be counted. The blood vessel branch angle can be calculated by imagej software and is 78.82 degrees and 65.51 degrees respectively. The morphology of the blood vessels is more obvious under the image, and micro blood vessels with the diameter less than 2 mu m can be counted.

(3) Observation and statistics of capillary vessel detector

The rats are anesthetized by isoflurane, back hairs are cleaned, back skin is fully exposed, image signal acquisition is carried out on the middle area of the back, and the specific acquisition mode is the same as that of the back. Then, an image is derived, the running of the capillary blood vessels is manually outlined, and the capillary blood vessel network and the node number of the capillary blood vessel network are counted by imagej software.

As can be seen from fig. 3g, under the scope of the capillary probe, the back blood vessels are in a net shape, and hair follicles are distributed in the middle of the blood vessel net (white arrows), fig. 3i shows 16 closed blood vessel nets and 9 non-closed blood vessel nets, the total number of the visible blood vessel nets is 25, fig. 3k shows the node statistics of the blood vessel net, and 64 nodes are provided. The number of vessel nets and the nodes of the vessel nets may both represent the distribution density of the vessels.

Observing blood vessel in Z-axis level

After all the living body data are collected, the skin in the middle of the back of the rat is taken down and is put into 4 percent paraformaldehyde for soaking for a week, and then the tissue is dehydrated, embedded and made into paraffin sections. Dewaxing and rehydrating the paraffin sections, washing with 0.01M PBS (phosphate buffer solution) for 5min multiplied by 3 times; sealing the normal goat serum, and carrying out 37 ℃ and 30 min; excess serum was decanted, primary antibody (abcam, Anti-CD31 antibody [ EPR17259] (ab182981) was added dropwise at a dilution ratio of 1:500), overnight at 4 °, washed with 0.01M PBS for 5min X3 times, secondary fluorescent antibody (Alexa Fluor 594 labeled goat Anti-rabbit IgG (H + L) at a dilution ratio of 1:500), incubated at room temperature in the dark for 1H, washed with 0.1M PBS for 5min X3 times, blocked with an Anti-quenching blocking tablet (Biyunyan, product No. P0131, Anti-fluorescence quenching blocking solution (containing DAPI)) and stored at 4 ℃ in the dark. And observing the vascular morphology and the distribution condition of the dermis under a two-photon microscope, observing the autofluorescence of the dermis, superposing the vascular fluorescence, the autofluorescence and the cell nucleus blue stain images, and then carrying out classification statistics on different vascular morphologies.

The positions and shapes of the blood vessels can be observed more clearly according to the superposed images 4a, 4d, 4g and 4j of fig. 4b, 4e, 4h and 4k, and the superposed images are circular or long-strip-shaped in fig. 4b, 4e, 4h and 4k, and by contrast, the blood vessels are counted by using imagej software to obtain fig. 4c, 4f, 4j and 4 l.

The conclusion can be drawn:

(1) comparing fig. 4i, 4l and 4c, 4f, it can be seen that the blood vessels in the deep dermis (including the blood vessels in the horizontal and vertical rows) are more numerous than those in the dermal layer at the interface with the epidermis, and the blood vessels in the deep dermis are roughly distributed (fig. 4i white dotted line);

(2) comparing fig. 4c and 4f, it can be seen that there are two kinds of vessels in the lower dermis layer of the epidermis, i.e. there are 25 vessels in the row (dots) in fig. 4c, and there are 9 vessels in the row (white dots) and 9 vessels in the column (dark dots) in fig. 4f, which indicate that the vessels in the row and the column are interlaced with each other and need to be separately counted according to the form;

(3) comparing fig. 4i and 4l, the deeper dermis also has two blood vessels, namely vertical and horizontal blood vessels, and large blood vessels, which need to be counted separately, 63 blood vessels in horizontal row (fig. 4 dot), 2 large blood vessels (fig. 4i white dotted line), 17 blood vessels in vertical row (fig. 4l dot line), and blood vessel branches can be seen (fig. 4l white dotted line).

By combining the observation and statistical results of the X-axis level and the Z-axis level, the indexes such as the trend, the number, the density, the diameter, the branch angle, the number of the mesh nodes, the blood perfusion amount and the like of the capillary vessel can be comprehensively and carefully represented, and a foundation is laid for the research of the blood vessel blood flow microcirculation system.

The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to the above-described embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A quantitative evaluation system of skin blood vessels and blood flow based on optical imaging is characterized in that,

the device comprises a laser speckle blood flow imaging unit, a two-photon imaging unit, a capillary detection unit and an immunofluorescence staining unit;

2. The system for quantitative evaluation of cutaneous blood vessels and blood flow based on optical imaging according to claim 1,

the system also comprises an information processing unit;

3. The system for quantitative evaluation of cutaneous blood vessels and blood flow based on optical imaging according to claim 1,

the diameter of the blood vessel observed by the laser speckle blood flow imaging unit is larger than 2 mu m.

4. A quantitative evaluation method of skin blood vessels and blood flow based on optical imaging is characterized in that,

quantitative assessment of cutaneous blood vessels and blood flow is carried out using the system according to any one of claims 1 to 3.

5. The method for quantitatively evaluating blood vessels and blood flow of skin based on optical imaging according to claim 4,

observing a capillary network through the capillary detection unit;

observing the transverse and longitudinal blood vessels at different detection depths by the immunofluorescence staining unit;