CN114010163A - Epidermal cell migration positioning system and method based on optical imaging - Google Patents
Epidermal cell migration positioning system and method based on optical imaging Download PDFInfo
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Abstract
The invention discloses an epidermal cell migration positioning system and method based on optical imaging, and belongs to the technical field of biological information processing. The epidermal cell migration positioning system based on optical imaging comprises a two-photon imaging unit, a capillary detection unit, a skin imaging unit and a laser speckle blood flow imaging unit; the two-photon imaging unit is used for positioning the migrating epidermal cells in a net structure, the migrating epidermal cells are stacked, and neogenetic hair follicles are arranged among the stacked migrating epidermal cells; the capillary detection unit is used for positioning a gray area, and capillary vessels are not imaged in the positioning area; the skin imaging unit is used for positioning a pink area, and capillary vessel-free imaging is carried out in the positioning area; the laser speckle blood flow imaging unit is used for positioning the area where the live-action image is gray and the blood flow perfusion image is green. The system is used for carrying out migration and positioning of epidermal cells, the wound surface edge can be defined, and a foundation is laid for collecting wound surface healing data.
Description
Technical Field
The invention belongs to the technical field of biological information processing, and particularly relates to an epidermal cell migration positioning system and method based on optical imaging.
Background
The wound healing is a long and complex process, the healing effect of the wound healing not only affects the skin aesthetic property, but also is directly related to the recovery of the normal function of the skin, and therefore, the establishment of a wound healing effect evaluation system is necessary.
Wound healing is a dynamic process, how to better dynamically track and evaluate the healing condition of a wound in real time becomes a big problem in the field of open wound treatment at present, and although the wound healing condition can be evaluated by methods such as one-dimensional measurement, area measurement, volume measurement, a three-dimensional wound reconstruction system, size change rate, wound healing time and the like, the evaluation process still has many factors influencing judgment, such as unclear boundary, difficult definition of irregular wound shape, difficult evaluation of early healing and the like.
Therefore, how to define the wound edge and further provide an analysis basis for the evaluation of wound healing is a technical problem to be solved in the field.
Disclosure of Invention
The invention discloses an epidermal cell migration positioning system and method based on optical imaging, which realize wound surface edge definition by positioning migrated epidermal cells.
In order to achieve the purpose, the invention adopts the following technical scheme:
the epidermal cell migration positioning system based on optical imaging comprises a two-photon imaging unit, a capillary detection unit, a skin imaging unit and a laser speckle blood flow imaging unit;
the two-photon imaging unit is used for positioning the migrating epidermal cells in a net structure, the migrating epidermal cells are stacked, and neogenetic hair follicles are arranged among the stacked migrating epidermal cells;
the capillary detection unit is used for positioning a gray area between a black opaque area in the center of the wound surface and an area with capillary distribution on the outer edge, and capillary-free imaging is performed in the positioning area;
the skin imaging unit is used for positioning a pink area between a red area in the middle of a wound surface and an area with capillary blood vessel distribution at the outer edge, and no capillary blood vessel is imaged in the positioning area;
the laser speckle blood flow imaging unit is used for positioning the area where the live-action image is gray and the blood flow perfusion image is green.
The skin consists of the epidermis, the dermis separated by a basement membrane, and the subcutaneous tissue; when severe damage occurs and parts of the dermis are destroyed, it must be repaired quickly to restore the protective barrier. The restoration of skin integrity is mainly accomplished by four consecutive but overlapping phases: coagulation, inflammation, re-epithelialization and remodeling. After injury, firstly, fibrin fiber and blood platelet form a clot to block the wound; subsequently, under the action of various growth factors, the neovasculature and fibroblasts form granulation tissue, keratinocytes migrate and proliferate at the wound margins to extend the newly formed epithelial carpet consisting of several layers of cells in the epidermis, a process called re-epithelialization, lasting two to three weeks; then, myofibroblasts transformed from fibroblasts contract and gather the wound margins together; finally, remodeling is performed to restore normal skin homeostasis.
During re-epithelialization, as the depth of granulation tissue increases, the boundary epithelial thickness and epithelial regeneration length change continuously. Dermal-derived fibroblasts establish granulation tissue, epidermis migrates on the temporary connective tissue layer, and the migration of epidermal cells and the contraction of a dermal connective tissue bed pull the wound edges together to further realize the closure of the wound; whereas epidermal migration involves locomotion by active pseudopodia of epidermal cells and purse-string contraction at the epidermal free boundary, basal layer epidermal cells are stacked on top of each other to transform a single layer of cells into a multi-layered cell. This can undoubtedly become a criterion for determining the wound edges in the course of irregular wound healing. Therefore, the invention mainly focuses on the process of re-epithelialization, applies various optical imaging technologies to in-vivo observation, observes the optical imaging form of epidermal cell migration, and realizes the positioning of the migrated epidermal cells, thereby completing the definition of the wound edge and providing an information acquisition basis for the evaluation of wound healing.
Preferably, the system further comprises an optical coherence tomography unit;
the optical coherence tomography unit is used for positioning the black cavity area at the edge of the longitudinal section of the wound.
Preferably, the system further comprises a pathological section detection unit;
the pathological section detection unit is used for positioning the migration epidermal cells accumulated on the longitudinal section of the wound, and the newly-born hair follicles are arranged among the accumulated migration epidermal cells.
Preferably, the system further comprises an information processing unit;
the information processing unit is used for processing the positioning information obtained by the other units and comprehensively analyzing to obtain the migrated epidermal cell positioning area.
The method for positioning the migration of the epidermal cells based on optical imaging uses any system to perform the positioning of the migration of the epidermal cells.
The epidermal cell migration positioning method based on optical imaging comprises the following steps:
the two-photon imaging unit is used for positioning the migrating epidermal cells in a net structure, the migrating epidermal cells are stacked, and neogenetic hair follicles are arranged among the stacked migrating epidermal cells;
positioning a gray area through a capillary detection unit, and imaging hairless blood vessels in the positioning area between a black lightless area in the center of the wound surface and an area with hairless blood vessels on the outer edge;
the pink area is positioned by the skin imaging unit and is between the red area in the middle of the wound surface and the area with capillary blood vessel distribution on the outer edge, and no capillary blood vessel is imaged in the positioning area;
positioning the gray area of the real scene image and the green area of the blood flow perfusion image through a laser speckle blood flow imaging unit, wherein the gray scale 2039-;
and realizing the positioning of the migrated epidermal cells by the correlation and correspondence of the positioning results of all units.
Preferably, the length of epidermal cell migration is in the range of 0.8-1.2 mm.
Preferably, the method further comprises positioning a black cavity region at the edge of the longitudinal section of the wound by the optical coherence tomography unit.
Preferably, the method further comprises positioning the migrating epidermal cells accumulated in the longitudinal section of the wound by the pathological section detection unit, wherein the newly-grown hair follicles are arranged between the accumulated migrating epidermal cells.
In summary, the invention uses various optical imaging technologies to characterize and position the migrated epidermal cells, uses the two-photon imaging unit, the capillary detection unit, the skin imaging unit and the laser speckle blood flow imaging unit to observe the wound margin on the XY axis level, uses the optical coherence tomography unit and the pathological section detection unit to observe the structure on the Z axis level, and performs the length range measurement and the correlation correspondence between the living body image information, and multiple verification results to obtain the conclusion, thereby locking the wound boundary and providing the basis for wound healing evaluation.
Drawings
Fig. 1 is a view showing migration of a wound surface;
a, cutting a wound surface picture (marking positions of suture lines by circles) after skin is completely cut;
b. wound surface pictures (circle marks suture line positions) 5 days after the model is made;
c. wound surface pictures (circle marks suture line position, outer layer dotted line marks wound surface contour after molding, middle layer dotted line marks wound surface contour after molding for 5 days, arrow marks migration direction of suture line along with wound) after molding for 10 days;
FIG. 2 shows a wound margin cross-sectional observation;
a, wound edge morphology under a two-photon mirror (an arrow marks inward-migrating epidermal cells, and hair follicles are in the middle);
b. is an enlarged image of the location indicated by the arrow in fig. 3a (the arrow in this figure indicates the neogenetic follicle);
c. live-action images of the speckle pattern (the gray area between the two dotted lines is the migrating epidermal cells, the length range is 1 mm);
d. blood flow perfusion image of the speckle machine (green area in the middle of two dotted lines indicates migrating epidermal cells);
e. wound edge images under the capillary detector mirror (dark grey areas between dotted lines indicate migrating epidermal cells);
f. dermoscopic wound edge images (meat pink areas between dashed lines indicate migrating epidermal cells);
FIG. 3 shows the results of a longitudinal section of the wound margin;
the method comprises the following steps of a, acquiring a central longitudinal section of a wound surface by an OCT (three circles respectively indicate a left wound edge, extravasated blood and a right wound edge from left to right, and a black line segment marks the length range of migrating epidermal cells);
acquiring a live-action image by the OCT image (three circles respectively mark a left wound edge, extravasated blood and a right wound edge from left to right, and black line segments mark the length range of the migrating epidermal cells);
masson staining wound surface center longitudinal section (three circles respectively mark a left wound edge, extravasated blood and a right wound edge from left to right, a white line segment marks a length range of migrating epidermal cells; the corresponding position of the extravasated blood and the OCT image are taken from the same section position);
d. an enlarged view of the right circle of FIG. 3c (inward migrating epidermal cells are visible, and the black line segment marks the extent of the length of the migrating epidermal cells).
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 epidermal cell migration positioning system based on optical imaging comprises a two-photon imaging unit, a capillary detection unit, a skin imaging unit and a laser speckle blood flow imaging unit;
the two-photon imaging unit comprises a two-photon confocal scanning microscope (Olympus FV1000) and a Mai Tai Deepsee laser (120fs, 80MHz), and is used for positioning migrating epidermal cells with a mesh structure at the edge of a wound, wherein the migrating epidermal cells are stacked, and newly-grown hair follicles are arranged among the stacked migrating epidermal cells;
the capillary vessel detection unit selects a capillary vessel detector (model: CAM1CVF) for positioning a gray area between a black opaque area at the center of the wound surface and an area with capillary vessels distributed at the outer edge, and no capillary vessel is imaged in the positioning area;
the skin imaging unit selects a skin mirror for positioning a wound edge pink area between a wound surface middle red area and an area with capillary blood vessel distribution at the outer edge, no capillary blood vessel imaging is carried out in the positioning area, and the migration length range is 0.8-1.2 mm;
the laser speckle blood flow imaging unit selects a laser speckle blood flow imaging instrument (model: SIM BFI HRPro) for positioning the gray area of the wound edge live-action image and the green area of the blood flow perfusion image, and the migration length range is 0.8-1.2 mm.
Furthermore, the device can also comprise an optical coherence tomography unit, wherein an Optical Coherence Tomography (OCT) scanner is selected for positioning the black cavity area at the edge of the longitudinal section of the wound, and the migration length is in the range of 0.8-1.2 mm;
the pathological section detection unit comprises a paraffin section processing material, a staining kit and a panoramic scanner, and is used for positioning the migration epidermal cells accumulated on the longitudinal section of the wound, wherein the newly-born hair follicles are arranged among the accumulated migration epidermal cells, and the migration length range is 0.8-1.2 mm.
And the information processing unit selects image processing software for processing the positioning information obtained by the rest units and comprehensively analyzes to obtain the migrated epidermal cell positioning area.
Example 2
Epidermal cell migration localization was performed using the system of example 1.
1. Modeling and wound surface migration experiment
3 male Wistar rats with a body weight of 190 + -20 g were selected.
Shaving off back hair with shaver, removing residual back hair with depilatory cream, drying with physiological saline, selecting middle part of back, and dryingFor the operation area, after the local sterilization of the operation area, a diameter of 1.4cm is subtracted from the 1cm position on the two sides of the rat spine, and the area of the wound surface is 1.54cm2Is circular. The skin is cut off in a full layer, the wound surface is opened and reaches the muscular layer, after sufficient hemostasis is achieved, surgical suture lines are sewn into the edge of the wound around the wound surface, the depth reaches the full-layer skin, and the purpose is to mark the migration condition of the skin around the wound. To prevent infection, appropriate amounts of antibiotics are applied to the wound area. The rats are raised in a single cage in a clean cage, and are fed with water freely and taken medicine every day. And respectively photographing at 0D, 5D and 10D after wound modeling to record the migration condition of the wound surface.
As shown in fig. 1, the suture line migrates toward the center of the wound surface along the wound healing direction as the wound surface heals, which means that the skin around the wound surface migrates toward the center of the wound surface along with the decrease of the wound margin during the wound healing process, i.e. the epidermal cells on the outermost layer of the skin migrate toward the center of the wound surface along with the wound healing.
2. Observation of wound margin cross section
(1) Observation by two-photon imaging technique
And (10D) anesthetizing the rat with isoflurane after the model building, cleaning hair and impurities around the wound, and injecting a rhodamine dextran solution (8ml/ml) into the tail vein, wherein the injection dose is 0.4ml/100 g.
Signals are collected from the back by using a two-photon confocal scanning microscope and a laser, and the skin at the edge of the wound surface is fixed at a proper height by using a special skin absorption fixer, so that the influence generated by the respiration of a rat is reduced as much as possible. Signal collection is started, the two-photon fluorescence excitation wavelength is 860nm, excitation light is focused to an observation part by an objective lens, a generated fluorescence signal is collected by 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 in a two-photon fluorescence channel, the wavelength range is 420 and 460nm, tissue is scanned in a full-layer Z-axis mode, the step is 1 mu m, the image collection speed is 10 mu m/pixel, and the obtained images are subjected to Z-axis superposition.
As a result, as shown in fig. 2a and 2b, under the two-photon microscope, the migrating epidermal cells were in a network structure, the epidermal cells were densely packed, and the newly-grown hair follicles were present between the packed migrating epidermal cells, and the capillary vessels were present at the outer edges of the migrating epidermal cells.
(2) Speckle appearance observation
And (5) anesthetizing the rat by using isoflurane after the model building at the 10 th step, cleaning hairs and impurities around the wound, finding the position of the wound surface under a microscope generally by using a laser speckle blood flow imager, connecting a computer image, carrying out real-time positioning and calibration, simultaneously acquiring a live-action image and a blood flow perfusion image, and acquiring the images when the live-action image and the blood flow perfusion image are relatively clear, wherein the acquisition time is 100 s. The length measurement was then made with imagej software according to the image ruler.
As shown in fig. 2c and 2d, under the live-action mirror, the migrating epidermal cells are represented by a circle of gray of the edge of the wound surface, and according to the color bar on the right, it can be determined that the gray level is about 2039-3067, and the migration length range is about 1 mm; the migrating epidermal cells in the blood flow perfusion image are one week green of the wound edge, and the relative blood flow perfusion amount range is approximately between 182 and 274 according to the right color bar, and the migration length range is 1 mm.
(3) Observation by capillary vessel detector
And (5) anesthetizing a rat by using isoflurane after the model is manufactured at the 10 th D, cleaning hairs and impurities around the wound, collecting image information at the junction of the wound and normal skin by using a capillary vessel detector, smearing white oil on the surface of the skin, aligning to a measurement area, starting laser, adjusting the focal length until the visual field is clear, collecting an image, and recording an image file by using a Capiscope processing unit.
As a result, as shown in fig. 2e, under the capillary detector, the center of the wound surface is a black opaque region, and outside the central region, the migrating epidermal cells become a dark gray fuzzy zone, no obvious capillary vessels appear, and the capillary vessels growing toward the center of the wound surface can be observed toward the outer edge.
(4) Observation with a skin mirror
And (5) anesthetizing the rat by using isoflurane after the model is made at the 10D, cleaning hair and impurities around the wound, and acquiring an image of the edge area of the wound surface by using a 20X lens of a skin mirror. The length measurement was then made with imagej software according to the image ruler.
As shown in fig. 2f, under the skin mirror, the center of the wound surface is a red area, the migrating epidermal cells are shown as a ring of meat pink area around the wound surface, the length range is about 1mm, no obvious capillary vessels appear in the area, and obvious blood vessel running can be observed outwards, which corresponds to the observation result of the capillary vessel detector.
3. Observation of wound edge in longitudinal section
(1) OCT (optical coherence tomography) Observation
After molding, 10D, rats were anesthetized with isoflurane, excess hair around the wound was removed with a shaver and depilatory cream, wiped with warm saline, and the wound and skin around the window were gently wiped dry with a sterile cotton swab, completely exposing the wound surface. The OCT is used for observation, the selection mode is line scanning, the line length is 10mm, the whole wound surface can be spanned, the height and the direction of a lens are adjusted, the line scanning area is aligned to the central area of the wound surface until the collected signals are kept at a higher level, the image is basically in a horizontal state, and the image is collected. The length measurement was then made with imagej software according to the image ruler.
As shown in fig. 3a, a longitudinal section of the central area of the wound surface is observed, the center of the wound surface has a black cavity area with a low reflection signal, after position correspondence comparison with the live-action image 3b, the central congestion part can be determined, the left wound edge and the right wound edge have a black cavity area with a low reflection signal, respectively, the area with a pale pink color at the edge of the wound surface can be corresponded on the live-action image, the new tissue which is not completely developed and mature and the inward migrating epidermal cells are marked, and the length range can be roughly measured to be 1mm according to a ruler.
Therefore, it can be concluded that the areas of low-reflection signals (i.e. black void areas) exhibited by the wound margin areas on OCT images during wound healing indicate new immature tissues and migrating epidermal cells.
(2) Masson stained section Observation
After all the living body data are collected, the wound skin tissue is taken down, fixed with 4% paraformaldehyde for one week, dehydrated and embedded in paraffin sections at 10D after the model is made. Paraffin sections were dewaxed to water, stained according to Solebao modified Masson trichrome staining kit (Cat # G1345) instructions, and finally dehydrated and mounted. And scanning the panoramic of the wound surface by using a panoramic scanner, and comparing the panoramic with the data acquired by the OCT living body. The length measurement was then made with imagej software according to the image ruler.
As shown in fig. 3c, newly generated tissues at the edge of the wound surface and a central congestion area are observed, and according to the shape of congestion and the position relative to the wound surface, the selected position of the section can be determined to be the same as the position of OCT living body data acquisition. From fig. 3d, it can be observed that there are inward migrating epidermal cells at the right wound edge, and there are significant increase and accumulation and thickening at the wound edge compared with normal skin, and the length range is roughly measured to be 1mm according to the ruler, and there are new hair follicles among the accumulated migrating epidermal cells.
Further, fig. 2 and 3 may correspond to each other. The thickened areas of epidermal cell accumulation at the wound edges of fig. 3a, 3b, 3c (areas indicated by circles on the left and right of fig. 3a, 3b, 3 c) correspond to the wound edge network of fig. 2a (indicated by white arrows in fig. 2 a); corresponding to FIG. 2c, the gray level around the wound surface is one week, and the gray level is probably 2039-3067 (as shown in the middle area of the two dotted lines in FIG. 2 c); corresponding to fig. 2d, the range of the relative blood perfusion amount is approximately 182-274 (as shown in the middle area between the two dotted lines in fig. 2 d); corresponding to the dark grey area around the wound in fig. 2e (as in the middle area between the two blue dashed lines in fig. 2 e); corresponding to the pink area of the flesh around the wound in fig. 2f (as shown in the middle area of the two dashed lines in fig. 2 f). And the length of the corresponding area is measured, and the length range of the epidermal cells migrating at the wound edge is represented by 1mm in the transverse and longitudinal sections. On the other hand, from a microscopic perspective, fig. 2b and 3d show the microscopic morphology of the migrating epidermal cells, and it can be observed that there are numerous new hair follicles among the migrating epidermal cells. Namely, the method realizes the positioning of the migrated epidermal cells through correlation and correspondence, and further can carry out wound margin definition according to the migrated epidermal cells, thereby being beneficial to the subsequent data statistics of the wound healing condition.
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 (8)
1. An epidermal cell migration positioning system based on optical imaging is characterized in that,
the device comprises a two-photon imaging unit, a capillary detection unit, a skin imaging unit and a laser speckle blood flow imaging unit;
the two-photon imaging unit is used for positioning the migrating epidermal cells in a net structure, the migrating epidermal cells are stacked, and neogenetic hair follicles are arranged among the stacked migrating epidermal cells;
the capillary vessel detection unit is used for positioning a gray area between a black opaque area in the center of the wound surface and an area with capillary vessels on the outer edge, and capillary vessel-free imaging is performed in the positioning area;
the skin imaging unit is used for positioning a pink area between a red area in the middle of a wound surface and an area with capillary blood vessel distribution at the outer edge, and no capillary blood vessel is imaged in the positioning area;
the laser speckle blood flow imaging unit is used for positioning an area where a live-action image is gray and a blood flow perfusion image is green.
2. The system for epidermal cell migration localization based on optical imaging according to claim 1,
further comprising an optical coherence tomography unit;
the optical coherence tomography unit is used for positioning the black cavity area at the edge of the longitudinal section of the wound.
3. The system for epidermal cell migration localization based on optical imaging according to claim 1,
the device also comprises a pathological section detection unit;
the pathological section detection unit is used for positioning the migration epidermal cells accumulated on the longitudinal section of the wound, and neogenetic hair follicles are arranged among the accumulated migration epidermal cells.
4. The system for epidermal cell migration localization based on optical imaging according to claim 1,
the system also comprises an information processing unit;
the information processing unit is used for processing the positioning information obtained by the other units and comprehensively analyzing to obtain the migrated epidermal cell positioning area.
5. An epidermal cell migration positioning method based on optical imaging is characterized in that,
localization of epidermal cell migration using the system of any one of claims 1-4.
6. The method for positioning epidermal cell migration based on optical imaging according to claim 5,
positioning the migrating epidermal cells in a net structure through the two-photon imaging unit, wherein the migrating epidermal cells are stacked, and neogenetic hair follicles are arranged among the stacked migrating epidermal cells;
positioning a gray area between a black opaque area in the center of the wound surface and an area with capillary vessels on the outer edge by the capillary vessel detection unit, and imaging without capillary vessels in the positioning area;
the pink area is positioned by the skin imaging unit and is between the red area in the middle of the wound surface and the area with capillary blood vessels on the outer edge, and no capillary blood vessel is imaged in the positioning area;
positioning an area where the live-action image is gray and the blood perfusion image is green through the laser speckle blood flow imaging unit;
and realizing the positioning of the migrated epidermal cells by the correlation and correspondence of the positioning results of all units.
7. The method for positioning epidermal cell migration based on optical imaging according to claim 5,
and the optical coherence tomography unit is used for positioning the black cavity area at the edge of the longitudinal section of the wound.
8. The method for positioning epidermal cell migration based on optical imaging according to claim 5,
the pathological section detection unit is used for positioning the migrating epidermal cells accumulated on the longitudinal section of the wound, and neogenetic hair follicles are arranged among the accumulated migrating epidermal cells.
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