Real-time, in-VIVO, skin imaging using quantitative oblique back-illumination microscopy

Abstract

An exemplary embodiment of the present disclosure provides a method of imaging skin to obtain information on dermal lesions, comprising: providing a quantitative oblique back- illumination microscopy (qOBM) system; and volumetrically imaging, with the qOBM system, at least a portion of skin of a subject in vivo.

Description

REAL-TIME, IN-VIVO, SKIN IMAGING USING QUANTITATIVE OBLIQUE BACK-ILLUMINATION MICROSCOPYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 575,11 1, filed on 5 April 2024, which is incorporated herein by reference in its entirety as if fully set forth belowGOVERNMENT LICENSE RIGHTS

[0002] This invention was made with government support under GM147437 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD OF THE DISCLOSURE

[0003] The various embodiments of the present disclosure relate generally to skin imaging system and methods.BACKGROUND

[0004] Skin cancer is the most common malignancy in the United States, and concurrently one of the most preventable. Lower mortality and morbidity rates are associated with early detection and diagnosis. Moreover, undetected malignant lesions in skin of color are associated with greater morbidity and mortality, necessitating accurate diagnosis in all skin tones. The gold standard for diagnosis of skin cancer is histopathological analysis; however, the selection of lesions for histopathology is at the discretion of the dermatologist in the clinic. In order to aid the dermatologist in this selection, various imaging technologies have been developed.

[0005] Dermatoscopy is the most commonplace method for imaging skin lesions in the dermatology clinic. Handheld dermatoscopes provide a clinician with a 1 Ox magnified image of a lesion of interest. Since imaging at cellular resolution is not possible with dermatoscopy, dermatologists must be trained to correlate features found on der-matoscopy with histopathological features. Despite its widespread usage, the diagnostic utility of dermatoscopy in determining cancerous lesions is equivocal and highly dependent on the expertise of the user.

[0006] Imaging modalities with higher spatial resolution have been developed, including optical coherence tomography (OCT) and confocal reflectance microscopy (RCM). AlthoughOCT provides extensive depth information, subcellular information cannot be gleaned laterally. RCM can provide subcellular, volumetric resolution of the skin, however its cost, complexity, and bulkiness prevent widespread clinical use. These presented limitations elucidate a need for a low-cost, convenient tool for imaging skin with cellular detail. The present disclosure addresses this need.BRIEF SUMMARY

[0007] An exemplary embodiment of the present disclosure provides a method of imaging skin to obtain information on dermal lesions, comprising: providing a quantitative oblique back- illumination microscopy (qOBM) system; and volumetrically imaging, with the qOBM system, at least a portion of skin of a subject in vivo.

[0008] In any of the embodiments disclosed herein, the qOBM system can be a handheld qOBM system.

[0009] In any of the embodiments disclosed herein, the qOBM system can be configured to image the at least a portion of the skin of the subject using light having a wavelength of 400- 1750 nm.

[0010] In any of the embodiments disclosed herein, volumetrically imaging the at least a portion of the skin can occur from a surface to a depth of about 3 mm.

[0011] In any of the embodiments disclosed herein, the qOBM system can comprise one or more light emitting diodes (LEDs) and one or more fibers. The one or more fibers can have first ends coupled to a respective LED and second ends disposed near a distal end of the qOBM system proximate the skin to be imaged.

[0012] In any of the embodiments disclosed herein, the one or more fibers can comprise four fibers having second ends disposed at the distal end of the qOBM system.

[0013] In any of the embodiments disclosed herein, the qOBM system can comprise a planar substrate disposed at a distal end of the qOBM system, and the planar substrate can be configured to maintain the at least a portion of the skin to be imaged at a constant imaging depth.

[0014] In any of the embodiments disclosed herein, volumetrically imaging the at least a portion of skin of the subject in vivo can comprise: illuminating the at least a portion of the skin with a first light source; imaging the at least a portion of the skin to generate a first image; and generating, based at least in part on the first image, a quantitative phase image of the atleast a portion of the skin, in which each pixel of the quantitative phase image is indicative of refractive index properties of the tissue corresponding to the respective pixel.

[0015] In any of the embodiments disclosed herein, illuminating the at least a portion of the skin with the first light source can comprise directing light from the first light source at a first oblique angle to the at least a portion of the skin.

[0016] In any of the embodiments disclosed herein, volumetrically imaging the at least a portion of skin of the subject in vivo can further comprise: illuminating the at least a portion of the skin with a second light source; and imaging the at least a portion of the skin to generate a second image, wherein generating the quantitative phase image of the at least a portion of the skin is further based at least in part on the second image.

[0017] In any of the embodiments disclosed herein, illuminating the at least a portion of the skin with the second light source can comprise directing light from the second light source at a second oblique angle different from the first oblique angle to the at least a portion of the skin.

[0018] In any of the embodiments disclosed herein, the first and second images can be at a same field of view.

[0019] In any of the embodiments disclosed herein, the light from the first light source and the light from the second light source can have the same wavelength.

[0020] In any of the embodiments disclosed herein, the at least a portion of skin can comprise one or more vessels having a plurality of blood cells contained therein, and the method can further comprising quantifying, based at least in part on the quantitative phase image, the plurality of blood cells.

[0021] In any of the embodiments disclosed herein, the skin can be volumetrically imaged at cellular resolution.

[0022] Another embodiment of the present disclosure provides a quantitative oblique back- illumination microscopy (qOBM) system configured to image at least a portion of skin of a subject in vivo. The system can comprise a planar substrate, at least one light source, a camera, and a processor. The planar substrate can be disposed at a distal end of the qOBM system and configured to maintain the at least a portion of the skin to be imaged at a constant imaging depth. The at least one light source can be configured to illuminate the at least a portion of the skin in at least one oblique angle. The camera can be configured to receive light from the at least a portion of the skin and generate at least one image. The processor can be configured to process the at least one image and generate a quantitative phase image based at least in part onthe at least one image, in which each pixel of the quantitative phase image can be indicative of refractive index properties of the tissue corresponding to the respective pixel.

[0023] In any of the embodiments disclosed herein, the at least one light source can be configured to emit light having a wavelength of 400-1750 nm.

[0024] In any of the embodiments disclosed herein, the qOBM system can be handheld.

[0025] In any of the embodiments disclosed herein, the system can be configured to volumetrically image the at least a portion of the skin at a depth of up to 3 mm.

[0026] In any of the embodiments disclosed herein, the at least one light source can comprise a plurality of LEDs, and each LED can be configured to illuminate the at least a portion of the skin at a distinct oblique angle.

[0027] In any of the embodiments disclosed herein, each of the plurality of LEDs can be connected to a first end of a corresponding optical fiber, each of the optical fibers can comprise a second end positioned proximate the planar substrate, and the second end can be configured to direct light to the at least a portion of the skin to be imaged at an oblique angle.

[0028] In any of the embodiments disclosed herein, the processor can be configured to: cause the at least one light source to illuminate the at least a portion of the skin at a first oblique angle; cause the camera to generate a first image at a first field of view during illumination at the first oblique angle; and generate the quantitative phase image based at least in part on the first image.

[0029] In any of the embodiments disclosed herein, the processor can be further configured to: cause the at least one light source to illuminate the at least a portion of the skin at a second oblique angle; cause the camera to generate a second image at the first field of view during illumination at the second oblique angle; and generate the quantitative phase image based at least in part on the first and second images.

[0030] In any of the embodiments disclosed herein, the processor can be further configured to: cause the at least one light source to illuminate the at least a portion of the skin at a third oblique angle; cause the camera to generate a third image at the first field of view during illumination at the third oblique angle; and generate the quantitative phase image based at least in part on the first, second, and third images.

[0031] In any of the embodiments disclosed herein, the processor can be further configured to: cause the at least one light source to illuminate the at least a portion of the skin at a fourth oblique angle; cause the camera to generate a fourth image at the first field of view duringillumination at the fourth oblique angle; and generate the quantitative phase image based at least in part on the first, second, third, and fourth images.

[0032] These and other aspects of the present disclosure are described in the Detailed Description below and the accompanying drawings. Other aspects and features of embodiments will become apparent to those of ordinary skill in the art upon reviewing the following description of specific, exemplary embodiments in concert with the drawings. While features of the present disclosure may be discussed relative to certain embodiments and figures, all embodiments of the present disclosure can include one or more of the features discussed herein. Further, while one or more embodiments may be discussed as having certain advantageous features, one or more of such features may also be used with the various embodiments discussed herein. In similar fashion, while exemplary embodiments may be discussed below as device, system, or method embodiments, it is to be understood that such exemplary embodiments can be implemented in various devices, systems, and methods of the present disclosure.BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The following detailed description of specific embodiments of the disclosure will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the disclosure, specific embodiments are shown in the drawings. It should be understood, however, that the disclosure is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0034] FIG. 1 provides a flow chart of a method of imaging skin, in accordance with some embodiments of the present disclosure.

[0035] FIGS. 2A-B provide systems for imaging skin, in accordance with some embodiments of the present disclosure.

[0036] FIGS. 3A-H provide epidermal images acquired with handheld qOBM skin imaging system prove along the Fitzpatrick scale at different z-depths. Images from (A, E) Fitzpatrick 2, (B, F) Fitzpatrick 3, (C, G) Fitzpatrick 4, (D, H) Fitzpatrick 6 skin, where the top row include images from the top of the basal layer of the epidermis and the bottom include images from areas of blood flow (indicated by boxes), in which scale bars are 50 microns.DETAILED DESCRIPTION

[0037] To facilitate an understanding of the principles and features of the present disclosure, various illustrative embodiments are explained below. The components, steps, and materials described hereinafter as making up various elements of the embodiments disclosed herein are intended to be illustrative and not restrictive. Many suitable components, steps, and materials that would perform the same or similar functions as the components, steps, and materials described herein are intended to be embraced within the scope of the disclosure. Such other components, steps, and materials not described herein can include, but are not limited to, similar components or steps that are developed after development of the embodiments disclosed herein.

[0038] An inventor of the present invention recently introduced quantitative oblique back- illumination microscopy (qOBM) which provides quantitative phase information in thick, highly scattering tissues. Such concepts are disclosed in US Patent Application Publication No. 2021 / 0025818, entitled “Cell-Imaging Systems and Methods,” and US Patent Application Publication No. 2023 / 0329532, entitled “Optical Phase Imaging Device Optimization Methods,” the entire contents of which are incorporated herein by reference in their entireties as if fully set forth below. Like quantitative phase imaging (QPI), qOBM, provides cellular and subcellular contrast based on the intrinsic refractive index properties of the sample with nanometer scale sensitivity. A critical difference, however, is that qOBM can provide 3D information of thick scattering samples using epi-illumination, while traditional QPI methods cannot. The inventors have applied qOBM, a label-free, non-invasive, and real-time imaging modality, to image a number of clinical samples at cellular and subcellular resolution. Moreover, qOBM can enable 3D imaging of complex biological structure and can be adapted into a handheld probe design for the use of imaging brain tumors in the operating room. Embodiments of the present disclosure provide systems and methods for fast, compact, low- cost, label-free and portable imaging that can bridge the gap between dermatoscopes and more complex and expensive RCM and OCT systems to provide dermatologists with real-time diagnostic information at the bedside.

[0039] The present disclosure relates to the use of qOBM to image skin in real-time to provide information regarding dermal lesions. The qOBM system could have an embodiment comprising any of the following: a compact handheld probe, a fiber based system, a compact chip-on-tip system, or a larger table top system. Information of the epidermis, dermis and other components of the skin (e.g., blood vessels and blood flowing inside) can be visualized and analyzed using qOBM for screening, monitoring or diagnostic purposes.

[0040] As shown in FIG. 1 , an exemplary embodiments of the present disclosure provides a method of imaging skin 100. The method can comprise providing qOBM system 105 and volumetrically imaging at least a portion of the skin of a subject in vivo. The qOBM system can be any of the qOBM systems disclosed herein, which are discussed below.

[0041] In some embodiments, volumetrically imaging the at least a portion of the skin of the subject in vivo 110 can comprise: illuminating the at least a portion of the skin with a light source 111, imaging the at least a portion of the skin to generate an image 112, and generating, based at least in part on the image, a quantitative phase image of the at least a portion of the skin. The quantitative phase image can comprise a plurality of pixels in which each pixel can be indicative of refractive index properties of the tissue corresponding to the respective pixel.

[0042] In some embodiments, some embodiments, multiple images can be taken in order to generate the quantitative phase image. For example, in some embodiments multiple images can be taken with various illumination patterns based on imaging while light is directed to the skin from different angles. For example, as discussed below, in some embodiments, the qOBM system can comprise a plurality of light sources. Accordingly, in some embodiments, a first image can be taken during illumination from a first light source and then a second image can be taken during illumination from a second light source. In some embodiments, additional images can be taken during illumination from additional light sources, e.g., third image with a third light source, fourth image with a fourth light source, and so on. Each of the images can be taken from the same field of view, e.g., the camera of the qOBM system is not moved relative to the skin between the first and second (or third, fourth, etc. images). Additionally, each of the images can be taken while light from the various light sources is illuminating the skin with the same wavelength of light, which can range from 400-1750nm. The quantitative phase image can then be generated based on each of these images through known techniques.

[0043] In some embodiments, volumetrically imaging the skin can occur throughout the thickness of the skin. For example, in some embodiments, volumetrically imaging the skin can occur from a surface of the ski to a depth of about 3 mm. Additionally, the volumetric imaging can be performed at a cellular resolution (i.e., the quantitative phase image can be at a resolution allowing visual distinction between adjacent cells).

[0044] In some embodiments, the quantitative phase image can be utilized to characterize (e.g., quantify) blood cells in a blood vessel (e.g., capillary) in the skin.

[0045] As shown in FIGs. 2A-B, the present disclosure also provides qOBM systems configured to image at least a portion of skin of a subject in vivo. In some embodiments, thesystem can comprise a planar substrate 205. The planar substrate 205 can be disposed at a distal end of the qOBM system. The planar substrate 205 can be configured to allow the system to be placed proximate the skin to be imaged and can maintain the portion of the skin to be imaged at a constant imaging depth. The substrate 205 can be made of many materials known in the art. In some embodiments, the substrate 205 can be made of a transparent material, e.g., glass, that allows light to pass through the substrate where the light can be captured by a camera 215. In some embodiments, the planar substrate 205 can be formed by one or more lens 206 which can maintain the portion of the skin to be imaged at a constant imaging depth. Additionally, in some embodiments, the planar substrate 205 can comprise both one or more lens 206 and a viewing window 207. As used herein, the substrate 205 is described as “planar” to mean that the substrate 205 has a substantially planar / flat surface proximate the skin to be imaged.

[0046] The qOBM system can further comprise one or more light sources 210A-B 211A-B. The light sources 210A-B 21 1 A-B can be any light sources known in the art capable of emitting light having a wavelength between 400 nm and 1750 nm. Exemplary light sources, include, but are not limited to, LEDs, optical fibers, light bulbs, lasers, and the like. For example, as shown in FIG. 2A, two LED light sources 210A 210B are provided on the system. Similarly, as shown in FIG. 2B, two LEDs 210A 210B are connected to respective optical fibers 211A 21 IB. light can be emitted from the LEDs 210A 210B into first ends of the optical fibers 211A 21 IB. The light can then travel down the optical fibers 211A 21 IB wherein the light exits at second ends and illuminates the skin proximate the substrate 205.

[0047] Though the embodiments shown in FIGs. 2A-B show only two light sources, the disclosure is not so limited. Rather, as those skilled in the art would appreciate, various embodiments can include any number of light sources, e.g., three, four, or more. Further, each light source can be positioned at a distinct location around the qOBM system (e.g., the distal end thereof) and configured to illuminate the skin proximate the substrate from a distinct angle as a result of the position of the light source. Illumination from each light source can create a different light scattering through the skin, thus allowing for multiple images to be taken and used to generate the quantitative phase image as discussed above.

[0048] The qOBM system can further comprise a camera 215 configured to receive light scattered by the skin (light that is emitted by the light sources to illuminate the skin which then scatters through and is then received by the camera) to generate an image. The camera 215 can be many cameras known in the art. As discussed above, in some embodiments, the camera 215 can take multiple images during illumination by multiple light sources.

[0049] The qOBM system can further comprise a processor 220. The processor 220 can be any processor known in the art and can include a combination of hardware and software for controlling the light source(s) 210A-B 211A-B and camera 215, receiving an image(s) from the camera 215, and generating the quantitative phase image based on the received image(s).

[0050] Below, an exemplary qOBM system is described. The system is exemplary only and should not be construed as limiting the scope of the present disclosure or claims submitted herewith.

[0051] Methods

[0052] A qOBM handheld probe developed for volumetric imaging. The probe comprises four 850nm LEDs coupled to multi-mode PMMA polymer fibers to deliver illumination to the sample from the four comers of the front of the probe. A piece of cover glass can be mounted on the probe front to flatten the sample and keep a constant imaging depth within the sample. The field of view at a certain depth (e.g. 50pm into the sample) can be imaged onto the camera with a Nikon 40x objective (CFI S Plan Fluor ELWD 40XC, 0.6NA) and a 50mm tube lens (Thorlabs AC127-050-AB). Lateral positioning can be dictated by probe position along the skin. Axial positioning can be achieved with an electronically tunable lens (Optotune EL- 12- 30-TC-VIS-16D) positioned behind the tube lens.

[0053] Results

[0054] The epidermis was volumetrically imaged for a range of Fitzpatrick skin types with the qOBM handheld probe. These types are assessed by the skin color and propensity for burning and freckling, where lower numbers can indicate a lighter skin tone. To assess equivalent differences between skin types, all skin was imaged at the reference z=0pm, set to be near the basal layer of the epidermis (FIGs. 3A-D). Cellular resolution was present for all skin types. Differences in image quality were apparent between participants, however these discrepancies do not seem to trend with skin type. Additionally, blood flow was observed in all skin types (FIGs. 3E-H), a promising sign for qOBM’s ability to image into the dermis. These results are encouraging for the use of qOBM as an real-time and inclusive skin imaging modality.

[0055] It is to be understood that the embodiments and claims disclosed herein are not limited in their application to the details of construction and arrangement of the components set forth in the description and illustrated in the drawings. Rather, the description and the drawings provide examples of the embodiments envisioned. The embodiments and claims disclosed herein are further capable of other embodiments and of being practiced and carried out invarious ways. Also, it is to be understood that the phraseology and terminology employed herein are for the purposes of description and should not be regarded as limiting the claims.

[0056] Accordingly, those skilled in the art will appreciate that the conception upon which the application and claims are based may be readily utilized as a basis for the design of other structures, methods, and systems for carrying out the several purposes of the embodiments and claims presented in this application. It is important, therefore, that the claims be regarded as including such equivalent constructions.

[0057] Furthermore, the purpose of the foregoing Abstract is to enable the United States Patent and Trademark Office and the public generally, and especially including the practitioners in the art who are not familiar with patent and legal terms or phraseology, to determine quickly from a cursory inspection the nature and essence of the technical disclosure of the application. The Abstract is neither intended to define the claims of the application, nor is it intended to be limiting to the scope of the claims in any way.

Claims

CLAIMSWhat is claimed is:

1. A method of imaging skin to obtain information on dermal lesions, comprising: providing a quantitative oblique back- illumination microscopy (qOBM) system; and volumetrically imaging, with the qOBM system, at least a portion of skin of a subject in vivo.

2. The method of claim 1, wherein the qOBM system is handheld qOBM system.

3. The method of any of claims 1-2, wherein the qOBM system is configured to image the at least a portion of the skin of the subject using light having a wavelength in the range of 400- 1750 nm.

4. The method of claim 1 , wherein volumetrically imaging the at least a portion of the skin occurs from a surface to a depth of about 3 mm.

5. The method of any of claims 1, wherein the qOBM system comprises: one or more light emitting diodes (LEDs); and one or more fibers having first ends couple to a respective LED and second ends disposed near a distal end of the qOBM system proximate the skin to be imaged.

6. The method of claim 5, wherein the one or more fibers comprise four fibers having second ends disposed at the distal end of the qOBM system.

7. The method of claim 5, wherein the qOBM system comprises a planar substrate disposed at a distal end of the qOBM system, the planar substrate configured to maintain the at least a portion of the skin to be imaged at a constant imaging depth.

8. The method of claim 1 , wherein volumetrically imaging the at least a portion of skin of the subject in vivo comprises: illuminating the at least a portion of the skin with a first light source; imaging the at least a portion of the skin to generate a first image; and generating, based at least in part on the first image, a quantitative phase image of the at least a portion of the skin, each pixel of the quantitative phase image indicative of refractive index properties of the tissue corresponding to the respective pixel.

9. The method of claim 8, wherein illuminating the at least a portion of the skin with the first light source, comprises directing light from the first light source at a first oblique angle to the at least a portion of the skin.

10. The method of claim 9, wherein volumetrically imaging the at least a portion of skin of the subject in vivo further comprises: illuminating the at least a portion of the skin with a second light source; and imaging the at least a portion of the skin to generate a second image, wherein generating the quantitative phase image of the at least a portion of the skin, is further based at least in part on the second image.

11. The method of claim 10, wherein illuminating the at least a portion of the skin with the second light source, comprises directing light from the second light source at a second oblique angle different from the first oblique angle to the at least a portion of the skin.

12. The method of claim 11, wherein the first and second images are at a same field of view.

13. The method of claim 11, wherein the light from the first light source and the light from the second light source have the same wavelength.

14. The method of claim 8, wherein the at least a portion of skin comprises one or more vessels having a plurality of blood cells contained therein, the method further comprising quantifying, based at least in part on the quantitative phase image, the plurality of blood cells.

15. The method of claims 1, wherein the skin is volumetrically imaged at cellular resolution.

16. A quantitative oblique back- illumination microscopy (qOBM) system configured to image at least a portion of skin of a subject in vivo, the system comprising: a planar substrate disposed at a distal end of the qOBM system and configured to maintain the at least a portion of the skin to be imaged at a constant imaging depth; a least one light source configured to illuminate the at least a portion of the skin in at least one oblique angle; a camera configured to receive light from the at least a portion of the skin and generate at least one image; and a processor configured to process the at least one image and generate a quantitative phase image based at least in part on the at least one image, each pixel of the quantitative phase image indicative of refractive index properties of the tissue corresponding to the respective pixel.

17. The qOBM system of claim 16, wherein the at least one light source is configured to emit light having a wavelength in the range of 400-1750 nm.

18. The qOBM system of claim 16, wherein the qOBM system is handheld.

19. The qOBM system of claim 16, wherein the system is configured to volumetrically image the at least a portion of the skin at a depth of up to 3 mm.

20. The qOBM system of claim 16, wherein the at least one light source comprises a plurality of LEDs, each LED configured to illuminate the at least a portion of the skin at a distinct oblique angle.

21. The qOBM system of claim 20, wherein each of the plurality of LEDs are connected to a first end of a corresponding optical fiber, each of the optical fibers comprising a second end positioned proximate the planar substrate, the second end configured to direct light to the at least a portion of the skin to be imaged at an oblique angle.

22. The qOBM system of claim 16, wherein the processor is configured to: cause the at least one light source to illuminate the at least a portion of the skin at a first oblique angle; cause the camera to generate a first image at a first field of view during illumination at the first oblique angle; and generate the quantitative phase image based at least in part on the first image.

23. The qOBM system of claim 22, wherein the processor is further configured to: cause the at least one light source to illuminate the at least a portion of the skin at a second oblique angle; cause the camera to generate a second image at the first field of view during illumination at the second oblique angle; and generate the quantitative phase image based at least in part on the first and second images.

24. The qOBM system of claim 23, wherein the processor is further configured to: cause the at least one light source to illuminate the at least a portion of the skin at a third oblique angle; cause the camera to generate a third image at the first field of view during illumination at the third oblique angle; and generate the quantitative phase image based at least in part on the first, second, and third images.

25. The qOBM system of claim 24, wherein the processor is further configured to: cause the at least one light source to illuminate the at least a portion of the skin at a fourth oblique angle;cause the camera to generate a fourth image at the first field of view during illumination at the fourth oblique angle; and generate the quantitative phase image based at least in part on the first, second, third, and fourth images.

Images