Non-invasive Imaging Techniques for Monitoring Cellular Response to Treatment in Stable Vitiligo.

Punch grafting procedures, where small pieces of normal skin are transplanted into stable vitiligo patches, results in repigmentation in only half of patients treated, yet the factors that determine whether a patient responds to treatment or not are still unknown. Reflectance confocal microscopy (RCM) is adept at visualizing melanocyte migration and epidermal changes over large areas while multiphoton microscopy (MPM) can capture metabolic changes in keratinocytes. With the overall goal of identifying optical biomarkers for early treatment response, we followed 12 vitiligo lesions undergoing punch grafting. Dendritic melanocytes adjacent to the graft site were observed before clinical evidence of repigmentation in treatment responsive patients but not in treatment non-responsive patients, suggesting that the early visualization of melanocytes is indicative of a therapeutic response. Keratinocyte metabolic changes in vitiligo skin adjacent to the graft site also correlated with treatment response, indicating that a keratinocyte microenvironment that more closely resembles normal skin is more hospitable for migrating melanocytes. Taken together, these studies suggest that successful melanocyte transplantation requires both the introduction of new melanocytes and modulation of the local tissue microenvironment.

Multimodal analyses of vitiligo skin identify tissue characteristics of stable disease.

Vitiligo is an autoimmune skin disease characterized by the destruction of melanocytes by autoreactive CD8+ T cells. Melanocyte destruction in active vitiligo is mediated by CD8+ T cells, but the persistence of white patches in stable disease is poorly understood. The interaction between immune cells, melanocytes, and keratinocytes in situ in human skin has been difficult to study due to the lack of proper tools. We combine noninvasive multiphoton microscopy (MPM) imaging and single-cell RNA-Seq (scRNA-Seq) to identify subpopulations of keratinocytes in stable vitiligo patients. We show that, compared with nonlesional skin, some keratinocyte subpopulations are enriched in lesional vitiligo skin and shift their energy utilization toward oxidative phosphorylation. Systematic investigation of cell-to-cell communication networks show that this small population of keratinocyte secrete CXCL9 and CXCL10 to potentially drive vitiligo persistence. Pseudotemporal dynamics analyses predict an alternative differentiation trajectory that generates this new population of keratinocytes in vitiligo skin. Further MPM imaging of patients undergoing punch grafting treatment showed that keratinocytes favoring oxidative phosphorylation persist in nonresponders but normalize in responders. In summary, we couple advanced imaging with transcriptomics and bioinformatics to discover cell-to-cell communication networks and keratinocyte cell states that can perpetuate inflammation and prevent repigmentation.

In vivo imaging with a fast large-area multiphoton exoscope (FLAME) captures the melanin distribution heterogeneity in human skin.

Melanin plays a significant role in the regulation of epidermal homeostasis and photoprotection of human skin. The assessment of its epidermal distribution and overall content is of great interest due to its involvement in a wide range of physiological and pathological skin processes. Among several spectroscopic and optical imaging methods that have been reported for non-invasive quantification of melanin in human skin, the approach based on the detection of two-photon excited fluorescence lifetime distinguishes itself by enabling selective detection of melanin with sub-cellular resolution, thus facilitating its quantification while also resolving its depth-profile. A key limitation of prior studies on the melanin assessment based on this approach is their inability to account for the skin heterogeneity due to the reduced field of view of the images, which results in high dispersion of the measurement values. Pigmentation in both normal and pathological human skin is highly heterogeneous and its macroscopic quantification is critical for reliable measurements of the epidermal melanin distribution and for capturing melanin-related sensitive dynamic changes as a response to treatment. In this work, we employ a fast large-area multiphoton exoscope (FLAME), recently developed by our group for clinical skin imaging, that has the ability to evaluate the 3D distribution of epidermal melanin content in vivo macroscopically (millimeter scale) with microscopic resolution (sub-micron) and rapid acquisition rates (minutes). We demonstrate significant enhancement in the reliability of the melanin density and distribution measurements across Fitzpatrick skin types I to V by capturing the intra-subject pigmentation heterogeneity enabled by the large volumetric sampling. We also demonstrate the potential of this approach to provide consistent measurement results when imaging the same skin area at different times. These advances are critical for clinical and research applications related to monitoring pigment modulation as a response to therapies against pigmentary skin disorders, skin aging, as well as skin cancers.

Research Techniques Made Simple: Emerging Imaging Technologies for Noninvasive Optical Biopsy of Human Skin.

Over the past few years, high-resolution optical imaging technologies such as optical coherence tomography (OCT), reflectance confocal microscopy (RCM), and multiphoton microscopy (MPM) have advanced significantly as new methodologies for clinical research and for real-time detection, diagnosis, and therapy monitoring of skin diseases. Implementation of these technologies into clinical research and practice requires clinicians to have an understanding of their capabilities, benefits, and limitations. This concise review provides insights on the application of OCT, RCM, and MPM for clinical skin imaging through images acquired in vivo from the same lesions. The presented data are limited to pigmented lesions and basal cell carcinoma.

Deep Learning-Assisted Multiphoton Microscopy to Reduce Light Exposure and Expedite Imaging in Tissues With High and Low Light Sensitivity.

Purpose: Two-photon excitation fluorescence (2PEF) reveals information about tissue function. Concerns for phototoxicity demand lower light exposure during imaging. Reducing excitation light reduces the quality of the image by limiting fluorescence emission. We applied deep learning (DL) super-resolution techniques to images acquired from low light exposure to yield high-resolution images of retinal and skin tissues. Methods: We analyzed two methods: a method based on U-Net and a patch-based regression method using paired images of skin (550) and retina (1200), each with low- and high-resolution paired images. The retina dataset was acquired at low and high laser powers from retinal organoids, and the skin dataset was obtained from averaging 7 to 15 frames or 70 frames. Mean squared error (MSE) and the structural similarity index measure (SSIM) were outcome measures for DL algorithm performance. Results: For the skin dataset, the patches method achieved a lower MSE (3.768) compared with U-Net (4.032) and a high SSIM (0.824) compared with U-Net (0.783). For the retinal dataset, the patches method achieved an average MSE of 27,611 compared with 146,855 for the U-Net method and an average SSIM of 0.636 compared with 0.607 for the U-Net method. The patches method was slower (303 seconds) than the U-Net method (<1 second). Conclusions: DL can reduce excitation light exposure in 2PEF imaging while preserving image quality metrics. Translational Relevance: DL methods will aid in translating 2PEF imaging from benchtop systems to in vivo imaging of light-sensitive tissues such as the retina.

A Clinical Perspective on the Automated Analysis of Reflectance Confocal Microscopy in Dermatology.

BACKGROUND AND OBJECTIVES: Non-invasive optical imaging has the potential to provide a diagnosis without the need for biopsy. One such technology is reflectance confocal microscopy (RCM), which uses low power, near-infrared laser light to enable real-time in vivo visualization of superficial human skin from the epidermis down to the papillary dermis. Although RCM has great potential as a diagnostic tool, there is a need for the development of reliable image analysis programs, as acquired grayscale images can be difficult and time-consuming to visually assess. The purpose of this review is to provide a clinical perspective on the current state of artificial intelligence (AI) for the analysis and diagnostic utility of RCM imaging. STUDY DESIGN/MATERIALS AND METHODS: A systematic PubMed search was conducted with additional relevant literature obtained from reference lists. RESULTS: Algorithms used for skin stratification, classification of pigmented lesions, and the quantification of photoaging were reviewed. Image segmentation, statistical methods, and machine learning techniques are among the most common methods used to analyze RCM image stacks. The poor visual contrast within RCM images and difficulty navigating image stacks were mediated by machine learning algorithms, which allowed the identification of specific skin layers. CONCLUSIONS: AI analysis of RCM images has the potential to increase the clinical utility of this emerging technology. A number of different techniques have been utilized but further refinements are necessary to allow consistent accurate assessments for diagnosis. The automated detection of skin cancers requires more development, but future applications are truly boundless, and it is compelling to envision the role that AI will have in the practice of dermatology. Lasers Surg. Med. © 2020 Wiley Periodicals LLC.

Rapid chemically selective 3D imaging in the mid-infrared.

The emerging technique of mid-infrared optical coherence tomography (MIR-OCT) takes advantage of the reduced scattering of MIR light in various materials and devices, enabling tomographic imaging at deeper penetration depths. Because of challenges in MIR detection technology, the image acquisition time is, however, significantly longer than for tomographic imaging methods in the visible/near-infrared. Here we demonstrate an alternative approach to MIR tomography with high-speed imaging capabilities. Through femtosecond nondegenerate two-photon absorption of MIR light in a conventional Si-based CCD camera, we achieve wide-field, high-definition tomographic imaging with chemical selectivity of structured materials and biological samples in mere seconds.

Fast, large area multiphoton exoscope (FLAME) for macroscopic imaging with microscopic resolution of human skin.

We introduce a compact, fast large area multiphoton exoscope (FLAME) system with enhanced molecular contrast for macroscopic imaging of human skin with microscopic resolution. A versatile imaging platform, FLAME combines optical and mechanical scanning mechanisms with deep learning image restoration to produce depth-resolved images that encompass sub-mm2 to cm2 scale areas of tissue within minutes and provide means for a comprehensive analysis of live or resected thick human skin tissue. The FLAME imaging platform, which expands on a design recently introduced by our group, also features time-resolved single photon counting detection to uniquely allow fast discrimination and 3D virtual staining of melanin. We demonstrate its performance and utility by fast ex vivo and in vivo imaging of human skin. With the ability to provide rapid access to depth resolved images of skin over cm2 area and to generate 3D distribution maps of key sub-cellular skin components such as melanocytic dendrites and melanin, FLAME is ready to be translated into a clinical imaging tool for enhancing diagnosis accuracy, guiding therapy and understanding skin biology.

Non-invasive optical biopsy by multiphoton microscopy identifies the live morphology of common melanocytic nevi.

Multiphoton microscopy (MPM) is a promising non-invasive imaging tool for discriminating benign nevi from melanoma. In this study, we establish a MPM morphologic catalogue of common nevi, information that will be critical in devising strategies to distinguish them from nevi that are evolving to melanoma that may present with more subtle signs of malignancy. Thirty common melanocytic nevi were imaged in vivo using MPM. Quantitative parameters that can distinguish between different types of nevi were developed and confirmed by examining the histology of eleven of the imaged nevi. MPM features of nevi examined included cytologic morphology of melanocytes in the epidermis and dermis, the size and distribution of nevomelanocytes both within and around nests, the size of rete ridges, and the presence of immune cells in the dermis. Distinguishing features include cytological morphology, the size of nevomelanocytes, the size of nevomelanocyte nests, and the distribution of nevomelanocytes. Notably, these distinguishing characteristics were not easily appreciated in fixed tissues, highlighting essential differences in the morphology of live skin. Taken together, this work provides a morphologic compendium of normal nevi, information that will be critical in future studies directed at identifying melanocytic nevi that are evolving to melanoma.

Feature characterization of scarring and non-scarring types of alopecia by multiphoton microscopy.

OBJECTIVES: Non-invasive visualization of hair follicles is important for proper diagnosis and management of alopecia; however, histological assessment remains the gold standard. Laser imaging technologies have made possible noninvasive in vivo evaluation of skin and hair follicle. The aim of this study was to evaluate the ability of multiphoton microscopy (MPM) to non-invasively identify morphological features that can distinguish scarring from non-scarring alopecia. METHODS: MPM images were obtained from areas on the scalp affected by alopecia. Investigators blinded to the diagnosis analyzed hair follicle and shaft sizes. Patients were recruited and imaged at the UC Irvine Health Medical Center and the University of California, Irvine Beckman Laser Institute. Patients with androgenetic alopecia (AGA) and alopecia areata (AA), and scarring alopecia, in particular frontal fibrosing alopecia (FFA) were recruited and imaged from July 2016 to July 2017. RESULTS: We imaged 5 normal scalp subjects and 12 patients affected by non-scarring (7 subjects) and scarring (5 subjects) alopecia. In normal and non-scarring alopecia patients, MPM identified presence of sebaceous glands associated with hair follicles. MPM images of scarring alopecia were characterized by the presence of inflammatory cells surrounding hair follicles. Measurements of hair follicle diameter sizes were found to be significantly smaller in scarring alopecia patients compared to normal (P < 0.001) and compared to non-scarring alopecia patients (P = 0.046); non-scarring hair follicles were also significantly smaller than normal hair follicles (P = 0.043). CONCLUSIONS: This study shows that MPM imaging can non-invasively identify morphological features that distinguish scarring from non-scarring alopecia. Further studies are needed to validate this technique and evaluate its potential to be used as an aid for guiding treatment. Lasers Surg. Med. 51:95-103, 2019. © 2018 Wiley Periodicals, Inc.

In vivo multiphoton microscopy of melasma.

Melasma is a skin disorder characterized by hyperpigmented patches due to increased melanin production and deposition. In this pilot study, we evaluate the potential of multiphoton microscopy (MPM) to characterize non-invasively the melanin content, location, and distribution in melasma and assess the elastosis severity. We employed a clinical MPM tomograph to image in vivo morphological features in melasma lesions and adjacent normal skin in 12 patients. We imaged dermal melanophages in most dermal melasma lesions and occasionally in epidermal melasma. The melanin volume fraction values measured in epidermal melasma (14% ± 4%) were significantly higher (p < 0.05) than the values measured in perilesional skin (11% ± 3%). The basal keratinocytes of melasma and perilesions showed different melanin distribution. Elastosis was predominantly more severe in lesions than in perilesions and was associated with changes in melanin distribution of the basal keratinocytes. These results demonstrate that MPM may be a non-invasive imaging tool for characterizing melasma.

Noninvasive assessment of skin structure by combined photothermal radiometry and optical spectroscopy: coregistration with multiphoton microscopy.

We are combining two optical techniques, pulsed photothermal radiometry (PPTR) and diffuse reflectance spectroscopy (DRS), for noninvasive assessment of the structure and composition of human skin in vivo. The analysis involves simultaneous multidimensional fitting of the measured PPTR signals and DRS spectra with predictions of a numerical model of light transport (Monte Carlo) in a four-layer model optical model of human skin, accounting for the epidermis, papillary and reticular dermis, and subcutis. The assessed epidermal thickness values were tested by coregistration with a multiphoton microscope, which provides vertical sectioning capability based on two-photon excited fluorescence and second-harmonic generation in selected skin components. The comparison shows that these values correspond well to the maximal epidermal thicknesses measured in the multiphoton microscopy images, the rete ridges.

In vivo multiphoton-microscopy of picosecond-laser-induced optical breakdown in human skin.

IMPORTANCE: Improvements in skin appearance resulting from treatment with fractionated picosecond-lasers have been noted, but optimizing the treatment efficacy depends on a thorough understanding of the specific skin response. The development of non-invasive laser imaging techniques in conjunction with laser therapy can potentially provide feedback for guidance and optimizing clinical outcome. OBJECTIVE: The purpose of this study was to demonstrate the capability of multiphoton microscopy (MPM), a high-resolution, label-free imaging technique, to characterize in vivo the skin response to a fractionated non-ablative picosecond-laser treatment. DESIGN, SETTING, AND PARTICIPANTS: Two areas on the arm of a volunteer were treated with a fractionated picosecond laser at the Dermatology Clinic, UC Irvine. The skin response to treatment was imaged in vivo with a clinical MPM-based tomograph at 3 hours and 24 hours after treatment and seven additional time points over a 4-week period. MAIN OUTCOMES AND MEASURES: MPM revealed micro-injuries present in the epidermis. Pigmented cells were particularly damaged in the process, suggesting that melanin is likely the main absorber for laser induced optical breakdown. RESULTS: Damaged individual cells were distinguished as early as 3 hours post pico-laser treatment with the 532 nm wavelength, and 24 hours post-treatment with both 532 and 1064 nm wavelengths. At later time points, clusters of cellular necrotic debris were imaged across the treated epidermis. After 24 hours of treatment, inflammatory cells were imaged in the proximity of epidermal micro-injuries. The epidermal injuries were exfoliated over a 4-week period. CONCLUSIONS AND RELEVANCE: This observational and descriptive pilot study demonstrates that in vivo MPM imaging can be used non-invasively to provide label-free contrast for describing changes in human skin following a fractionated non-ablative laser treatment. The results presented in this study represent the groundwork for future longitudinal investigations on an expanded number of subjects to understand the response to treatment in different skin types with different laser parameters, critical factors in optimizing treatment outcome. Lasers Surg. Med. 49:555-562, 2017. © 2017 Wiley Periodicals, Inc.

Imaging mitochondrial dynamics in human skin reveals depth-dependent hypoxia and malignant potential for diagnosis.

Active changes in mitochondrial structure and organization facilitate cellular homeostasis. Because aberrant mitochondrial dynamics are implicated in a variety of human diseases, their assessment is potentially useful for diagnosis, therapy, and disease monitoring. Because current techniques for evaluating mitochondrial morphology are invasive or necessitate mitochondria-specific dyes, their clinical translation is limited. We report that mitochondrial dynamics can be monitored in vivo, within intact human skin by relying entirely on endogenous two-photon-excited fluorescence from the reduced metabolic coenzyme nicotinamide adenine dinucleotide (NADH). We established the sensitivity of this approach with in vivo, fast temporal studies of arterial occlusion-reperfusion, which revealed acute changes in the mitochondrial metabolism and dynamics of the lower human epidermal layers. In vitro hypoxic-reperfusion studies validated that the in vivo outcomes were a result of NADH fluorescence changes. To demonstrate the diagnostic potential of this approach, we evaluated healthy and cancerous human skin epithelia. Healthy tissues displayed consistent, depth-dependent morphological and mitochondrial organization patterns that varied with histological stratification and intraepithelial mitochondrial protein expression. In contrast, these consistent patterns were absent in cancerous skin lesions. We exploited these differences to successfully differentiate healthy from cancerous tissues using a predictive classification approach. Collectively, these results demonstrate that our label-free, automated, near real-time assessments of mitochondrial organization-relying solely on endogenous contrast-could be useful for accurate, noninvasive in vivo diagnosis.

Rapid mesoscale multiphoton microscopy of human skin.

We present a multiphoton microscope designed for mesoscale imaging of human skin. The system is based on two-photon excited fluorescence and second-harmonic generation, and images areas of ~0.8x0.8 mm2 at speeds of 0.8 fps (800x800 pixels; 12 frame averages) for high signal-to-noise ratio, with lateral and axial resolutions of 0.5µm and 3.3µm, respectively. The main novelty of this instrument is the design of the scan head, which includes a fast galvanometric scanner, optimized relay optics, a beam expander and high NA objective lens. Computed aberrations in focus are below the Marechal criterion of 0.07λ rms for diffraction-limited performance. We demonstrate the practical utility of this microscope by ex-vivo imaging of wide areas in normal human skin.

Sub-40 fs, 1060-nm Yb-fiber laser enhances penetration depth in nonlinear optical microscopy of human skin.

Advancing the practical utility of nonlinear optical microscopy requires continued improvement in imaging depth and contrast. We evaluated second-harmonic generation (SHG) and third-harmonic generation images from ex vivo human skin and showed that a sub-40 fs, 1060-nm Yb-fiber laser can enhance SHG penetration depth by up to 80% compared to a >100 fs, 800 nm Ti:sapphire source. These results demonstrate the potential of fiber-based laser systems to address a key performance limitation related to nonlinear optical microscopy (NLOM) technology while providing a low-barrier-to-access alternative to Ti:sapphire sources that could help accelerate the movement of NLOM into clinical practice.