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.
The combined use of nonlinear optical microscopy and broadband reflectance techniques to assess melanin concentration and distribution thickness in vivo over the full range of Fitzpatrick skin types is presented. Twelve patients were measured using multiphoton microscopy (MPM) and spatial frequency domain spectroscopy (SFDS) on both dorsal forearm and volar arm, which are generally sun-exposed and non-sun-exposed areas, respectively. Both MPM and SFDS measured melanin volume fractions between (skin type I non-sun-exposed) and 20% (skin type VI sun exposed). MPM measured epidermal (anatomical) thickness values ~30-65 μm, while SFDS measured melanin distribution thickness based on diffuse optical path length. There was a strong correlation between melanin concentration and melanin distribution (epidermal) thickness measurements obtained using the two techniques. While SFDS does not have the ability to match the spatial resolution of MPM, this study demonstrates that melanin content as quantified using SFDS is linearly correlated with epidermal melanin as measured using MPM (R² = 0.8895). SFDS melanin distribution thickness is correlated to MPM values (R² = 0.8131). These techniques can be used individually and/or in combination to advance our understanding and guide therapies for pigmentation-related conditions as well as light-based treatments across a full range of skin types.
Monitoring of atypical nevi is an important step in early detection of melanoma, a clinical imperative in preventing the disease progression. Current standard diagnosis is based on biopsy and histopathological examination, a method that is invasive and highly dependent upon the physician’s experience. In this work, we employed a clinical multiphoton microscope to image in vivo and non-invasively melanocytic nevi at three different stages: common nevi without dysplastic changes, dysplastic nevi with structural and architectural atypia, and melanoma. We analyzed multiphoton microscopy (MPM) images corresponding to 15 lesions (5 in each group) both qualitatively and quantitatively. For the qualitative analysis, we identified the morphological features characteristic of each group. MPM images corresponding to dysplastic nevi and melanoma were compared with standard histopathology in order to determine correlations between tissue constituents and morphology and to evaluate whether standard histopathology criteria can be identified in the MPM images. Prominent qualitative correlations included the morphology of epidermal keratinocytes, the appearance of nests of nevus cells surrounded by collagen fibers, and the structure of the epidermal-dermal junction. For the quantitative analysis, we defined a numerical “multiphoton melanoma index (MMI)” based on 3D in vivo image analysis that scores signals derived from two-photon excited fluorescence, second harmonic generation, and melanocyte morphology features on a continuous 9-point scale. Indices corresponding to common nevi (0–1), dysplastic nevi (1–4) and melanoma (5–8) were significantly different (p<0.05), suggesting the potential of the method to distinguish between melanocytic nevi in vivo.
We demonstrate a fiber-based probe for maximum collection of the coherent anti-Stokes Raman scattering (CARS) signal in biological tissues. We discuss the design challenges including capturing the back-scattered forward generated CARS signal in the sample and the effects of fiber nonlinearities on the propagating pulses. Three different single mode fibers (fused silica fiber, photonic crystal fiber and double-clad photonic crystal fiber) were tested for the probe design. We investigated self-phase modulation, stimulated Raman scattering (SRS) and four-wave-mixing (FWM) generation in the fiber: nonlinear processes expected to occur in a two-beam excitation based probe. While SPM and SRS induced spectral broadening was negligible, a strong non phase-matched FWM contribution was found to be present in all the tested fibers for excitation conditions relevant to CARS microscopy of tissues. To spectrally suppress this strong contribution, the probe design incorporates separate fibers for excitation light delivery and for signal detection, in combination with dichroic optics. CARS images of the samples were recorded by collecting the back-scattered forward generated CARS signal in the sample through a multi-mode fiber. Different biological tissues were imaged ex vivo in order to assess the performance of our fiber-delivered probe for CARS imaging, a tool which we consider an important advance towards label-free, in vivo probing of superficial tissues.
Importance Basal cell carcinomas (BCCs) are diagnosed by clinical evaluation, which can include dermoscopic evaluation, biopsy, and histopathologic examination. Recent translation of multiphoton microscopy (MPM) to clinical practice raises the possibility of noninvasive, label-free in vivo imaging of BCCs that could reduce the time from consultation to treatment. Objectives To demonstrate the capability of MPM to image in vivo BCC lesions in human skin, and to evaluate if histopathologic criteria can be identified in MPM images. Design, Setting, and Participants Imaging in patients with BCC was performed at the University of California–Irvine Health Beckman Laser Institute & Medical Clinic, Irvine, between September 2012 and April 2014, with a clinical MPM-based tomograph. Ten BCC lesions were imaged in vivo in 9 patients prior to biopsy. The MPM images were compared with histopathologic findings. Main Outcomes and Measures MPM imaging identified in vivo and noninvasively the main histopathologic feature of BCC lesions: nests of basaloid cells showing palisading in the peripheral cell layer at the dermoepidermal junction and/or in the dermis. Results The main MPM feature associated with the BCC lesions involved nests of basaloid cells present in the papillary and reticular dermis. This feature correlated well with histopathologic examination. Other MPM features included elongated tumor cells in the epidermis aligned in 1 direction and parallel collagen and elastin bundles surrounding the tumors. Conclusions and Relevance This study demonstrates, in a limited patient population, that noninvasive in vivo MPM imaging can provide label-free contrast that reveals several characteristic features of BCC lesions. Future studies are needed to validate the technique and correlate MPM performance with histopathologic examination.
We employ a clinical multiphoton microscope to monitor in vivo and noninvasively the changes in reduced nicotinamide adenine dinucleotide (NADH) fluorescence of human epidermal cells during arterial occlusion. We correlate these results with measurements of tissue oxy-and deoxyhemoglobin concentration during oxygen deprivation using spatial frequency domain imaging. During arterial occlusion, a decrease in oxyhemoglobin corresponds to an increase in NADH fluorescence in the basal epidermal cells, implying a reduction in basal cell oxidative phosphorylation. The ischemia-induced oxygen deprivation is associated with a strong increase in NADH fluorescence of keratinocytes in layers close to the stratum basale, whereas keratinocytes from epidermal layers closer to the skin surface are not affected. Spatial frequency domain imaging optical property measurements, combined with a multilayer Monte Carlo-based radiative transport model of multiphoton microscopy signal collection in skin, establish that localized tissue optical property changes during occlusion do not impact the observed NADH signal increase. This outcome supports the hypothesis that the vascular contribution to the basal layer oxygen supply is significant and these cells engage in oxidative metabolism. Keratinocytes in the more superficial stratum granulosum are either supplied by atmospheric oxygen or are functionally anaerobic. Based on combined hemodynamic and two-photon excited fluorescence data, the oxygen consumption rate in the stratum basale is estimated to be ~0.035 mmoles/10 6 cells/h.
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 3h and 24h after treatment and 7 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 3h post pico-laser treatment with the 532nm wavelength, and 24h post treatment with both 532nm and 1064nm wavelengths. At later time points, clusters of cellular necrotic debris were imaged across the treated epidermis. After 24h 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.
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