Heating has been known to cure cancer for over 2000 years1, and recent studies have confirmed this in the treatment of basal cell carcinomas (BCC)2. The application of uncontrolled heating often results in unacceptable scarring. Our ability to control the delivery of heat using a forward-looking infrared (FLIR) camera and a modulated long pulsed (LP) Nd:YAG 1064nm laser has resulted in better outcomes in comparison to traditional destruction techniques3. Use of optical coherence tomography (OCT) enhances our ability to define the tumor margins prior to treatment. The combination of FLIR and OCT allows very precise treatment of BCCs with at least comparable outcomes to conventional LP Nd:YAG treatments.
Our group and others have demonstrated the strong potential of the multiphoton microscopy for a broad range of applications from advancing the understanding of skin biology to non-invasive diagnosis of skin diseases and monitoring therapy effects. We have recently reported on a fast large area multiphoton exoscope for rapidly mapping out macroscopic tissue areas with microscopic resolution and enhanced contrast for selective melanin detection. We will describe the technical abilities of this instrument and demonstrate its feasibility for early melanoma diagnosis based on a pilot study on ex-vivo and in-vivo imaging of pigmented lesions suspicious of melanoma in human skin.
KEYWORDS: Skin, In vivo imaging, Associative arrays, Tumors, Melanoma, Image resolution, Diagnostics, Denoising, Convolutional neural networks, Chemical elements
Multiphoton microscopy can provide sub-micron resolution images of living tissues in their
native environment with chemical contrast. We recently reported on a fast large area multiphoton
exoscope (FLAME) for rapidly mapping out macroscopic tissue areas (cm-scale) with microscopic
resolution. In this presentation we demonstrate the imaging capability and the clinical utility of this
system by performing a pilot study on ex vivo imaging of benign and malignant pigmented lesions of
human skin. We identify morphological features such as cytological atypia, lentiginous hyperplasia,
migration of melanocytes and demonstrate the value of sampling large tissue volumes for capturing the
lesion heterogeneity.
Multiphoton microscopy (MPM) can provide sub-micron resolution images of living tissues in their native environment with contrast from multiple modalities, including second harmonic generation (SHG) and two-photon excited fluorescence (TPEF). Recent advances of MPM in clinical skin imaging demonstrated the unique potential of this technology as a label-free research and clinical tool for a broad area of applications such as melanoma and non-melanoma skin cancer detection, monitoring pigmentary skin disorders, characterizing keratinocyte metabolism, etc. In this contribution we demonstrate the ability of this microscope to provide sub-micrometer resolution ex-vivo images of large areas of skin tissue (up to 5x5 mm2) in <1 minute. We demonstrate the importance of high-speed, high-resolution mesoscopic imaging on cancerous skin tissues that present heterogeneous morphology to show the ability of the instrument to capture both benign and malignant areas of the lesion.
We use a multiphoton microscopy (MPM)-based clinical microscope (MPTflex, JenLab, Germany) to describe changes in human skin following treatment with a fractional non-ablative laser (PicoWay, Candela).
The treatment was based on a fractionated picosecond Nd:YAG laser (1064 and 532nm, 3mJ and 1.5mJ (no attenuation), respectively maximum energy/pulse, 100 microbeams/6mmx6mm). Improvements in skin appearance resulting from treatment with this laser have been noted but optimizing the efficacy depends on a thorough understanding of the specific skin response to treatment.
MPM is a nonlinear laser scanning microscopy technique that features sub-cellular resolution and label-free molecular contrast. MPM contrast in skin is derived from second-harmonic generation of collagen and two-photon excited fluorescence of NADH/FAD+, elastin, keratin, melanin.
In this pilot study, two areas on the arm of a volunteer (skin type II) were treated with the picoWay laser (1064nm, 3mJ; 532nm, 1.5mJ; 1pass). The skin response to treatment was imaged in-vivo at 8 time points over the following 4 weeks. MPM revealed micro-injuries present in epidermis. Damaged individual cells were distinguished after 3h and 24h from treatment with both wavelengths. Pigmented cells were particularly damaged in the process, suggesting that melanin is the main absorber and the primary target for laser induced optical breakdown. At later time points, clusters of cellular necrotic debris were imaged across the treated epidermis. These results represent the groundwork for future longitudinal studies on expanded number of subjects to understand the response to treatment in different skin types at different laser parameters, critical factors in optimizing treatment outcomes.
In vivo imaging of pigmented lesions in human skin was performed with a clinical multiphoton microscopy (MPM)-based tomograph (MPTflex, JenLab, Germany). Two-photon excited fluorescence was used for visualizing endogenous fluorophores such as NADH/FAD, keratin, melanin in the epidermal cells and elastin fibers in the dermis. Collagen fibers were imaged by second harmonic generation. Our study involved in vivo imaging of benign melanocytic nevi,
atypical nevi and melanoma. The goal of this preliminary study was to identify in vivo the characteristic features and
their frequency in pigmented lesions at different stages (benign, atypical and malignant) and to evaluate the ability of in
vivo MPM to distinguish atypical nevi from melanoma. Comparison with histopathology was performed for the biopsied
lesions. Benign melanocytic nevi were characterized by the presence of nevus cell nests at the epidermal-dermal
junction. In atypical nevi, features such as lentiginous hyperplasia, acanthosis and architectural disorder were imaged.
Cytological atypia was present in all the melanoma lesions imaged, showing the strongest correlation with malignancy. The MPM images demonstrated very good correlation with corresponding histological images, suggesting that MPM
could be a promising tool for in vivo non-invasive pigmented lesion diagnosis, particularly distinguishing atypical nevi
from melanoma.
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