PROJECT SUMMARY
Multiphoton microscopy (MPM) can provide sub-micron resolution images of living tissues in their native
environment with label-free molecular contrast from multiple modalities, including second harmonic generation
(SHG) and two-photon excited fluorescence (TPEF). Several endogenous tissue components can be
visualized, including collagen (from SHG) and reduced nicotinamide adenine dinucleotide (NADH), flavin
adenosine dinucleotide (FAD), keratin, melanin and elastin fibers (from TPEF). We have advanced label-free
MPM technologies in skin clinical/translational studies for characterizing keratinocyte metabolism, diagnosing
melanoma, understanding melanocyte biology, detecting basal cell carcinoma, quantifying skin pigmentation,
and assessing the effects of cutaneous laser therapy. Many of these studies have been completed using a
commercial multi-photon microscope for clinical skin imaging that has limitations in terms of field-of-view
(FOV), speed, footprint, and cost. In order to address these barriers to clinical adoption, we propose to build
a “next-generation” clinical multiphoton microscope that integrates advanced benchtop technologies into a
compact, practical, and cost-effective bedside device. This new instrument will have comparable FOV,
resolution, and scanning features to standard-of-care reflectance confocal microscopes (RCM), yet provide
unique structural and metabolic contrast from multiple modalities (TPEF and SHG) that can only be achieved
with MPM. We will establish the clinical safety of this device in a light dose escalation study that assesses
DNA and cellular damage, and establish key performance benchmarks in a 12-patient clinical study of healthy
volunteers across a range of skin types. In addition, we will conduct pilot studies of wound re-epithelialization
and melanocyte migration in the context of vitiligo micro-grafts, a clinical procedure where pigmented skin is
transplanted into skin affected by vitiligo, which is devoid of melanocytes. Melanocytes migrating out of
engrafted skin and keratinocytes turning over within engrafted skin can be visualized by measuring the TPEF
of cellular melanin and co-factors (NADH, FAD+) in and around the grafts, effectively identifying different cell
populations involved in wound healing. Our broad, long term goal is to develop ev-MPM as a practical
approach for rapid, in vivo characterization of cellular morphologic and metabolic imaging endpoints in
patients. These can be used to understand and optimize wound healing and provide a practical beside platform
for detecting, diagnosing, and optimizing therapeutic response in skin diseases.
