Development of a fast scanning, extended field-of-view multiphoton microscope for clinical skin imaging

Summary
The study of mammalian immune cells and their interactions with tissue in situ is critical for understanding
how they regulate processes ranging from wound healing to autoimmune disease initiation to cancer and for
designing better therapeutic strategies to treat these prevalent conditions. Intravital multiphoton microscopy
(MPM) combined with a rich repertoire of fluorescent reporter mouse models and in vivo cell and tissue labeling
techniques have made it possible to visualize immune cell-tissue interactions at a subcellular level in skin and
other organs. However, there are significant differences in the structure and immune milieu of human skin that
limits the translatability of these findings to the human cutaneous immune response. Our group has recently
developed a fast large area multiphoton exoscope (FLAME), a unique imaging platform optimized for efficient
clinical skin imaging to rapidly generate macroscopic images (mm to cm-scale) with microscopic resolution (0.5-
1µm) based on label-free molecular contrast (fluorescence intensity and lifetime). In this application, we leverage
our extensive experience in MPM technology development and clinical imaging of more than 400 patients over
the past several years to develop the first MPM-based clinical device (iFLAME) as a research imaging tool
optimized for, and dedicated to, in vivo label-free imaging of immune cell populations and their dynamics in
human skin. In Aim 1, we develop iFLAME as a clinical research tool for efficient in vivo label-free imaging of
dermal cell populations and their dynamics in human skin. This work involves development of detection and
analytic approaches as well as optical and computational methods to enable rapid fluorescence lifetime detection
and analysis necessary to automate measurements of the cellular morphological and metabolic signatures. In
Aim 2, we validate iFLAME performance by demonstrating in vivo characterization of immune cells in normal
and inflamed human skin. In Aim 3, we develop quantitative morphological and metabolic MPM imaging
endpoints to assess immune infiltrates and their dynamics in human skin in the context of monitoring wound
healing. This work represents the first attempt to use intrinsic sources of MPM contrast to image, identify, and
quantify key immune cells in human skin in vivo based on their optical signatures and migratory behavior. Our
long-term goal is to develop iFLAME as a clinical research tool for rapid, label-free imaging of immune cells in
skin based on cellular morphologic and metabolic imaging endpoints. These can be used to better understand,
evaluate and optimize wound healing, autoimmune skin diseases and therapeutic responses.

Grant Number: 5R01EB026705-08
NIH Institute/Center: NIH
Principal Investigator: Mihaela Balu