Deep‐skin multiphoton microscopy in vivo excited at 1600 nm: A comparative investigation with silicone oil and deuterium dioxide immersion

Abstract

Multiphoton microscopy (MPM) excited at the 1700-nm window has enabled deep-tissue penetration in biological tissue, especially brain. MPM of skin may also benefit from this deep-penetration capability. Skin is a layered structure with varying refractive index (from 1.34 to 1.5). Consequently, proper immersion medium should be selected when imaging with high numerical aperture objective lens. To provide guidelines for immersion medium selection for skin MPM, here we demonstrate comparative experimental investigation of deep-skin MPM excited at 1600 nm in vivo, using both silicone oil and deuterium dioxide (D 2 O) immersion. We specifically characterize imaging depths, signal levels and spatial resolution. Our results show that both immersion media give similar performance in imaging depth and spatial resolution, while signal levels are slightly better with silicone oil immersion. We also demonstrate that local injection of fluorescent beads into the skin is a viable technique for spatial resolution characterization in vivo.Skin is the outside layer of tissue covering the body of many animals including human beings, which plays important roles in protection, regulation and sensation. Research on skin structures and even functions leverage on various imaging modalities. Among them, multiphoton microscopy (MPM) finds a unique niche by combining reasonable penetration depth (>100 μm) and subcellular resolution. Consequently, MPM has found successful applications in skin imaging of both animals and more importantly, human subjects in clinical environments [1][2][3][4][5][6][7][8][9][10][11][12][13].Even MPM itself comprises a variety of modalities, examples include 2-/3-/4-photon fluorescence (i.e., 2PF/3PF/4PF)