High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution

Abstract

High-resolution four-dimensional (4-D) optical tomography of human skin based on multiphoton autofluorescence imaging and second harmonic generation (SHG) was performed with the compact femtosecond laser imaging system DermaInspect as well as a modified multiphoton microscope. Femtosecond laser pulses of 80 MHz in the spectral range of 750 to 850 nm, fast galvoscan mirrors, and a time-correlated single-photon counting module have been used to image human skin in vitro and in vivo with subcellular spatial and 250-ps temporal resolution. The nonlinear induced autofluorescence originates from naturally endogenous fluorophores and protein structures such as reduced nicotinamide adenine dinucleotide phosphate, flavins, collagen, elastin, porphyrins, and melanin. Second harmonic generation was used to detect collagen structures. Tissues of patients with dermatological disorders such as psoriasis, fungal infections, nevi, and melanomas have been investigated. Individual intratissue cells and skin structures could be clearly visualized. Intracellular components and connective tissue structures could be further characterized by fluorescence excitation spectra, by determination of the fluorescence decay per pixel, and by fluorescence lifetime imaging. The novel noninvasive multiphoton autofluorescence-SHG imaging technique provides 4-D (x,y,z,tau) optical biopsies with subcellular resolution and offers the possibility of introducing a high-resolution optical diagnostic method in dermatology.

Two‐photon microscopy of deep intravital tissues and its merits in clinical research

“…Excitation: 800-860 nm; emission: one half of the excitation wavelength (Zoumi et al, 2002;König & Riemann, 2003;Zipfel et al, 2003b;Wang et al, 2008a).…”

“…The first 2-photon excitation of electronic states was found for blue fluorescence emission with CaF 2 :Eu 2+ crystals based on the availability of lasers in 1961 (Kaiser & Garrett, 1961). To date, tunable mode-locked Ti:sapphire lasers operating at high repetition rate (typically 80-90 MHz) and a ultrashort pulse duration (less than 200 fs) are typically used as near infrared (NIR) sources in multiphoton excitation imaging (König & Riemann, 2003;Zipfel et al, 2003b;Rubart, 2004;Wang et al, 2005a). The required transient high irradiance can be obtained by tightly focusing ultrashort laser pulses within a sub-femtolitre focal volume provided by a special objective with high numerical aperture.…”

Multi-photon excitation fluorescence microscopy of brain-tumour tissue and analysis of cell density

“…Further, the human tumour samples underwent similar processing compared to the experimental tissue obtained from immuno-deficient NMRI mice, which lacked these cell types. The problem of unidentified cell [13,14,19,30]. Furthermore, experimental glioma tissue showed significantly longer fluorescence lifetimes than normal cortex or white matter [8].…”

Fluorescence lifetime imaging in biosciences: technologies and applications

“…interactions between proteins in living cells 48,[82][83][84][85][86] , photophysics of complexes involved in plant photosynthesis 87 or the redox metabolism 38,50,[88][89][90] . In the last years, FLIM gained on interest in the biomedicine, as well 27,49,78,[91][92][93][94][95][96] . For instance, a FLIM device for the clinical diagnosis of malign skin tissue is commercially available (DermaInspect, JenLab, Jena, Germany) 27 .…”

“…In the last years, FLIM gained on interest in the biomedicine, as well 27,49,78,[91][92][93][94][95][96] . For instance, a FLIM device for the clinical diagnosis of malign skin tissue is commercially available (DermaInspect, JenLab, Jena, Germany) 27 . In the following we do not intend to review all these applications, more we focus our attention on some experiments based on time-gated FLIM where single-beam as well as multi-focal TPLSM were used.…”