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.

The influence of the pulse length, tau , of ultrashort laser pulses at 780 and 920 nm on cell vitality and cellular reproduction has been studied. A total of 2400 nonlabeled cells were exposed to a highly focused scanning beam from a mode-locked 80-MHz Ti:sapphire laser with 60-micros pixel dwell time. For the same pulse energy, destructive effects were more pronounced for shorter pulses. The damage behavior was found to follow approximately a P(2)/tau dependence (P , mean power), indicating that cell destruction is likely based on a two-photon excitation process rather than a one- or a three-photon event. Therefore, femtosecond as well as picosecond pulses provide approximately the same relative optical window for safe two-photon fluorescence microscopy.

No abstract

Multiphoton excited tissue fluorescence summarises the emission of all naturally occurring endogenous fluorescent bio-molecules with their often overlapping fluorescence spectra. Common fluorescence intensity measurements could not be utilised to distinguish between different fluorophores or metabolic states. To overcome this limitation, we investigated new procedures of selective melanin imaging and spectral fluorescence lifetime imaging in combination with high resolution multiphoton laser tomography. Overall 46 melanocytic lesions of human skin were analysed. We suggested that fluorescence light, detected in such a way, may yield additional information for melanoma diagnostics. Remarkable differences in lifetime behaviour of keratinocytes in contrast to melanocytes were observed. Fluorescence lifetime distribution was found in correlation with the intracellular amount of melanin. Spectral analysis of melanoma revealed a main fluorescence peak around 470 nm in combination with an additional peak close to 550 nm throughout all epidermal layers. Excitation at 800 nm shows a selectively observable fluorescence of melanin containing cells and offers the possibility of cell classification. Procedures of selective imaging as well as spectral fluorescence lifetime imaging by means of multiphoton laser tomography support diagnostic decisions and may improve the process of non-invasive early detection of melanoma.Key words: melanoma -melanocytic skin lesions -multiphoton laser tomography -selective melanin imaging -spectral fluorescence lifetime imaging Please cite this paper as: Spectral fluorescence lifetime detection and selective melanin imaging by multiphoton laser tomography for melanoma diagnosis.

Two-photon medical imaging has found its way into dermatology as an excellent method for noninvasive skin cancer detection without need of contrast agents as well as for in situ drug screening of topically-applied cosmetical and pharmaceutical components. There is an increasing demand to apply the multiphoton technology also for deep-tissue skin imaging as well as for intracorporal imaging. We report on the first clinical use of multiphoton endoscopes, in particular of a miniaturized rigid two-photon GRIN lens endoscope. The microendoscope was attached to the multiphoton tomograph DermaInspect and employed to detect the extracellular matrix proteins collagen and elastin in the human dermis of volunteers and patients with ulcera by in vivo second harmonic generation and in vivo two-photon autofluorescence.