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 $(x,y,z,τ)$ optical biopsies with subcellular resolution and offers the possibility of introducing a high-resolution optical diagnostic method in dermatology. © 2003 Society of Photo-Optical Instrumentation Engineers.

In recent years, fluorescence microscopy based on two-photon excitation has become a popular tool for biological and biomedical imaging. Among its advantages is the enhanced depth penetration permitted by fluorescence excitation with the near-infrared photons, which is particularly attractive for deep-tissue imaging. To fully utilize two-photon fluorescence microscopy as a three-dimensional research technique in biology and medicine, it is important to characterize the two-photon imaging parameters in a turbid medium. We investigated the two-photon point spread functions (PSFs) in a number of scattering samples. Gel samples containing 0.1-μm fluorescent microspheres and Liposyn III were used as phantoms mimicking the turbid environment often found in tissue. A full characterization of the two-photon PSFs of a water and oil immersion objective was completed in samples composed of 0, 0.25, 0.5, 1, and 2% Liposyn III. Our results show that up to depths of about 100 (oil) and 200 μm (water), the presence of scatterers (up to 2% Liposyn III) does not appreciably degrade the PSF widths of the objectives. © 2003 Society of Photo-Optical Instrumentation Engineers.

The intrinsic fluorescence of various cell cultures in the blue and green spectral range has been attributed mainly to hydrated nicotinamide adenine dinucleotide (NADH) and flavin molecules. Their fluorescence decay curves were measured with subnanosecond resolution. The reduced coenzymes NADH and hydrated nicotinamide adenine dinucleotide phosphate NADPH, both showed a biexponential decay pattern in solution with similar time constants, but different relative intensities of the two components. They could thus be distinguished from one another as well as from their oxidized forms. The NAD(P)H fluorescence of Saccharomyces cerevisiae was located within the cytoplasm and its organelles and was by about a factor 4 higher for respiratory-deficient than for intact yeast strains. Intracellular flavin fluorescence showed a triexponential behavior-probably due to a superposition of protein-bound and free flavin molecules. The lifetime of the shortest component varied within the time range of 0.20 to 0.50 ns between respiratory-deficient and intact yeast strains, and the relative intensity of this component as most pronounced for the intact strain DL1. Time-resolved fluorescence seems therefore to be an appropriate method to probe the function of the respiratory chain and-in the further step-to differentiate between various types of cells and tissues in medical diagnosis or environmental research.

Fluorescence Lifetime Imaging (FLIM) in life sciences based on ultrashort laser scanning microscopy and time-correlated single photon counting (TCSPC) started 30 years ago in Jena/East-Germany. One decade later, first two-photon FLIM images of a human finger were taken with a lab microscope based on a tunable femtosecond Ti:sapphire laser. In 2002/2003, first clinical non-invasive two-photon FLIM studies on patients with dermatological disorders were performed using a novel multiphoton tomograph. Current in vivo two-photon FLIM studies on human subjects are based on TCSPC and focus on (i) patients with skin inflammation and skin cancer as well as brain tumors, (ii) cosmetic research on volunteers to evaluate anti-ageing cremes, (iii) pharmaceutical research on volunteers to gain information on in situ pharmacokinetics, and (iv) space medicine to study non-invasively skin modifications on astronauts during long-term space flights. Two-photon FLIM studies on volunteers and patients are performed with multiphoton FLIM tomographs using near infrared femtosecond laser technology that provide rapid non-invasive and label-free intratissue autofluorescence biopsies with picosecond temporal resolution.