Tiemo Anhut

Fluorescence lifetime imaging of human skin and hair

Multiphoton imaging has developed into an important technique for in-vivo research in life sciences. With the laser System DermaInspect (JenLab, Germany) laser radiation from a Ti:Sapphire laser is used to generate multiphotonabsorption deep in the human skin in vivo. The resulting autofluorescence radiation arises from endogenous fluorophores such as NAD(P)H, flavines, collagen, elastin, porphyrins und melanin. Second harmonic generation (SHG) was used to detect collagen structures in the dermal layer. Femtosecond laser multiphoton imaging offers the possibility of high resolution optical tomography of human skin as well as fluorescence lifetime imaging (FLIM) with picosecond time resolution. In this work a photon detector with ultrashort rise time of less than 30ps was applied to FLIM measurements of human skin and hair with different pigmentation. Fluorescence lifetime images of different human hair types will be discussed.

Fluorescence correlation spectroscopy on dielectric surfaces in total internal reflection geometries

Fluorescence correlation spectroscopy (FCS) has evolved to a valuable tool for biomolecular analysis on the single molecule level. Measurements on a single molecule level can only be performed if the measurement volume is small enough to contain on average only very few molecules. Common FCS-systems are therefore based on a confocal geometry in which a laser spot is focused into a liquid sample. This illumination concept in combination with a pinhole in the detection path leads to an observation volume in the order of one femtoliter. On the other hand, many biomolecular interactions need to be measured on surfaces. To study such interactions or the fluctuating signal of surface bound molecules itself, as for instance during single molecule enzyme catalysis, evanescent field based excitation seems advantageous as compared to confocal FCS. We discuss different schemes for evanescent field FCS and present an efficient excitation-detection scheme in an objective-based TIR-FCS configuration.

Nonlinear Laser-Scanning-Microscopy and Microprocessing of Biological and Technical Materials Using a New Diode Pumped Solid State Femtosecond Laser with Cavity Dumping

A diode-pumped Yb:glass femtosecond laser oscillator with electro-optical cavity-dumping has been applied for nonlinear laser-scanning microscopy and processing of biomaterials. The high-energy pulses delivered by this source in combination with its unique parameters proved very efficient for micro-processing of biomaterials.

Multiphoton tomography of epidermis and dermis

Multiphoton imaging has developed into an important technique for in-vivo research in life sciences in the last few years. A near-infrared laser beam is focused into a sample such that multiphoton-absorption can be generated which stimulates a fluorescence signal as well as second harmonic generation (SHG). Recently it has been shown that it is possible to image the epidermis in vivo with a resolution of about 1 μm. It was possible to produce 3-dimensional autofluorescence maps of the investigated tissue. However, the depth range of this technique is limited through the working distance of the focusing optics mostly to the epidermal part of the skin. Gradient index lenses offer possibilities to expand the imaging depth into the dermal layer. With typical diameters of up to 2mm and lengths between 2 and 6 cm they are capable of transmitting the laser, fluorescence and SHG radiation and to be integrated into an imaging system. First results of the applicability of gradient index lenses for imaging of skin are presented.