Jörg Martini

Two-photon laser scanning microscopy on native cartilage and collagen membranes for tissue engineering

In our experiments 2-Photon laser scanning microscopy (2PLSM) has been used to acquire 3-dimensional structural information on native unstained biological samples for tissue engineering purposes. Using near infrared (NIR) femtosecond laser pulses for 2-photon excitation and second harmonic generation (SHG) it was possible to achieve microscopic images at great depths in strongly (light) scattering collagen membranes (depth up to 300 μm) and cartilage samples (depth up to 460 μm). With the objective of optimizing the process of chondrocyte growth on collagen scaffolding materials for implantation into human knee joints, two types of samples have been investigated. (1) Both arthritic and non-arthritic bovine and human cartilage samples were examined in order to differentiate between these states and to estimate the density of chondrocytes. In particular, imaging depth, fluorescence intensity and surface topology appear promising as key information for discriminating between the non-arthritic and arthritic states. Human chondrocyte densities between 2-106/cm3 and 20-106/cm3, depending on the relative position of the sample under investigation within the cartilage, were measured using an automated procedure. (2) Chondrocytes which had been sown out on different types of I/III-collagen membranes, were discriminated from the scaffolding membranes on the basis of their native fluorescence emission spectra. With respect to the different membranes, either SHG signals from the collagen fibers of the membranes or differences in the emission spectra of the chondrocytes and the scaffolding collagenes were used to identify chondrocytes and membranes.

Combined TIRF-AFM Setup: Controlled Quenching of Individual Quantum Dots

Single molecules can nowadays be investigated by means of optical, mechanical and electrical methods. Fluorescence imaging and spectroscopy yield valuable and quantitative information about the optical properties and the spatial distribution of single molecules. Force spectroscopy by atomic force microscopy (AFM) or optical tweezers allows addressing, manipulation and quantitative probing of the nanomechanical properties of individual macromolecules. We present a combined AFM and total internal reflection fluorescence (TIRF) microscopy setup that enables ultrasensitive laser induced fluorescence detection of individual fluorophores, control of the AFM probe position in x, y and z-direction with nanometer precision, and simultaneous investigation of optical and mechanical properties at the single molecule level. Here, we present the distance-controlled quenching of semiconductor quantum dot clusters with an AFM tip. In future applications, fluorescence resonant energy transfer between single donor and acceptor molecules will be investigated.

Multifocal multispectral descanned detection in TPLSM

We present a new detection method for multifocal two-photon laser scanning microscopy (TPLSM) that allows a fast and easy access to spectrally resolved, three-dimensional images. In our setup eight fluorescent foci are directed through a descanned tube lens combination and a straight vision prism. This prism spectrally splits up the fluorescence beamlets, resulting in eight parallel spectral fluorescence lines. These lines are imaged onto a slit block array in front of a 8x8 multi anode PMT. Each PMT row detects different spectral characteristics from a special point in the sample whereas each column represents one focus. The eight exciting foci are scanned in the region of interest inside the sample by the two scanning mirrors in x- and y-direction. As a result of this imaging technique eight spectrally resolved images of slightly shifted sample regions are generated simultaneously and added up after the measurement, maintaining the spectral information. We present spectrally resolved 3D-data of various biological samples like pollen grains, tobacco cells and orange peel cells.