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 2-10<sup>6</sup>/cm<sup>3</sup> and 20-10<sup>6</sup>/cm<sup>3</sup>, 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.
One of the most serious problems relevant to the use of optical fiber sensors in real-world environments is surface fouling, that is, the cumulative build-up of undesirable material on the working surface of a sensor. This paper present the results of anti-biofouling tests on coated fiber optic probes for reflectance spectroscopy in blood- simulating foul media, namely Bovine Serum Albumin (BSA) and Fibrinogen. The anti-biofouling coating, a proprietary invention of Biocompatibles Ltd., was a cross-linkable Phosphorylcholine (PC) polymer with Silane functionality, to improve adhesion to silica-containing substrates. All tests in BSA and Fibrinogen showed that PC-1036 coating was efficient in avoiding the build-up of biological material. In fact, optical signal variations of un-coated probes showed fluctuations in the 6-20% range, while coated probes exhibited a nearly-stable optical signal. These results were also confirmed by a microscopic check, which showed adhesions of biological material to un-coated probes.