In molecular medicine, the manipulation of cells is prerequisite to evaluate genes as therapeutic targets or to transfect cells to develop cell therapeutic strategies. To achieve these purposes it is essential that given transfection techniques are capable of handling high cell numbers in reasonable time spans. To fulfill this demand, an alternative nanoparticle mediated laser transfection method is presented herein. The fs-laser excitation of cell-adhered gold nanoparticles evokes localized membrane permeabilization and enables an inflow of extracellular molecules into cells. The parameters for an efficient and gentle cell manipulation are evaluated in detail. Efficiencies of 90% with a cell viability of 93% were achieved for siRNA transfection. The proof for a molecular medical approach is demonstrated by highly efficient knock down of the oncogene HMGA2 in a rapidly proliferating prostate carcinoma in vitro model using siRNA. Additionally, investigations concerning the initial perforation mechanism are conducted. Next to theoretical simulations, the laser induced effects are experimentally investigated by spectrometric and microscopic analysis. The results indicate that near field effects are the initial mechanism of membrane permeabilization. This methodical approach combined with an automated setup, allows a high throughput targeting of several 100,000 cells within seconds, providing an excellent tool for in vitro applications in molecular medicine. NIR fs lasers are characterized by specific advantages when compared to lasers employing longer (ps/ns) pulses in the visible regime. The NIR fs pulses generate low thermal impact while allowing high penetration depths into tissue. Therefore fs lasers could be used for prospective in vivo applications.

We present a time-resolved photographic analysis of the pulse-to-pulse interaction. In particular, we studied the influence of the cavitation bubble induced by a fs-pulse on the optical focusing of the consecutive pulse and its cavitation bubble dynamics in dependence on temporal pulse separation in water. As a first result, by decreasing the temporal separation of laser pulses, there is a diminishment of the laser-induced optical breakdown (LIOB) efficiency in terms of energy conversion, caused by disturbed focusing into persisting gas bubbles at the focal volume. A LIOB at the focal spot is finally suppressed by impinging the expanding or collapsing cavitation bubble of the preceding pulse. These results could be additionally confirmed in porcine gelatin solution with various concentrations. Hence, the interaction between the laser and transparent ophthalmic tissue may be accompanied by a raised central laser energy transmission, which could be observed in case of a temporal pulse overlap. In conclusion, our experimental results are of particular importance for the optimization of the prospective ophthalmic surgical process with future generation fs-lasers.

We present a high-speed photographic analysis of the interaction of cavitation bubbles generated in two spatially separated regions by femtosecond laser-induced optical breakdown in water. Depending on the relative energies of the femtosecond laser pulses and their spatial separation, different kinds of interactions, such as a flattening and deformation of the bubbles, asymmetric water flows, and jet formation were observed. The results presented have a strong impact on understanding and optimizing the cutting effect of modern femtosecond lasers with high repetition rates (>1 MHz).

Optical coherence tomography (OCT) provides structural information of laryngeal tissue which is comparable to histopathological analysis of biopsies taken under general anesthesia. In awake patients, movements impede clinically useful OCT acquisition. Therefore, an automatic compensation of movements was implemented into a swept source OCT-laryngoscope. Video and OCT beam path were combined in one tube of 10-mm diameter. Segmented OCT images served as distance sensor and a feedback control adjusted the working distance between 33 and 70 mm by synchronously translating the reference mirror and focusing lens. With this motion compensation, the tissue was properly visible in up to 88% of the acquisition time. During quiet respiration, OCT contrasted epithelium and lamina propria. Mean epithelial thickness was measured to be 109 and $135μm$ in female and male, respectively. Furthermore, OCT of mucosal wave movements during phonation enabled estimation of the oscillation frequency and amplitude. Regarding clinical issues, the OCT-laryngoscope with automated working distance adjustment may support the estimation of the depth extent of epithelial lesions and contribute to establish an indication for a biopsy. Moreover, OCT of the vibrating vocal folds provides functional information, possibly giving further insight into mucosal behavior during the vibratory cycle.