Abstract:In two-photon laser-scanning microscopy using femtosecond laser pulses, the dependence of the photobleaching rate on excitation power may have a quadratic, cubic or even biquadratic order. To date, there are still many open questions concerning this so-called high-order photobleaching. We studied the photobleaching kinetics of an intrinsic (enhanced Green Fluorescent Protein (eGFP)) and an extrinsic (Hoechst 33342) fluorophore in a cellular environment in two-photon microscopy. Furthermore, we examined the correlation between bleaching and the formation of reactive oxygen species. We observed bleaching-orders of three and four for eGFP and two and three for Hoechst increasing step-wise at a certain wavelength. An increase of reactive oxygen species correlating with the bleaching over time was recognized. Comparing our results to the mechanisms involved in intracellular ablation with respect to the amount of interacting photons and involved energetic states, we found that a low-density plasma is formed in both cases with a smooth transition in between. Photobleaching, however, is mediated by sequential-absorption and multiphoton-ionization, while ablation is dominated by the latter and cascade-ionization processes.

Femtosecond (fs) laser-based intracellular nanosurgery has become an important tool in cell biology, albeit the mechanisms in the so-called low-density plasma regime are largely unknown. Previous calculations of free-electron densities for intracellular surgery used water as a model substance for biological media and neglected the presence of dye and biomolecules. In addition, it is still unclear on which time scales free-electron and free-radical induced chemical effects take place in a cellular environment. Here, we present our experimental study on the influence of laser parameters and staining on the intracellular ablation threshold in the low-density plasma regime. We found that the ablation effect of fs laser pulse trains resulted from the accumulation of single-shot multiphoton-induced photochemical effects finished within a few nanoseconds. At the threshold, the number of applied pulses was inversely proportional to a higher order of the irradiance, depending on the laser repetition rate and wavelength. Furthermore, fluorescence staining of subcellular structures before surgery significantly decreased the ablation threshold. Based on our findings, we propose that dye molecules are the major source for providing seed electrons for the ionization cascade. Consequently, future calculations of free-electron densities for intracellular nanosurgery have to take them into account, especially in the calculations of multiphoton ionization rates.

Since the birth of "Dolly" as the first mammal cloned from a differentiated cell, somatic cell cloning has been successful in several mammalian species, albeit at low success rates. The highly invasive mechanical enucleation step of a cloning protocol requires sophisticated, expensive equipment and considerable micromanipulation skill. We present a novel noninvasive method for combined oocyte imaging and automated functional enucleation using femtosecond (fs) laser pulses. After three-dimensional imaging of Hoechst-labeled porcine oocytes by multiphoton microscopy, our self-developed software automatically identified the metaphase plate. Subsequent irradiation of the metaphase chromosomes with the very same laser at higher pulse energies in the low-density-plasma regime was used for metaphase plate ablation (functional enucleation). We show that fs laser-based functional enucleation of porcine oocytes completely inhibited the parthenogenetic development without affecting the oocyte morphology. In contrast, nonirradiated oocytes were able to develop parthenogenetically to the blastocyst stage without significant differences to controls. Our results indicate that fs laser systems have great potential for oocyte imaging and functional enucleation and may improve the efficiency of somatic cell cloning.

Cardiac tissue engineering is a promising strategy for regenerative therapies to overcome the shortage of donor organs for transplantation. Besides contractile function, the stiffness of tissue engineered constructs is crucial to generate transplantable tissue surrogates with sufficient mechanical stability to withstand the high pressure present in the heart. Although several collagen cross-linking techniques have proven to be efficient in stabilizing biomaterials, they cannot be applied to cardiac tissue engineering, as cell death occurs in the treated area. Here, we present a novel method using femtosecond (fs) laser pulses to increase the stiffness of collagen-based tissue constructs without impairing cell viability. Raster scanning of the fs laser beam over riboflavin-treated tissue induced collagen cross-linking by two-photon photosensitized singlet oxygen production. One day post-irradiation, stress-strain measurements revealed increased tissue stiffness by around 40% being dependent on the fibroblast content in the tissue. At the same time, cells remained viable and fully functional as demonstrated by fluorescence imaging of cardiomyocyte mitochondrial activity and preservation of active contraction force. Our results indicate that two-photon induced collagen cross-linking has great potential for studying and improving artificially engineered tissue for regenerative therapies.

Femtosecond (fs) laser-based cell surgery is typically done in two different regimes, at kHz or MHz repetition rate. Formation of reactive oxygen species (ROS) is an often predicted effect due to illumination with short laser pulses in biological tissue. We present our study on ROS formation in single cells in response to irradiation with fs laser pulses depending on the repetition rate while focusing into the cell nucleus. We observed a significant increase of ROS concentration directly after manipulation followed by a decrease in both regimes at kHz and MHz repetition rate. In addition, effects of consecutive exposures at MHz and kHz repetition rate and vice versa on ROS production were studied. Irradiation with a MHz pulse train followed by a kHz pulse train resulted in a significantly higher increase of ROS concentration than in the reversed case and often caused cell death. In the presence of the antioxidant ascorbic acid, accumulation of ROS and cell death were strongly reduced. Therefore, addition of antioxidants during fs laser-based cell surgery experiments could be advantageous in terms of suppressing photochemical damage to the cell.