Vital imaging of molecular processes in organs and intact animals by two-photon (lifetime) laser scanning microscopy.

Rapid progress in the understanding of the biology of cells and tissues, both in health and disease, thrives on new cell biological and genetic methodologies. Technical developments in (confocal) fluorescence microscopy, particularly in combination with digital imaging techniques and the availability of fluorescence probes for use in living cells, have significantly contributed to this knowledge. In combination with advanced transfection technologies, avenues of possibilities have been opened to monitor the dynamics of many molecular processes in isolated cells and in cells in superficial tissues (e.g., transcription of genes, intracellular signalling and trafficking, and intercellular communication). However, conventional bright-field and laser scanning microscopy lack sufficient penetration depth to appropriately image deeper in organs, even when modern algorithms are used for image restoration. Classical confocal microscopy is also hampered by out-of focus bleaching and the cytotoxic effects of ultraviolet irradiation. These limitations are largely overcome by the new technique of tow-photon (multi-photon) laser scanning microscopy (TPLSM). Here, a mode-locked femto/picosecond pulsed laser is used to excite fluorescence probes by absorption of two re more photons in a co-operative process. Accordingly, red or infrared illumination is used to exite fluorescence probes that are normally excitable by ultraviolet or blue light. The non-linearity of the two-photon excitation process significantly decreases the exitation region of the laser beam. As a result, fluorescence images are collected with a resolution as high as with conventional (confocal) single-photon laser scanning microscopy, though without the use of pinholes. As demonstrated, the images can be collected from deeper in tissues after prolonged illumination. Use of these pulsed lasers in two-photon microscopy renders the possibility of fluorescence lifetime imaging (FLI). The fluorescence lifetime characteristics of many fluorochromes alter with changes in the immediate environment (levels of Ca2+, H+, proteins, etc.), independently of probe concentration. Quantitative FLI can thus provide new information on the intracellular state and add an extra (molecular) dimension to three-dimensional vital imaing studies in vivio. Furthermore, FLI can reveal contrast when this is not possible by spectral analysis, and can discriminate between tissue autofluorescence and probe fluorescence. Various institues at the universities of Maastricht, Eindhoven and Utrecht consider TPLSM/FLI as an indispensable techniwue to extend current studies towards imaging of the molecular dynamics of vital cells in their natural environment in tissues, organs and intact animals. In Maastricht, these are the Cardiovascular Research Institure Maastricht (CARIM), the Nutrition and Toxicology Research Institute Maastricht (NUTRIM), the Institute of Growth and Development (GREO) and the Interantional Institute of Brain aned Behaviour (EURON). Vital imaging in tissues is also one of the central activities of the Faculty of Biomedical Engineering (a joint entity of the universities of Eindhoven and Maastricht), and plays an important role in the mutual integration of research. Investigators form the Debye Institue (Utrecht) participate in further development of the FLI technique for use in the biomedical field. The combined TPLSM/FLI system will be placed in the laboratories of CARIM in Maastrich, and managed by its Core-unit Vital Imaging. The proposed investigations concern vital imaging of various inter- and intracellular processes in the cardiovascular and other tissue systems, which can not yet be monitored by other means. Our major goal with the TPLSM/FLI system is to translate present knowledge of molecular dynamics in the isolated cell towards understanding of the functioning of tissues and organs in health/disease and in intact animal models.