Two-photon microscopy for biomedical studies

Fluorescence optical microscopy is one of the most important tools to investigate biological samples and to observe structures in living cells. Based on the principle of the simultaneous absorption of two-photons and subsequent emission of fluorescent light we have developed a Two-Photon Laser Scanning Fluorescence Microscope that allow us to perform optical sectioning and three-dimensional reconstructions of structures in biological samples such as dopaminergic clusters of neurons and tracheal branches in Drosophila brain. The spatial confinement of the excitation light to the focal plane combined with the temporal compression of photons into short packets during femtosecond pulses make our system suitable for in vivo imaging. The study of Mycobacterial infection of macrophages is another interesting problem we have been addressing in our laboratory. It involves fluorescence resonance energy transfer (FRET), a mechanism between two cromophores that is very sensitive to the distance between them. We have developed a method to quantify Mycobacterial rupture of a subcellular compartment which allows these pathogens to proliferate and eventually cause cellular death