Measuring serotonin distribution in live cells with three photon excitation. Tryptophan and serotonin were imaged with infrared illumination by three photon excitation of their native ultraviolet fluorescence. This technique circumvents the limitations imposed by photodamage, scattering and indiscriminate background encountered in other UV microscopies. Three dimensionally-resolved images are presented along with measurements of the serotonin concentration and content of individual secretory granules. Three photon excitation thus enables quantitative imaging of cellular processes by harnessing the UV fluorescence of native molecules.
Photons add up to better microsocopy.
The system currently available to users has a pulsed laser source of 1047nm which can excite most blue and red, and some green emitting fluorophores. After aldehydic fixation, the cells can be permeabilized and processed with a specific antibody and a DNA stain for conventional or confocal microscopy. This allows simultaneous three-channel imaging of mitochondria, DNA and an antibody of choice. We used the above procedure to pre-label the mitochondria in the tails of live bull sperm that were subsequently used to fertilized bovine oocytes. The procedure allowed us to follow the fate of paternal mitochondria in fertilized eggs concomitantly with the observations of microtubule-cytoskeleton rearrangement and pronuclear development.