@article {2081, title = {High-speed, low-photodamage nonlinear imaging using passive pulse splitters. (With commentary)}, journal = {Nature Methods}, volume = {5}, year = {2008}, month = {2008 Feb}, pages = {197-202}, abstract = {

Pulsed lasers are key elements in nonlinear bioimaging techniques such as two-photon fluorescence excitation (TPE) microscopy. Typically, however, only a percent or less of the laser power available can be delivered to the sample before photoinduced damage becomes excessive. Here we describe a passive pulse splitter that converts each laser pulse into a fixed number of sub-pulses of equal energy. We applied the splitter to TPE imaging of fixed mouse brain slices labeled with GFP and show that, in different power regimes, the splitter can be used either to increase the signal rate more than 100-fold or to reduce the rate of photobleaching by over fourfold. In living specimens, the gains were even greater: a ninefold reduction in photobleaching during in vivo imaging of Caenorhabditis elegans larvae, and a six- to 20-fold decrease in the rate of photodamage during calcium imaging of rat hippocampal brain slices.

Commentary: Na Ji came to me early in her postdoc with an idea to reduce photodamage in nonlinear microscopy by splitting the pulses from an ultrafast laser into multiple subpulses of reduced energy. In six weeks, we constructed a prototype pulse splitter and obtained initial results confirming the validity of her vision. Further experiments with Jeff Magee demonstrated that the splitter could be used to increase imaging speed or reduce photodamage in two photon microscopy by one to two orders of magnitude. This project is a great example of how quickly one can react and exploit new ideas in the Janelia environment.

}, keywords = {Animals, Caenorhabditis elegans, Cell Survival, Cells, Cultured, Equipment Design, Equipment Failure Analysis, Fluorescence, Image Enhancement, Lasers, Light, Lighting, Mice, Microscopy, Nonlinear Dynamics, Rats}, issn = {1548-7105}, doi = {10.1038/nmeth.1175}, url = {http://www.ncbi.nlm.nih.gov/pubmed/18204458}, author = {Ji, Na and Magee, Jeffrey C and Betzig, Eric} }