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. 2008 Feb;5(2):197-202.
doi: 10.1038/nmeth.1175. Epub 2008 Jan 20.

High-speed, Low-Photodamage Nonlinear Imaging Using Passive Pulse Splitters

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High-speed, Low-Photodamage Nonlinear Imaging Using Passive Pulse Splitters

Na Ji et al. Nat Methods. .

Erratum in

  • Nat Methods. 2009 Feb;6(2):179

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.

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