High-speed, low-photodamage nonlinear imaging using passive pulse splitters

Nature Methods
Na JiEric Betzig

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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Citations

Oct 30, 2012·Nature·Mark T HarnettJeffrey C Magee
Jan 11, 2011·Nature Methods·Adrian ChengCarlos Portera-Cailliau
Apr 30, 2011·Nature Methods·Mikhail DrobizhevAleksander Rebane
Jul 19, 2011·Nature Methods·Thai V TruongScott E Fraser
Jan 15, 2013·Nature Methods·Jonathan S MarvinLoren L Looger
Dec 4, 2013·Nature Methods·Vivien Marx
Sep 15, 2012·Nature Protocols·Xiaowei ChenArthur Konnerth
Nov 10, 2010·Proceedings of the National Academy of Sciences of the United States of America·Vladislav V YakovlevMarlan O Scully
Dec 23, 2011·Proceedings of the National Academy of Sciences of the United States of America·Na JiEric Betzig
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