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

A Corrigendum to this article was published on 01 February 2009

This article has been updated


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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Figure 1: Construction of pulse splitters.
Figure 2: Pulse splitting increases TPE signal rates.
Figure 3: Effect of pulse splitting on photobleaching of fixed GFP-labeled brain slices.
Figure 4: Effect of 64 × pulse splitting on in vivo photobleaching in a GFP-labeled C.elegans larva.
Figure 5: Effect of 64 × pulse splitting on photodamage during Ca2+ imaging of CA1 pyramidal neurons injected with Oregon Green BAPTA-1.

Change history

  • 31 December 2008

    NOTE: In the version of this article initially published, the traces in Figure 2 were incorrectly labeled. The error has been corrected in the HTML and PDF versions of the article.


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We thank our colleagues at Janelia Farm Research Campus, Howard Hughes Medical Institute, T. Sato and T.-Y. Mao for providing the GFP-labeled brain slice samples, V. Jayaraman and J. Seelig for sharing their Ti:sapphire laser, R. Kerr and H.-C. Peng for guidance with the C. elegans samples, and K. Svoboda for helpful suggestions.

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Correspondence to Na Ji.

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Ji, N., Magee, J. & Betzig, E. High-speed, low-photodamage nonlinear imaging using passive pulse splitters. Nat Methods 5, 197–202 (2008). https://doi.org/10.1038/nmeth.1175

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