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

Na Ji1, Jeffrey C Magee, Eric Betzig

  • 1Janelia Farm Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, Virginia 20147, USA. jin@janelia.hhmi.org

Nature Methods
|January 22, 2008
PubMed
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A novel passive pulse splitter enhances nonlinear bioimaging by dividing laser pulses. This technology significantly boosts signal rates and reduces photobleaching and photodamage in biological samples.

Area of Science:

  • Optics and Photonics
  • Biomedical Imaging
  • Laser Physics

Background:

  • Pulsed lasers are crucial for nonlinear bioimaging like two-photon fluorescence excitation (TPE) microscopy.
  • Current limitations restrict laser power delivery to samples, risking photoinduced damage.
  • Efficient energy utilization remains a challenge in high-resolution biological imaging.

Purpose of the Study:

  • To develop and evaluate a passive pulse splitter for enhancing TPE microscopy.
  • To investigate the splitter's ability to increase signal rates and reduce photodamage.
  • To assess the performance of the pulse splitter in both fixed and living biological specimens.

Main Methods:

  • A passive pulse splitter was designed to divide each laser pulse into multiple sub-pulses of equal energy.

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  • The splitter was integrated into TPE microscopy setups for imaging fixed mouse brain slices and live Caenorhabditis elegans larvae.
  • Photobleaching rates and photodamage were quantified during imaging of fixed and live samples, including rat hippocampal slices for calcium imaging.

    • Main Results:

    • The pulse splitter increased signal rates by over 100-fold in certain power regimes.
    • Photobleaching was reduced by over fourfold in fixed mouse brain slices.
    • In vivo imaging of C. elegans larvae showed a ninefold reduction in photobleaching.
    • Calcium imaging of rat hippocampal slices demonstrated a six- to 20-fold decrease in photodamage rates.

    Conclusions:

    • The passive pulse splitter is an effective tool for improving nonlinear bioimaging techniques.
    • This technology offers significant advantages in increasing signal acquisition and minimizing photodamage in biological samples.
    • The pulse splitter has broad applicability for advanced bioimaging, particularly in sensitive live-specimen studies.