Widefield Two-Photon Excitation without Scanning: Live Cell Microscopy with High Time Resolution and Low Photo-Bleaching

PLoS One. 2016 Jan 29;11(1):e0147115. doi: 10.1371/journal.pone.0147115. eCollection 2016.

Abstract

We demonstrate fluorescence imaging by two-photon excitation without scanning in biological specimens as previously described by Hwang and co-workers, but with an increased field size and with framing rates of up to 100 Hz. During recordings of synaptically-driven Ca(2+) events in primary rat hippocampal neurone cultures loaded with the fluorescent Ca(2+) indicator Fluo-4 AM, we have observed greatly reduced photo-bleaching in comparison with single-photon excitation. This method, which requires no costly additions to the microscope, promises to be useful for work where high time-resolution is required.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aniline Compounds
  • Animals
  • Animals, Newborn
  • Calcium / metabolism
  • Fluorescent Dyes
  • Hippocampus / metabolism
  • Hippocampus / ultrastructure*
  • Microscopy, Fluorescence, Multiphoton / instrumentation
  • Microscopy, Fluorescence, Multiphoton / methods*
  • Molecular Imaging / instrumentation
  • Molecular Imaging / methods*
  • Neurons / metabolism
  • Neurons / ultrastructure*
  • Photobleaching
  • Primary Cell Culture
  • Rats
  • Rats, Sprague-Dawley
  • Synapses / metabolism
  • Time-Lapse Imaging / instrumentation
  • Time-Lapse Imaging / methods*
  • Xanthenes

Substances

  • Aniline Compounds
  • Fluo 4
  • Fluorescent Dyes
  • Xanthenes
  • Calcium