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In vivo two-photon laser-scanning microscopy of Ca2+dynamics in visual motion-sensitive neurons

Elsevier Inc.
Publication Date
DOI: 10.1016/j.bbrc.2004.02.047
  • Calcium
  • Fluo-4
  • Fluorescence Imaging
  • Fly
  • Motion Vision
  • Neuronal Processing
  • Synapse
  • Tangential Cell
  • Two-Photon Microscopy
  • Physics


Abstract We applied two-photon laser-scanning microscopy (TPLSM) to motion-sensitive visual interneurons of the fly to study Ca 2+ dynamics in vivo at a higher spatial and temporal resolution than possible with conventional fluorescence microscopy. Based on a custom-built two-photon microscope, we performed line scans to measure changes in presynaptic Ca 2+ concentrations elicited by visual stimulation. We used a fast avalanche photodiode (APD) with a high quantum efficiency to detect even low levels of emitted fluorescence. Our experiments show that our in vivo preparation is amenable to TPLSM: with excitation intensities low enough not to cause photodamage, activity-dependent fluorescence changes of Ca 2+-sensitive dyes can be detected in small neuronal branches. The performance of two-photon and conventional Ca 2+ imaging carried out consecutively at the same neuron is compared and it is demonstrated that two-photon imaging allows us to detect differences in Ca 2+ dynamics between individual neurites.

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