In vivo two-photon laser-scanning microscopy of Ca2+ dynamics in visual motion-sensitive neurons.
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.
Kalb
Julia
J
Lehrstuhl für Neurobiologie, Fakultät für Biologie, Universität Bielefeld, Postfach 100131, D-33501 Bielefeld, Germany.
Nielsen
Tim
T
Fricke
Matthias
M
Egelhaaf
Martin
M
Kurtz
Rafael
R
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D004175:Diptera / Q000378:metabolism*; D005260:Female; D007395:Interneurons / Q000166:cytology / Q000378:metabolism*; D007700:Kinetics; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence; D009039:Motion Perception; D016501:Neurites / Q000378:metabolism; D010775:Photic Stimulation; D017785:Photons
D002118:Calcium