Application of multiline two-photon microscopy to functional in vivo imaging.
High spatial resolution and low risks of photodamage make two-photon laser-scanning microscopy (TPLSM) the method of choice for biological imaging. However, the study of functional dynamics such as neuronal calcium regulation often also requires a high temporal resolution. Hitherto, acquisition speed is usually increased by line scanning, which restricts spatial resolution to structures along a single axis. To overcome this gap between high spatial and high temporal resolution we performed TPLSM with a beam multiplexer to generate multiple laser foci inside the sample. By detecting the fluorescence emitted from these laser foci with an electron-multiplying camera, it was possible to perform multiple simultaneous linescans. In addition to multiline scanning, the array of up to 64 laser beams could also be used in x-y scan mode to collect entire images at high frame rates. To evaluate the applicability of multiline TPLSM to functional in vivo imaging, calcium signals were monitored in visual motion-sensitive neurons in the brain of flies. The capacity of our method to simultaneously acquire signals at different cellular locations is exemplified by measurements at branched neurites and 'spine'-like structures. Calcium dynamics depended on branch size, but 'spines' did not systematically differ from their 'parent neurites'. The spatial resolution of our setup was critically evaluated by comparing it to confocal microscopy and the negative effect of scattering of emission light during image detection was assessed directly by running the setup in both imaging and point-scanning mode.
Kurtz
Rafael
R
Lehrstuhl für Neurobiologie, Universität Bielefeld, Postfach 100131, D-33501 Bielefeld, Germany. rafael.kurtz@uni-bielefeld.de
Fricke
Matthias
M
Kalb
Julia
J
Tinnefeld
Philip
P
Sauer
Markus
M
D000818:Animals; D020013:Calcium Signaling / Q000502:physiology*; D004175:Diptera / Q000502:physiology*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D007089:Image Enhancement / Q000295:instrumentation / Q000379:methods; D018613:Microscopy, Confocal / Q000295:instrumentation* / Q000379:methods; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation* / Q000379:methods; D009039:Motion Perception / Q000502:physiology*; D009475:Neurons, Afferent / Q000502:physiology*; D014793:Visual Cortex / Q000502:physiology*