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Biophys J. 2015 Feb 3;108(3):520-9. doi: 10.1016/j.bpj.2014.12.005.

Multifocal fluorescence microscope for fast optical recordings of neuronal action potentials.

Author information

1
Department of Neurobiology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California; Department of Neurology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California.
2
Department of Neurology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California.
3
Department of Neurobiology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California.
4
Department of Neurobiology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California; Department of Psychology, University of California Los Angeles, Los Angeles, California.
5
Department of Physics, University of California Los Angeles, Los Angeles, California.
6
Department of Neurobiology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California. Electronic address: otist@ucla.edu.

Abstract

In recent years, optical sensors for tracking neural activity have been developed and offer great utility. However, developing microscopy techniques that have several kHz bandwidth necessary to reliably capture optically reported action potentials (APs) at multiple locations in parallel remains a significant challenge. To our knowledge, we describe a novel microscope optimized to measure spatially distributed optical signals with submillisecond and near diffraction-limit resolution. Our design uses a spatial light modulator to generate patterned illumination to simultaneously excite multiple user-defined targets. A galvanometer driven mirror in the emission path streaks the fluorescence emanating from each excitation point during the camera exposure, using unused camera pixels to capture time varying fluorescence at rates that are ∼1000 times faster than the camera's native frame rate. We demonstrate that this approach is capable of recording Ca(2+) transients resulting from APs in neurons labeled with the Ca(2+) sensor Oregon Green Bapta-1 (OGB-1), and can localize the timing of these events with millisecond resolution. Furthermore, optically reported APs can be detected with the voltage sensitive dye DiO-DPA in multiple locations within a neuron with a signal/noise ratio up to ∼40, resolving delays in arrival time along dendrites. Thus, the microscope provides a powerful tool for photometric measurements of dynamics requiring submillisecond sampling at multiple locations.

PMID:
25650920
PMCID:
PMC4317551
DOI:
10.1016/j.bpj.2014.12.005
[Indexed for MEDLINE]
Free PMC Article

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