Computer-generated holography enhances voltage dye fluorescence discrimination in adjacent neuronal structures.

Amanda J Foust,Eirini Papagiakoumou,Dimitrii Tanese,Valentina Emiliani,Valeria Zampini

doi:10.1117/1.nph.2.2.021007

Abstract

Voltage-sensitive fluorescence indicators enable tracking neuronal electrical signals simultaneously in multiple neurons or neuronal subcompartments difficult to access with patch electrodes. However, efficient widefield epifluorescence detection of rapid voltage fluorescence transients necessitates that imaged cells and structures lie sufficiently far from other labeled structures to avoid contamination from out of focal plane and scattered light. We overcame this limitation by exciting dye fluorescence with one-photon computer-generated holography shapes contoured to axons or dendrites of interest, enabling widefield detection of voltage fluorescence with high spatial specificity. By shaping light onto neighboring axons and dendrites, we observed that dendritic back-propagating action potentials were broader and slowly rising compared with axonal action potentials, differences not measured in the same structures illuminated with a large "pseudowidefield" (pWF) spot of the same excitation density. Shaped illumination trials showed reduced baseline fluorescence, higher baseline noise, and fractional fluorescence transient amplitudes two times greater than trials acquired with pWF illumination of the same regions.

Highlights

Understanding neuronal input–output transformations requires experimental characterization of how electrical signals generate and propagate in axonal and dendritic arbors
We utilized computer-generated holography (CGH) to confine Voltage-sensitive dyes (VSDs) fluorescence excitation light to an axon or dendrite of interest [Figs. 1(c), 2(b), and 2(c)], comparing signals obtained in this fashion to those illuminated in “pWF,” that is with a large spot of light with poor axial confinement [Figs. 1(d) and 2(a)]
We found that CGH-shaped voltage dye fluorescence excitation in neighboring axons and dendrites enabled discrimination of differing action potential kinetics not possible with large-field illumination

Summary

Introduction

Understanding neuronal input–output transformations requires experimental characterization of how electrical signals generate and propagate in axonal and dendritic arbors. Voltage-sensitive dyes (VSDs) provide an alternative method to track membrane potential and have been effectively imaged with one-photon epifluorescence microscopy to characterize action potential propagation in small diameter axons and dendrites.[1,2,3,4,5,6] Due to the close spatial mingling of neuronal substructures, improving lateral and axial confinement with confocal microscopy could enable discrimination of signals arising from adjacent or overlapping structures; the relatively low fractional sensitivity of voltage sensors (dF∕F ∼5% to 20% per 100 mV in brain slices) necessitates excitation densities and collection efficiencies sufficient to overcome high fractional shot noise.[7,8,9] Loss of photon flux through confocal pinhole and lens arrays stipulates extensive signal averaging[10] or long integration times[11] to increase the S/N.

Methods

Results

Conclusion