Abstract
Voltage imaging of many neurons simultaneously at single-cell resolution is hampered by the difficulty of detecting small voltage signals from overlapping neuronal processes in neural tissue. Recent advances in genetically encoded voltage indicator (GEVI) imaging have shown single-cell resolution optical voltage recordings in intact tissue through imaging naturally sparse cell classes, sparse viral expression, soma restricted expression, advanced optical systems, or a combination of these. Widespread sparse and strong transgenic GEVI expression would enable straightforward optical access to a densely occurring cell type, such as cortical pyramidal cells. Here we demonstrate that a recently described sparse transgenic expression strategy can enable single-cell resolution voltage imaging of cortical pyramidal cells in intact brain tissue without restricting expression to the soma. We also quantify the functional crosstalk in brain tissue and discuss optimal imaging rates to inform future GEVI experimental design.
Highlights
Over the last decade, functional fluorescence imaging has become a key technology in cellular and systems neurosciences (Knöpfel et al, 2006; Scanziani and Häusser, 2009; Knöpfel, 2012; Allen et al, 2017; Chen et al, 2017; Otis et al, 2017; Yang and Yuste, 2017)
As explained in the introduction, it is difficult to optically resolve fluorescent plasma membranes if a genetically encoded voltage indicator (GEVI) is targeted to all cells in a dense population of neurons, such as cortical pyramidal cells
We have shown that destabilized Cre recombinase based expression strategies enable single-cell resolution voltage imaging of cortical pyramidal cells in acute brain slices
Summary
Functional fluorescence imaging has become a key technology in cellular and systems neurosciences (Knöpfel et al, 2006; Scanziani and Häusser, 2009; Knöpfel, 2012; Allen et al, 2017; Chen et al, 2017; Otis et al, 2017; Yang and Yuste, 2017). In contrast to calcium indicators which are localized in the cytosol of the cells of interest, voltage indicators are localized to their plasma membranes, which account for a tiny fraction of their volume This limits the number of indicator molecules that can be employed and the flux of signaling photons that can be generated. Voltage signals of interest are Sparse Transgenic Voltage Imaging typically much faster than the signals provided by calcium indicators and must be imaged at higher frame rates This is an instrumentation challenge and translates, along with the limited number of dye molecules, into a signal-tonoise ratio (SNR) challenge, as a sufficiently high SNR requires a large number of photons sampled per spatiotemporal bin (e.g., 10,000 photons are required in order for a fluorescence change of 1% to have an SNR of 1)
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