Abstract
Transmembrane voltage and intracellular calcium concentration are coupled parameters essential to the function of neurons, cardiomyocytes, and other excitable cells. Here we introduce the Firefly-HR microscope for simultaneous optogenetic stimulation and voltage and calcium imaging with fluorescent proteins using three spectrally distinct visible color bands. Firefly-HR combines patterned stimulation, near-total internal reflection laser excitation through a prism located between the sample and a water-immersion objective, and concurrent imaging of three color channels. The microscope has efficient light collection, low fluorescent background, and a large field of view (0.24 x 1.2 mm @ 1000 frames/sec). We characterize optical crosstalk and demonstrate capabilities with three applications: (1) probing synaptically connected neuronal microcircuits, (2) examining the coupling between neuronal action potentials and calcium influx, and (3) studying the pharmacology of paced human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) via simultaneous recordings of voltage, calcium, and contraction.
Highlights
Recent decades have seen a rapid expansion in the number of protein-based optical tools for exploring cellular systems, both optogenetic actuators [1,2] and fluorescent sensors [3,4,5,6]
The microscope can be built fully equipped for a little over $100k. It is compatible with a variety of coverslip-bottomed dishes, fused silica and cyclic olefin copolymer coverslips offer the lowest background for demanding voltage-sensing applications
Attempts have been made to automate the patch-clamp process to achieve throughputs of 10’s of cells an hour [31], but simultaneous calcium recording is challenging, no more than 1 cell can be recorded at a time, and extensive human intervention is require to swap out patch pipettes
Summary
Recent decades have seen a rapid expansion in the number of protein-based optical tools for exploring cellular systems, both optogenetic actuators [1,2] and fluorescent sensors [3,4,5,6]. Combining tools yielded the all-optical electrophysiology platform Optopatch, which pairs the blue-light gated channelrhodopsin CheRiff with the red-light excited voltage sensor QuasAr [11]. We pair the Optopatch platform with orange cytosolic calcium sensor jRGECO1a [15] (Fig. 1(A)). We build on optical design element from the Firefly microscope, designed for high-throughput screening applications with Optopatch, which can simultaneously stimulate and record action potentials in hundreds of neurons [21]. Firefly-HR pairs patterned optogenetic stimulation with simultaneous voltage and calcium recordings from tens of neurons in parallel at a 1 kHz frame rate with a high signal-to-noise ratio (SNR). We show optogentetic stimulation paired with voltage and calcium imaging in neurons (Fig. 5) and examine how simultaneous voltage and calcium signals can be used to access biological information unavailable with either recording alone.
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