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Biomed Opt Express. 2019 Jan 23;10(2):789-806. doi: 10.1364/BOE.10.000789. eCollection 2019 Feb 1.

Simultaneous voltage and calcium imaging and optogenetic stimulation with high sensitivity and a wide field of view.

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Q-State Biosciences, Cambridge, MA 02139, USA.


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.

Conflict of interest statement

CAW: Q-State Biosciences (I,E,P), All other authors: Q-State Biosciences (I,E).

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