Abstract
The rapid increase in the number and quality of fluorescent reporters and optogenetic actuators has yielded a powerful set of tools for recording and controlling cellular state and function. To achieve the full benefit of these tools requires improved optical systems with high light collection efficiency, high spatial and temporal resolution, and patterned optical stimulation, in a wide field of view (FOV). Here we describe our 'Firefly' microscope, which achieves these goals in a Ø6 mm FOV. The Firefly optical system is optimized for simultaneous photostimulation and fluorescence imaging in cultured cells. All but one of the optical elements are commercially available, yet the microscope achieves 10-fold higher light collection efficiency at its design magnification than the comparable commercially available microscope using the same objective. The Firefly microscope enables all-optical electrophysiology ('Optopatch') in cultured neurons with a throughput and information content unmatched by other neuronal phenotyping systems. This capability opens possibilities in disease modeling and phenotypic drug screening. We also demonstrate applications of the system to voltage and calcium recordings in human induced pluripotent stem cell derived cardiomyocytes.
Highlights
Recent advances in fluorescent reporters and optogenetic actuators have enhanced our ability to detect and control cellular function with light [1,2]
We anticipate that the ease and low cost of assembly, high optical quality, and modular design of the Firefly microscope system will give it broad application in functional biological imaging and screening
The Firefly microscope has three main optical systems: (1) a high-NA, large field of view (FOV) imaging path, (2) patterned illumination using a digital micromirror device (DMD), and (3) near-total internal reflection (TIR) illumination with a high-powered red laser coupled into the sample with a prism (Fig. 1)
Summary
Recent advances in fluorescent reporters and optogenetic actuators have enhanced our ability to detect and control cellular function with light [1,2]. To fully characterize neuronal excitability typically requires 30 s of recording under a variety of stimulus patterns This requirement for extended observation strongly disfavors tiling multiple fields of view with conventional high-magnification imaging. Sub-cellular targeting of reporters can provide physiological information on cellular microcompartments (e.g. inside organelles) without the need for sub-cellular optical resolution In these examples, imaging at low magnification increases the number of cells measured in parallel, without sacrificing the relevant biological information. The Firefly microscope meets the needs of wide-field optogenetic applications, with good light collection efficiency, sub-cellular spatial resolution for recording and stimulation, high temporal resolution, and many wavelengths available for combinatorial application of different reporters and actuators. The Firefly microscope provides arbitrarily reconfigurable patterned light illumination for optogenetic stimulation, with 20 kHz update rate and 7 μm spatial resolution. We anticipate that the ease and low cost of assembly, high optical quality, and modular design of the Firefly microscope system will give it broad application in functional biological imaging and screening
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