Sensitivity optimization of a rhodopsin-based fluorescent voltage indicator

Neuron · 2023 · 128 citations

• Biophysics
• Biology
• Neuroscience

The ability to optically image cellular transmembrane voltages at millisecond-timescale resolutions can offer unprecedented insight into the function of living brains in behaving animals. Here, we present a point mutation that increases the sensitivity of Ace2 opsin-based voltage indicators. We use the mutation to develop Voltron2, an improved chemigeneic voltage indicator that has a 65% higher sensitivity to single APs and 3-fold higher sensitivity to subthreshold potentials than Voltron. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, although with lower baseline fluorescence. In multiple in vitro and in vivo comparisons with its predecessor across multiple species, we found Voltron2 to be more sensitive to APs and subthreshold fluctuations. Finally, we used Voltron2 to study and evaluate the possible mechanisms of interneuron synchronization in the mouse hippocampus. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability.

Large-scale voltage imaging in behaving mice using targeted illumination

iScience · 2021 · 47 citations

• Nanotechnology
• Chemistry
• Environmental science

. To perform high-speed voltage imaging of many neurons simultaneously over a large anatomical area, widefield microscopy remains an essential tool. However, the lack of optical sectioning makes widefield microscopy prone to background cross-contamination. We implemented a digital-micromirror-device-based targeted illumination strategy to restrict illumination to the cells of interest and quantified the resulting improvement both theoretically and experimentally with SomArchon expressing neurons. We found that targeted illumination increased SomArchon signal contrast, decreased photobleaching, and reduced background cross-contamination. With the use of a high-speed, large-area sCMOS camera, we routinely imaged tens of spiking neurons simultaneously over minutes in behaving mice. Thus, the targeted illumination strategy described here offers a simple solution for widefield voltage imaging of many neurons over a large field of view in behaving animals.

High-contrast multifocus microscopy with a single camera and z-splitter prism

Optica · 2020 · 60 citations

• Computer Science
• Artificial Intelligence
• Computer Science

Optical microscopy has been an indispensable tool for studying complex biological systems, but is often hampered by problems of speed and complexity when performing 3D volumetric imaging. Here, we present a multifocus imaging strategy based on the use of a simple z-splitter prism that can be assembled from off-the-shelf components. Our technique enables a widefield image stack to be distributed onto a single camera and recorded simultaneously. We exploit the volumetric nature of our image acquisition by further introducing a novel extended-volume 3D deconvolution strategy to suppress far-out-of-focus fluorescence background to significantly improve the contrast of our recorded images, conferring to our system a capacity for quasi-optical sectioning. By swapping in different z-splitter configurations, we can prioritize high speed or large 3D field-of-view imaging depending on the application of interest. Moreover, our system can be readily applied to a variety of imaging modalities in addition to fluorescence, such as phase-contrast and darkfield imaging. Because of its simplicity, versatility, and performance, we believe our system will be a useful tool for general biological or biomedical imaging applications.

Simultaneous multiplane imaging with reverberation two-photon microscopy

Nature Methods · 2020 · 116 citations

• Computer Science
• Artificial Intelligence
• Optics