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Updated: Jun 17, 2025

Direct Imaging of Laser-driven Ultrafast Molecular Rotation

Direct Imaging of Laser-driven Ultrafast Molecular Rotation

Published on: February 4, 2017

Optical constraints on two-photon voltage imaging.

F Phil Brooks¹, Hunter C Davis¹, J David Wong-Campos¹

¹Harvard University, Department of Chemistry and Chemical Biology, Cambridge, Massachusetts, United States.

|August 14, 2024

Summary

This summary is machine-generated.

Two-photon (2P) voltage imaging in vivo requires significantly more illumination power than one-photon (1P) imaging, limiting simultaneous recordings to few neurons. Advancements in genetically encoded voltage indicators (GEVIs) or novel imaging techniques are needed for high-SNR 2P imaging of many neurons deep within the brain.

Keywords:

shot noise two-photon voltage imaging

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Area of Science:

Neuroscience
Biophysics
Optical Imaging

Background:

Genetically encoded voltage indicators (GEVIs) are crucial for in vivo neural circuit studies.
The comparative performance of one-photon (1P) and two-photon (2P) voltage imaging remains poorly understood.

Purpose of the Study:

To characterize the optical and biophysical constraints of 1P and 2P voltage imaging.
To compare the imaging properties of common GEVIs under both 1P and 2P excitation.

Main Methods:

Measured brightness and voltage sensitivity of GEVIs under 1P and 2P illumination.
Assessed fluorescence decay with depth in mouse brain tissue.
Developed a model for predicting measurable cells based on reporter properties, imaging parameters, and SNR.

Main Results:

2P excitation demands significantly higher illumination power per cell for comparable photon rates versus 1P.
With current technology, 2P voltage imaging in mouse cortex (1 kHz bandwidth, 200 mW laser, 500 μm depth, SNR 10) can image a maximum of ~100 neurons.

Conclusions:

Existing GEVIs exhibit modest voltage sensitivity, creating a trade-off between shot noise and photodamage in 2P voltage imaging.
Achieving high-SNR 2P imaging of hundreds of neurons at depth necessitates substantial improvements in GEVIs or innovative imaging approaches.