Advancements in Voltage Imaging: Evaluating One-Photon and Two-Photon Techniques

In the intricate world of neuroscience, understanding how neurons communicate and process information hinges on advancements in imaging technologies. Recently, genetically encoded voltage indicators (GEVIs) have emerged as pivotal tools for visualizing electrical activity within brain circuits. By allowing scientists to observe voltage changes in real-time, these indicators present an unprecedented opportunity to explore neuronal dynamics. However, a looming question remains: Are one-photon (1P) or two-photon (2P) voltage imaging techniques more effective in capturing this vital data?

An important study conducted by Harvard University researchers has sought to dissect the advantages and drawbacks inherent to both 1P and 2P imaging modalities. Published in Neurophotonics, this research meticulously compares these two methodologies with an emphasis on their optical and biophysical limitations. By examining parameters such as brightness, voltage sensitivity of commonly utilized GEVIs, and the fluorescence decay as depth increases within the mouse brain, the study sheds light on the practical exports of each technique.

The research team’s innovative modeling approach allows them to predict how many cells can be effectively tracked under given imaging conditions. This model considers the unique properties of different GEVIs, imaging parameters, and signal-to-noise ratio (SNR) requirements. Such an analysis is critical for refining our understanding of how voltage imaging can be optimized for in vivo brain studies.

A striking conclusion of the study highlights the significant power disparity between the two imaging methods. When using 2P excitation, researchers found that approximately 10,000 times more illumination power per cell is needed compared to the requirements for 1P excitation to achieve equivalent photon counts. This disparity brings to light inherent challenges, notably concerning tissue photodamage and detectability due to shot noise.

For example, when employing the JEDI-2P indicator within the mouse cortex, the constraints around laser power limit the feasible data collection from neurons at greater depths—often restricting registration to only a small subset of neurons. With parameters set to achieve a SNR of 10, bandwidth of 1 kHz, and a laser power cap at 200 mW, the ability to conduct successful 2P imaging becomes even more laborious, often confining the breadth of study to around a dozen neurons.

This research underscores both the complexities and limitations faced in the realm of voltage imaging, particularly when utilizing 2P methods in vivo. The current generation of GEVIs presents challenges related to their voltage sensitivity and photon counting efficacy, making it evident that researchers must push for innovations that expand the capabilities of voltage imaging. In-depth explorations can lead to designs of enhanced 2P GEVIs or even entirely novel imaging systems that may revolutionize the way in which neural circuits are studied.

Ultimately, by juxtaposing the strengths and weaknesses of 1P and 2P voltage imaging, this study serves as a critical pivot point for the scientific community. It highlights the ongoing struggle faced by researchers as they endeavor to capture the intricate dance of neuronal interactions—an endeavor that, with continued technological evolution, may one day unveil profound insights into the brain’s workings.