The complexity of neuronal circuits in the brain has long piqued scientific curiosity, driving innovations in imaging methodologies. Among the techniques that have revolutionized our understanding of neuronal activity are genetically encoded voltage indicators (GEVIs). These tools are essential for visualizing the electrical signals that underlie communication between neurons. Yet, as researchers delve deeper into the mechanisms of the brain, the choice between one-photon (1P) and two-photon (2P) voltage imaging has sparked significant debate. A recent study conducted by researchers at Harvard University offers critical insights into the comparison of these two imaging modalities, elucidating both their strengths and weaknesses.
In a thorough exploration published in Neurophotonics, the Harvard team meticulously evaluated the performance of 1P and 2P voltage imaging. Their research was rooted in understanding the optical and biophysical limitations inherent to each technique. The team examined various GEVIs to assess their brightness and voltage sensitivity, fundamental metrics that determine imaging efficacy. An essential part of their approach included measuring how fluorescence signals attenuate with increasing depth in the mouse brain—an aspect vital for in vivo imaging applications.
The researchers’ innovative methodology involved creating a predictive model that assesses the number of detectable cells based on three critical factors: the properties of the reporter, the imaging parameters, and the expected signal-to-noise ratio (SNR). This analytical framework enabled them to benchmark the two techniques under comparable conditions.
One particularly striking outcome of the study is the revelation that achieving equivalent photon counts in 2P imaging necessitates approximately 10,000 times more illumination power per cell compared to 1P imaging. This discrepancy raises significant concerns regarding the practical application of 2P voltage imaging, especially the risks of tissue photodamage and elevated shot noise. For instance, utilizing the JEDI-2P sensor in the mouse cortex revealed limitations; under realistic experimental conditions, 2P imaging could monitor only around 12 neurons at depths exceeding 300 micrometers with a target SNR of 10.
This limitation poses substantial challenges for neuroscientists aiming to capture robust data from multiple neurons simultaneously, particularly when exploring deeper brain regions. Consequently, both the stringent photon-count requirements and the relatively low sensitivity of current GEVIs render 2P voltage imaging a daunting task in living organisms.
The Harvard study encapsulates the intricate balance between the scalability of 1P and 2P methods while underscoring the pressing need for innovation in this field. As the demand for high-resolution understanding of neural circuitry intensifies, the current limitations of 2P imaging cannot be overlooked. Achieving satisfactory SNR across hundreds of neurons located at considerable depths will necessitate either significant enhancements in existing GEVIs or a pivot towards novel imaging techniques.
While 1P imaging continues to offer various advantages, the pursuit of enhanced 2P imaging capabilities is crucial for advancing our grasp of complex neural interactions. The findings from this pivotal study provide a roadmap for future research endeavors, guiding scientists toward the development of more effective imaging technologies and, ultimately, a deeper understanding of brain function. The journey of unraveling the mysteries of neural circuits remains a challenging yet essential pursuit in neuroscience.