Brain functions stem from and rely on electrical signals created and propagated by neurons. As such, scientists measure electrical signals to study neuronal function at both single-cell and systemic levels. Researchers are looking for technologies that let them delve deeper into neuronal diversity and brain network complexity.
In this Innovation Spotlight, Jimmy Fong, a senior products and technology manager at Bruker, discusses voltage imaging, a newer strategy that can detect and visualize small voltage changes within a cell.
Senior Products and Technology Manager
Bruker
What is voltage imaging?
Fluorescent voltage imaging is an emerging method in neuroscience research that enables visualization of neural activity by detecting small voltage changes in the cell. Many of the advances in this technique were accelerated by the development of fluorescent voltage indicators, which have improved dramatically in brightness, stability, and responsivity to voltage changes. The indicators come in the form of dyes injected into a specimen or genetically encoded in an animal, which can then be imaged with a microscope capable of imaging the voltage signals. Microscope instrumentation also needed to advance to leverage the new developments of the probes. The indicators are now able to report millisecond events from many cells simultaneously, which also necessitates microscope technology with sufficient imaging frequency and sensitivity to capture the data. This was our goal when creating the OptoVolt module.
How does voltage imaging address limitations of conventional microscopy techniques?
Measuring neuronal voltage signals has traditionally been done through electrophysiology techniques. Highly skilled scientists can use glass electrodes when performing patch-clamp experiments to record the fast electrical activity from one or a few cells at a time. However, it has been difficult to use this strategy to record from many cells simultaneously.
Scientists have also used fluorescent calcium imaging to measure neural activity. Here, calcium probes, such as GCaMP, are expressed in a specimen to visualize the activity of large populations of cells. Many discoveries have been unlocked with calcium imaging, but the technique depends on calcium influx into a cell and subsequent binding to a fluorescent reporter. This process is slow, making it a delayed proxy of the underlying voltage signal. To that end, voltage imaging has the potential to combine the advantages of both of these techniques.
How does imaging frequency affect voltage imaging?
Neuronal voltage “spikes,” or action potentials, in a mouse brain can be as short as a few milliseconds in duration. To capture this, the imaging frequency needs to be high enough, or the event could be missed.
One way to obtain higher imaging frequencies is to use newer and faster cameras capable of higher frame rates. While developing OptoVolt, we chose a different method to achieve higher frequencies because we built the module onto our two-photon microscope. With a two-photon microscope, we scan near-infrared laser spots and detect with a sensitive photomultiplier tube (PMT). This allows deeper imaging than camera-based approaches. Working with collaborators at Boston University, we created the ability to combine high-speed resonant scan mirrors with a series of microlenses to steer the laser spots faster than what was previously possible.
With these higher frame rates, researchers are not only able to capture fast voltage dynamics of cells, but also fast dynamics of other biological processes, including neurotransmitter activity, blood flow, locomotion, and others.
What fields are best served by voltage imaging?
Even though much of the excitement around voltage imaging is in neuroscience research, cells other than neurons also exhibit voltage activity. For example, cardiomyocytes in heart tissue can potentially take advantage of these developments.
What applications does OptoVolt enable or improve?
We created OptoVolt as a modular addition to our two- and three-photon microscope called the Ultima 2Pplus. This microscope is widely used in neuroimaging experiments where deep, live animal imaging of brain activity is combined, often with behavioral measurements and optogenetic photostimulation of neurons. Many of our collaborators study large-population neural networks, and with the release of the OptoVolt module and improved probes, the hope is that scientists will be able to continue their work at a higher temporal resolution. We are particularly excited about the potential to combine this work with optogenetics, where voltage imaging and photostimulation can provide an avenue to study true input-output connectivity in the brain.
