Brain function originates from and relies on electrical signals generated and propagated by neurons. Therefore, scientists measure electrical signals to study neuron function at the single cell and system levels. Researchers are looking for techniques that will allow them to study more deeply the diversity of neurons and the complexity of brain networks.
In this Innovation Spotlight, Bruker senior product and technology manager Jimmy Fong discusses voltage imaging, a new strategy that can detect and visualize tiny voltage changes within cells.
Jimmy Fang
Senior Product and Technology Manager
Brooke
What is voltage imaging?
Fluorescence voltage imaging is an emerging method in neuroscience research that can visualize neural activity by detecting small voltage changes in cells. Many advances in this technology have been accelerated by the development of fluorescent voltage indicators, which have significantly improved their brightness, stability, and responsiveness to voltage changes. These indicators come in the form of dyes that are injected into samples or genetically encoded in animals, and can then be imaged using microscopes capable of imaging voltage signals. Microscopy instrumentation also needs to advance to take advantage of new developments in probes. These indicators are now able to report millisecond events from many cells simultaneously, which also requires microscopy techniques with sufficient imaging frequency and sensitivity to capture the data. This was our goal when creating the OptoVolt mod.
How does voltage imaging address the limitations of traditional microscopy techniques?
Measuring neuronal voltage signals has traditionally been accomplished through electrophysiological techniques. Highly skilled scientists performing patch-clamp experiments can use glass electrodes to record the rapid electrical activity of one or a few cells at a time. However, recording from multiple units simultaneously is difficult using this strategy.
Scientists also use fluorescent calcium imaging to measure neural activity. Here, calcium probes (e.g. GCaMP) are expressed in the sample to visualize the activity of large numbers of cells. Imaging through calcium has unlocked many discoveries, but the technique relies on calcium influx into cells and subsequent binding to fluorescent reporters. This process is slow, making it a delayed proxy for the underlying voltage signal. To this end, voltage imaging has the potential to combine the advantages of both techniques.
How does imaging frequency affect voltage imaging?
Measuring the action potentials of multiple cells simultaneously helps scientists collect more data and identify multicellular networks.
The duration of neuronal voltage “spikes,” or action potentials, in mouse brains can be as short as a few milliseconds. To capture this, the imaging frequency needs to be high enough, otherwise the event may be missed.
One way to achieve higher imaging frequencies is to use newer, faster cameras with higher frame rates. When developing OptoVolt, we chose a different approach to achieve higher frequencies because we built the module into a two-photon microscope. Using two-photon microscopy, we scan a near-infrared laser spot and detect it using a sensitive photomultiplier tube (PMT). This enables deeper imaging compared to camera-based methods. Working with collaborators at Boston University, we created the ability to combine a high-speed resonant scanning mirror with an array of microlenses to guide a laser spot faster than previously possible.
With higher frame rates, researchers can capture not only the fast voltage dynamics of cells, but also the rapid dynamics of other biological processes, including neurotransmitter activity, blood flow, movement, and more.
Where is voltage imaging best suited?
Although much of the excitement in voltage imaging is in neuroscience research, cells other than neurons also exhibit voltage activity. For example, cardiomyocytes in heart tissue could potentially exploit these developments.
What applications does OptoVolt support or improve?
We created OptoVolt as a modular complement to our two- and three-photon microscopes called Ultima 2Pplus. This microscope is widely used in neuroimaging experiments, where deep in vivo animal imaging of brain activity is combined with behavioral measurements and optogenetic light stimulation of neurons. Many of our collaborators work on large-scale neural networks, and with the release of OptoVolt modules and improved probes, scientists hope to continue their work with higher temporal resolution. We are particularly excited about the potential of this work combined with optogenetics, where voltage imaging and light stimulation can provide avenues to study real input-output connections in the brain.
