SUMMARY
A major goal of the BRAIN initiative is to develop neural imaging technologies enabling whole-brain imaging of
specific molecular signals such as neuronal calcium. Currently, fluorescent genetically encoded calcium
indicators (GEGIs) combined with advanced microscopy techniques enable single-neuron Ca2+ imaging in
volumes smaller than 1 mm3, typically at depths shallower than 1 mm. Alternatively, GECIs combined with
implanted fiber photometry enable point-measurements of the aggregate activity of genetically defined neuronal
populations in deep brain regions with dimensions on the order of 200 µm. While both of these optical approaches
have proven their great value in enabling neuroscience discoveries, they fall short of providing simultaneous
whole-brain access to neural signals. If a technology could be developed for whole-brain Ca2+ imaging it would
have a transformative impact on neuroscience research. In this project, we will address this ambitious goal by
developing ultrasonic genetically encoded calcium indicators (UGECIs) and showing that we can use them to
image brain-wide calcium in mice. Ultrasound has unique advantages as a modality for neural imaging due to its
ability to penetrate much deeper than light (several cm) while providing relatively high spatial (tens of µm) and
temporal (ms) resolution. This potential has been demonstrated by hemodynamic functional ultrasound (fUS),
which uses ultrafast Doppler imaging of blood flow to visualize neural activity with 100 µm and 100 ms resolution.
fUS has shown that ultrasound imaging of neural activity is possible in species ranging from mice to humans and
is compatible with awake, behaving, freely moving animals. However, hemodynamics provide only an indirect
measure of neural activity. In contrast, the measurement of calcium with GECIs provides access to a molecular
signal integral to neuronal excitation, and allows the probing of specific cellular populations. This project will
combine the whole-brain coverage of ultrasound with the molecular and genetic specificity of GECIs by
developing the ultrasound versions of these tools. Our proposal to develop UGECIs arises from a long-standing
research program in our lab to develop the first genetically encoded reporters and sensors for ultrasound. These
constructs are based on gas vesicles (GVs), a unique class of genetically encoded air-filled protein
nanostructures derived from buoyant bacteria, which we discovered are capable of scattering sound waves and
thereby producing ultrasound contrast. We recently showed that GVs can be engineered to incorporate
molecular binding domains allowing their protein shells to change their mechanical properties and resulting
ultrasound contrast in response to molecules such as calcium. Building on these advances, we will develop
UGECIs, express them in the mouse brain and use them to image brain-wide calcium signals using new
ultrasound techniques allowing rapid 2-dimensional and 3-dimensional molecular imaging. The resulting
technology will provide neuroscience researchers with revolutionary capabilities for whole-brain molecular neural
imaging.