Rapid volumetric optoacoustic imaging of neural dynamics across the mouse brain

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Abstract

Ongoing efforts to scale neuroimaging towards direct visualization of mammalian brain-wide neural activity face major challenges, and a large gap still exists between localized optical microscopy looking at rapid cellular-resolved neuronal activities and whole-brain observations of slow hemodynamics and metabolism provided by macroscopic imaging modalities. Optoacoustic imaging holds inherent advantages for deep tissue observations, but to date has not been applied towards direct activity observation in the mammalian brain. Here we demonstrate in vitro and in vivo functional optoacoustic neuroimaging from mice expressing the genetically encoded calcium indicators GCaMP6, effectively bridging the gap between functional microscopy and whole-brain macroscopic neuroimaging. We yielded instantaneous high-resolution 3D snapshots of whole-brain activity maps with single optoacoustic excitations and enabled non-invasive detection of fast neural responses to sensory stimuli in the presence of strong hemoglobin background absorption. These results demonstrate a new enabling technique towards scalable direct neuroimaging at unprecedented penetration depths and spatio-temporal resolutions.

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Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics

Jasper Akerboom, Nicole Carreras Calderón, Lin Tian … (2013)

Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Here we describe red, single-wavelength GECIs, “RCaMPs,” engineered from circular permutation of the thermostable red fluorescent protein mRuby. High-resolution crystal structures of mRuby, the red sensor RCaMP, and the recently published red GECI R-GECO1 give insight into the chromophore environments of the Ca2+-bound state of the sensors and the engineered protein domain interfaces of the different indicators. We characterized the biophysical properties and performance of RCaMP sensors in vitro and in vivo in Caenorhabditis elegans, Drosophila larvae, and larval zebrafish. Further, we demonstrate 2-color calcium imaging both within the same cell (registering mitochondrial and somatic [Ca2+]) and between two populations of cells: neurons and astrocytes. Finally, we perform integrated optogenetics experiments, wherein neural activation via channelrhodopsin-2 (ChR2) or a red-shifted variant, and activity imaging via RCaMP or GCaMP, are conducted simultaneously, with the ChR2/RCaMP pair providing independently addressable spectral channels. Using this paradigm, we measure calcium responses of naturalistic and ChR2-evoked muscle contractions in vivo in crawling C. elegans. We systematically compare the RCaMP sensors to R-GECO1, in terms of action potential-evoked fluorescence increases in neurons, photobleaching, and photoswitching. R-GECO1 displays higher Ca2+ affinity and larger dynamic range than RCaMP, but exhibits significant photoactivation with blue and green light, suggesting that integrated channelrhodopsin-based optogenetics using R-GECO1 may be subject to artifact. Finally, we create and test blue, cyan, and yellow variants engineered from GCaMP by rational design. This engineered set of chromatic variants facilitates new experiments in functional imaging and optogenetics.

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Thy1-GCaMP6 Transgenic Mice for Neuronal Population Imaging In Vivo

Hod Dana, Tsai-Wen Chen, Amy Hu … (2014)

Genetically-encoded calcium indicators (GECIs) facilitate imaging activity of genetically defined neuronal populations in vivo. The high intracellular GECI concentrations required for in vivo imaging are usually achieved by viral gene transfer using adeno-associated viruses. Transgenic expression of GECIs promises important advantages, including homogeneous, repeatable, and stable expression without the need for invasive virus injections. Here we present the generation and characterization of transgenic mice expressing the GECIs GCaMP6s or GCaMP6f under the Thy1 promoter. We quantified GCaMP6 expression across brain regions and neurons and compared to other transgenic mice and AAV-mediated expression. We tested three mouse lines for imaging in the visual cortex in vivo and compared their performance to mice injected with AAV expressing GCaMP6. Furthermore, we show that GCaMP6 Thy1 transgenic mice are useful for long-term, high-sensitivity imaging in behaving mice.

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Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms

Matthew Bouchard, Venkatakaushik Voleti, César Mendes … (2015)

We report a new 3D microscopy technique that allows volumetric imaging of living samples at ultra-high speeds: Swept, confocally-aligned planar excitation (SCAPE) microscopy. While confocal and two-photon microscopy have revolutionized biomedical research, current implementations are costly, complex and limited in their ability to image 3D volumes at high speeds. Light-sheet microscopy techniques using two-objective, orthogonal illumination and detection require a highly constrained sample geometry, and either physical sample translation or complex synchronization of illumination and detection planes. In contrast, SCAPE microscopy acquires images using an angled, swept light-sheet in a single-objective, en-face geometry. Unique confocal descanning and image rotation optics map this moving plane onto a stationary high-speed camera, permitting completely translationless 3D imaging of intact samples at rates exceeding 20 volumes per second. We demonstrate SCAPE microscopy by imaging spontaneous neuronal firing in the intact brain of awake behaving mice, as well as freely moving transgenic Drosophila larvae.