Kiryl Piatkevich
Molecular BioEngineering Group, Westlake University
Abstract
Holistic understanding of the brain, from intracellular processes to cell-cell interactions across the whole organ, will require integrated methodologies that can simultaneously map neuronal computations on both functional and structural levels in intact brain tissue. Kiryl Piatkevich will present the development and validation of the toolset of advanced imaging techniques for mapping functional and structural computations in the brain of the model species. For functional dissection of local neuronal circuits, we developed and validated an opsin-derived voltage sensor, named Archon. Archon is characterized by submillisecond temporal resolution (~0.6 ms), high voltage sensitivity (40% of ΔF/F per action potential), no photocurrent and high photostability allowing for long-term (over 5 min) imaging of neuronal activity with no detectable phototoxicity. Archon exhibits excellent localization upon long-term expression in vivo and allowed for a single spike detection in multiple neurons in behaving animals, such as C.elegans, zebrafish, and mice. For the whole-brain functional imaging, we developed a cell body-targeted variant of the fluorescent calcium indicators of the GCaMP family, called SomaGCaMPs. In the densely labeled brain tissue, SomaGCaMPs overcome major problems of non-targeted GCaMP sensors by reducing crosstalk between cell bodies and the surrounding neuropil. One-photon imaging of soma-targeted GCaMP6f in densely labeled neural circuits reported fewer artifactual spikes from neuropil, increased signal-to-noise ratio, and decreased artifactual correlation across neurons. Thus, soma-targeting of fluorescent calcium indicators increases neuronal signal fidelity and may facilitate even greater usage of simple, powerful, one-photon methods of population imaging of neural calcium dynamics. For the structural mapping of neuronal circuits in the brain tissue, we modified the recently developed expansion microscopy technique, a method that enables 3D super-resolution imaging of thick biological samples, to retain intrinsic signals from fluorescent proteins and conventional fluorescently labeled secondary antibodies, as well as preserve RNA molecules enabling their staining with FISH probes. Using improved ExM protocol, we demonstrate multi-color super-resolution (~70 nm) imaging of neurons in the brain tissue using conventional confocal microscope. Altogether, we have developed molecular and imaging technologies that are compatible with each other, allowing dynamic and structural imaging to be performed on the same subset of neurons. This can eventually enable comprehensive mapping of brain circuits at the activity level (i.e., dynomics) and the anatomical level (i.e., connectomics).