BLIMPS: a technique for tandem biosensor imaging across multiple populations of presynaptic terminals, using lattice light sheet microscopy

Within neuronal circuits, ordered neurotransmission is contingent upon balance between excitatory glutamatergic and inhibitory GABAergic signaling. To study circuit-level processes, the paradigm of 4D cellular physiology has been developed, where, single cells and subcellular structures are studied as individual units in three-dimensional space over a continuous interval rather than as a single moment in time, or as a population-level average. Neurons are excitable cells expressing voltage-gated Ca²⁺ channels and Ca²⁺ fluxes subsequent to action potential firing are widely used as markers of neuronal activity. While the imaging of Ca²⁺ dynamics at the soma is often performed, the imaging of Ca²⁺ fluxes at presynaptic terminals has often proven to be an experimental challenge: existing imaging modalities suffer from inadequate acquisition speeds, insufficient penetration depths, insufficient spatial resolution to identify axonal structures, or spectral crosstalk issues. To visualize presynaptic Ca²⁺ dynamics in both excitatory and inhibitory neurons, here we combine advanced lattice light-sheet microscopy with viral delivery of two genetically encoded calcium indicators (GECIs)- jRGECO1a and jGCaMP8f, to perform sequential imaging of Ca²⁺ dynamics within acute <i>ex vivo</i> slice preparations. Our methodology, Biosensor Lattice light-sheet Imaging of Multidimensional Presynaptic Structure (BLIMPS), includes acute brain slice preparation, mounting on a temperature-controlled flow chamber within a LLSM, and imaging of electrically evoked Ca²⁺ signals, with high adaptability to a range of genetic and pharmacological disease models. Our technique offers high spectral separation between evoked signals from each of the two GECIs and fast acquisition speeds of 0.1-0.3 KHz. Included within the BLIMPS technique is a robust, open-source data analysis pipeline to track highly responsive neuronal structures such as presynaptic terminals and quantify both the amplitudes and decay rates of evoked fluxes.