Neurons and neural networks often extend hundreds of micrometers in three dimensions. Capturing the calcium transients associated with their activity requires volume imaging methods with subsecond temporal resolution. Such speed is a challenge for conventional two-photon laser-scanning microscopy, because it depends on serial focal scanning in 3D and indicators with limited brightness. Here we present an optical module that is easily integrated into standard two-photon laser-scanning microscopes to generate an axially elongated Bessel focus, which when scanned in 2D turns frame rate into volume rate. We demonstrated the power of this approach in enabling discoveries for neurobiology by imaging the calcium dynamics of volumes of neurons and synapses in fruit flies, zebrafish larvae, mice and ferrets in vivo. Calcium signals in objects as small as dendritic spines could be resolved at video rates, provided that the samples were sparsely labeled to limit overlap in their axially projected images.
Video-rate volumetric functional imaging of the brain at synaptic resolution
Example application of Bessel focus scanning to a volume of GCaMP6s+ neurites in awake mouse cortex at 30 Hz. (c) Mean intensity projection of a 60-μm-thick image stack collected with Gaussian focus scanning at 1-μm z steps, with structures color-coded by depth.
Max Planck Florida Institute for Neuroscience
