131 — 50 Hz volumetric functional imaging with continuously adjustable depth of focus

One critical bottleneck in connectomics research is imaging functional data at high spatiotemporal resolution. Often, methods with high spatial acuity — such as electron microscopy — have no temporal information, and methods with high temporal resolution, such as light microscopy, lack spatial acuity.

One very potent and very popular compromise is two-photon light-sheet micrscopy, but even 2PLSM suffers from low temporal resolution because it “scans” its focus through a volume of tissue serially.

This newly proposed system addresses this limitation by providing a lens control which can be adjusted along its axis to change the Bessel focus to a desired state during image recording. The authors used this new technology, dubbed “Continuously Adjustable Depth of Focus”, or CADoF, to image zebrafish spinal projection neurons in live zebrafish larvae.

Using a conventional Gaussian focus, the zebrafish images required 28 distinct, discrete image slices. In contrast, the new CADoF technology enabled the visualization of approximatly 3× as many neurons all simultaneously, without having to adjust the Bessel focus.

This technique enables the visualization and differentiation of labeled neurons at a variety of scales; densely labeled neurons benefit from a short Bessel focus, whereas larger, sparsely labeled volumes can take advantage of a longer Bessel focus.

This is a very exciting technique because it will enable the in vivo exploration of full neural circuits of labeled neurons in realtime, rather than relying on scanning Gaussian methods that are lossier and less reliable.