Fast optical recording of neuronal activity by three-dimensional custom-access serial holography.

Optical recording of neuronal activity in three-dimensional (3D) brain circuits at cellular and millisecond resolution in vivo is essential for probing information flow in the brain. While random-access multiphoton microscopy permits fast optical access to neuronal targets in three dimensions, the method is challenged by motion artifacts when recording from behaving animals. Therefore, we developed three-dimensional custom-access serial holography (3D-CASH). Built on a fast acousto-optic light modulator, 3D-CASH performs serial sampling at 40 kHz from neurons at freely selectable 3D locations. Motion artifacts are eliminated by targeting each neuron with a size-optimized pattern of excitation light covering the cell body and its anticipated displacement field. Spike rates inferred from GCaMP6f recordings in visual cortex of awake mice tracked the phase of a moving bar stimulus with higher spike correlation between intra compared to interlaminar neuron pairs. 3D-CASH offers access to the millisecond correlation structure of in vivo neuronal activity in 3D microcircuits.

Single Molecule Imaging in Live Embryos Using Lattice Light-Sheet Microscopy.

In the past decade, live-cell single molecule imaging studies have provided unique insights on how DNA-binding molecules such as transcription factors explore the nuclear environment to search for and bind to their targets. However, due to technological limitations, single molecule experiments in living specimens have largely been limited to monolayer cell cultures. Lattice light-sheet microscopy overcomes these limitations and has now enabled single molecule imaging within thicker specimens such as embryos. Here we describe a general procedure to perform single molecule imaging in living Drosophila melanogaster embryos using lattice light-sheet microscopy. This protocol allows direct observation of both transcription factor diffusion and binding dynamics. Finally, we illustrate how this Drosophila protocol can be extended to other thick samples using single molecule imaging in live mouse embryos as an example.

Switch-like Arp2/3 activation upon WASP and WIP recruitment to an apparent threshold level by multivalent linker proteins in vivo.

Actin-related protein 2/3 (Arp2/3) complex activation by nucleation promoting factors (NPFs) such as WASP, plays an important role in many actin-mediated cellular processes. In yeast, Arp2/3-mediated actin filament assembly drives endocytic membrane invagination and vesicle scission. Here we used genetics and quantitative live-cell imaging to probe the mechanisms that concentrate NPFs at endocytic sites, and to investigate how NPFs regulate actin assembly onset. Our results demonstrate that SH3 (Src homology 3) domain-PRM (proline-rich motif) interactions involving multivalent linker proteins play central roles in concentrating NPFs at endocytic sites. Quantitative imaging suggested that productive actin assembly initiation is tightly coupled to accumulation of threshold levels of WASP and WIP, but not to recruitment kinetics or release of autoinhibition. These studies provide evidence that WASP and WIP play central roles in establishment of a robust multivalent SH3 domain-PRM network in vivo, giving actin assembly onset at endocytic sites a switch-like behavior.