Fast 3D Imaging of Spine, Dendritic, and Neuronal Assemblies in Behaving Animals

Abstract

SummaryUnderstanding neural computation requires methods such as 3D acousto-optical (AO) scanning that can simultaneously read out neural activity on both the somatic and dendritic scales. AO point scanning can increase measurement speed and signal-to-noise ratio (SNR) by several orders of magnitude, but high optical resolution requires long point-to-point switching time, which limits imaging capability. Here we present a novel technology, 3D DRIFT AO scanning, which can extend each scanning point to small 3D lines, surfaces, or volume elements for flexible and fast imaging of complex structures simultaneously in multiple locations. Our method was demonstrated by fast 3D recording of over 150 dendritic spines with 3D lines, over 100 somata with squares and cubes, or multiple spiny dendritic segments with surface and volume elements, including in behaving animals. Finally, a 4-fold improvement in total excitation efficiency resulted in about 500 × 500 × 650 μm scanning volume with genetically encoded calcium indicators (GECIs).

Wide-area all-optical neurophysiology in acute brain slices

“…Point-scanning based mesoscopes have achieved pixel rates of ~2×10 7 /s over 0.6 × 0.6 mm FOVs but the requirement to translate the beam long distances limits pixel rates over large FOVs (4.4 × 4.2 mm) to 5.6×10 6 /s. Acousto-optical steering allows fast 2P random-access imaging 38 For precisely targeted single-cell stimulation, 2P optics are essential, but for wide-area optogenetic stimulation, 1P optics are preferable, as follows: 2P optogenetic stimulation requires timeaverage optical powers of 20 -80 mW/cell, [2][3][4] . Maximal safe steady-state 2P optical power into intact brain tissue is ~200 mW 39 , limiting simultaneous 2P stimulation to at most a few tens of neurons at a time.…”

Deformable mirror-based two-photon microscopy for axial mammalian brain imaging

“…Because their settling times are on the order of a few milliseconds, these technologies are not fast enough to capture relevant in vivo depth-imaging with high z-resolution, limiting their use to in neural physiological imaging only a few defined z planes. Other systems use a high-speed axial scanner such as an acoustic-optical scanner [15]or an ultrasound-driven lens [16], but they can suffer from focusing aberrations. Another solution to imaging in depth is the technique of implanting microprisms in the brain to image orthogonal to the skull surface while still using a traditional xy scanner [17,18].…”

Locomotion-Related Population Cortical Ca2+ Transients in Freely Behaving Mice

“…The major advancement in measuring Ca 2+ signals was the invention and the application of two-photon microscopy in the nervous system (Denk et al, 1990; Yuste and Denk, 1995). Over many years, particularly with the help of the continuous development of Ca 2+ indicators, two-photon Ca 2+ imaging has become widely used for detecting neural activities on multiple scales ranging from networks to single synapses in both anesthetized and behaving animals (Stosiek et al, 2003; Chen et al, 2011, 2013; Nadella et al, 2016; Szalay et al, 2016). Another commonly used approach for in vivo brain Ca 2+ imaging is based on the use of charged coupled detector/complementary metal-oxide-semiconductor-based cameras, which are particularly useful for recording large-field Ca 2+ dynamics in the superficial cortical layers (Berger et al, 2007).…”