Optimizing and extending light-sculpting microscopy for fast functional imaging in neuroscience

Abstract

A number of questions in system biology such as understanding how dynamics of neuronal networks are related to brain function require the ability to capture the functional dynamics of large cellular populations at high speed. Recently, this has driven the development of a number of parallel and high speed imaging techniques such as light-sculpting microscopy, which has been used to capture neuronal dynamics at the whole brain and single cell level in small model organisms. However, the broader applicability of light-sculpting microcopy is limited by the size of volumes for which high speed imaging can be obtained and scattering in brain tissue. Here, we present strategies for optimizing the present tradeoffs in light-sculpting microscopy. Various scanning modalities in light-sculpting microscopy are theoretically and experimentally evaluated, and strategies to maximize the obtainable volume speeds, and depth penetration in brain tissue using different laser systems are provided. Design-choices, important parameters and their trade-offs are experimentally demonstrated by performing calcium-imaging in acute mouse-brain slices. We further show that synchronization of line-scanning techniques with rolling-shutter read-out of the camera can reduce scattering effects and enhance image contrast at depth. Rózsa, "Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes," Nat. Methods 9(2), 201-208 (2012). 6. G. Duemani Reddy, K. Kelleher, R. Fink, and P. Saggau, "Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity," Nat. Neurosci. 11(6), 713-720 (2008). 7. R. Prevedel, Y.-G. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. ©2015 Optical Society of AmericaZimmer, E. S. Boyden, and A. Vaziri, "Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy," Nat. Methods 11(7), 727-730 (2014 19645-19655 (2010)

A Guide to Emerging Technologies for Large-Scale and Whole-Brain Optical Imaging of Neuronal Activity

“…To improve this, the synchronization of a line-shaped illumination with the rolling shutter readout of modern CMOS cameras can be exploited (Baumgart & Kubitscheck 2012, Spiecker 2011). Our laboratory has demonstrated the full potential of this method by combining the line-scanning acquisition scheme with light sculpting using TeFo (Rupprecht et al 2015). We could demonstrate high-speed planar imaging of 200 x 200 mu; m FOV at 75 fps down to a depth of 75 μ m in scattering brain tissue.…”

Rapid adaptive remote focusing microscope for sensing of volumetric neural activity

“…System performance in SNR-limited samples can be further optimized by refining the illumination pattern to obtain more efficient time-averaged two photon emission [ 30 ]. Widefield detection of fluorescence can lead to increased background signal when imaging deeper through scattering tissue or in even more scattering samples.…”

High speed functional imaging with source localized multifocal two-photon microscopy

“…We generated the PSF using Monte Carlo simulation in a similar manner to [ 31 ] by simulating the propagation of photons from a point source at ( x , y , z ) = 0 through a slab of scattering tissue extending from 0 ≤ z ≤ z h . We simulated photons with an initial propagation angle drawn from a uniform distribution over the elevation and azimuthal angles corresponding to a NA of 1 to match the objective used in this experiment.…”

“…The photons were propagated through the tissue for a length drawn from an exponential distribution with a characteristic scattering length μ = 20 mm −1 [ 31 ]. If z < z h , we simulated a scattering event by drawing a new propagation angle from a uniform distribution between 0 and 2 π in azimuth and from a Henyey-Greenstein phase function [ 32 ] with a forward scattering anisotropy of g = 0.9 in elevation [ 31 ]. The propagation and scattering steps were then repeated until the photon left the tissue.…”