  A number of questions in systems 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 organism. However, the broader applicability of light-sculpting
microscopy 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.
