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.
Highlights
There are major efforts in different areas of biology and neuroscience to image volumes up to the level of whole organisms and brains at high spatial and temporal resolution [1,2,3,4,5,6,7,8]
In addition we provide a simple strategy for minimizing the effects of tissue light scattering in light-sculpting microscopy and quantify its performance in Ca2+-imaging experiments
This further highlights the advantages of our high-speed temporal focusing (TeFo) techniques for fast functional imaging, demonstrating sufficient signal-to-noise and temporal resolution to record dynamic Ca2+-signals
Summary
There are major efforts in different areas of biology and neuroscience to image volumes up to the level of whole organisms and brains at high spatial and temporal resolution [1,2,3,4,5,6,7,8]. It has remained a challenge to functionally image large volumes at single cell resolution and physiological time scales. Given that in almost all current imaging techniques, there is a more or less straightforward tradeoff between the imaging speed, spatial resolution, volume size and shape, signal strength and imaging duration, it is important to choose these parameters appropriately for the biological question in mind. We discuss the details of such tradeoffs for the emerging technique of light sculpting based on temporal focusing and provide guidelines for optimally choosing various imaging parameters under different conditions and available laser sources. In addition we provide a simple strategy for minimizing the effects of tissue light scattering in light-sculpting microscopy and quantify its performance in Ca2+-imaging experiments
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