Abstract
Understanding how neural circuits control behavior requires monitoring a large population of neurons with high spatial resolution and volume rate. Here we report an axicon-based Bessel beam module with continuously adjustable depth of focus (CADoF), that turns frame rate into volume rate by extending the excitation focus in the axial direction while maintaining high lateral resolutions. Cost-effective and compact, this CADoF Bessel module can be easily integrated into existing two-photon fluorescence microscopes. Simply translating one of the relay lenses along its optical axis enabled continuous adjustment of the axial length of the Bessel focus. We used this module to simultaneously monitor activity of spinal projection neurons extending over 60 µm depth in larval zebrafish at 50 Hz volume rate with adjustable axial extent of the imaged volume.
Highlights
Understanding how neural networks integrate inputs and generate outputs is a central goal of neuroscience
Capturing calcium dynamics of all labeled neurons in a volume within tens of milliseconds is challenging for 2PLSM because it images a volume by serially scanning the excitation focus in 3D
The axicon-based continuously adjustable depth of focus (CADoF) module was built between a Ti-Sapphire laser (Mai Tai HP, Spectra-Physics) and a custom two-photon fluorescence microscope equipped with a resonant galvanometer and controlled by ScanImage2016 (Vidrio Technologies, LLC)
Summary
Understanding how neural networks integrate inputs and generate outputs is a central goal of neuroscience. Neurons within these networks are distributed in three dimensions (3D). Monitoring their activity dynamics requires an imaging modality capable of rapid volumetric rates. Two-photon laser-scanning microscopy (2PLSM), combined with calcium indicators, has become the gold standard tool for in vivo monitoring of neural activity [1,2,3]. Capturing calcium dynamics of all labeled neurons in a volume within tens of milliseconds is challenging for 2PLSM because it images a volume by serially scanning the excitation focus in 3D
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