Abstract
We have configured a widefield fast imaging system that allows imaging at 1000 frames per second (512x512 pixels). The system was extended with custom processing tools including a time correlation method to facilitate the analysis of static subcellular compartments (e.g. neuronal varicosities) with enhanced contrast, as well as a dynamic intensity processing (DIP) algorithm that aids in data size reduction and fast visualization and interpretation of timing and directionality in neuronal circuits. This system, together with our custom developed processing tools enables efficient detection of fast physiological events, such as action potential dependent calcium steps. We show, using a specific blocker of nerve communication, that with this setup it is possible to discriminate between a pre and post synaptic event in an all optical way.
Highlights
To capture all facets of cellular communication in an optical way, it is of utmost importance to record with high temporal resolution as many biological interactions occur faster than the human eye can see or a classic camera can capture
Action potential firing and the consecutive intracellular calcium signaling events in neurons, which occur at the millisecond timescale, is a typical example of such fast cellular signaling
Live fluorescence microscopy is a powerful and heavily used tool in biomedical research mainly because it yields an optical readout from many cells simultaneously, which creates the possibility to visualize cellular interactions and circuits
Summary
To capture all facets of cellular communication in an optical way, it is of utmost importance to record with high temporal resolution as many biological interactions occur faster than the human eye can see or a classic camera can capture. In this study we report the successful development of a microscopy configuration based on a widefield approach with a dual camera system that allows recording images both at fast (up to 2000 Hz) and standard (2-10 Hz) frame rates with sufficient spatial resolution. With this setup it is possible to resolve individual neurotransmitter release sites and record their activity as they operate in a neuronal network. The approach yields high resolution feedback from sub-millisecond stimuli and allows calculating the propagation speed of the calcium wave front
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