Widefield Targeted Illumination Microscopy Enables Optically‐Sectioned, Motion‐Resilient Imaging of Neuronal Fibers and Their Dynamics

In widefield fluorescence imaging of neurons, out‐of‐focus and scattered light from the bright cell body often obscures nearby dim fibers and degrades their contrast. Scanning techniques can solve this problem but are limited by reduced imaging speed and increased cost. In this study, stray light in widefield imaging is greatly reduced by modulating the illumination intensity to different structures. An iterative approach is used to identify fibers by real‐time image processing and target illumination to fibers by a digital micromirror device add‐on to a common widefield microscope. Bright cell bodies are illuminated with minimal light intensity, and in‐focus fibers with high light intensity. This procedure minimizes the background and enhances the visibility of fibers while maintaining a fast imaging speed, low photobleaching rate, and low cost. By updating the targeting pattern, illumination is maintained on the structures of interest, even in moving samples. Using this targeted illumination approach, high contrast, optically sectioned imaging of complex neurons is demonstrated in anesthetized C. elegans , ex vivo mouse brain slice, and restrained zebrafish larva, as well as high‐speed imaging of dynamic changes in C. elegans .