Real-time targeted illumination in widefield microscopy achieves confocal quality neuronal images

Abstract

Widefield fluorescence imaging has significant challenges in visualizing neuronal fibers near cell bodies. Specifically, out-of-focus and scattered light from the bright cellbody often obscures nearby dim fibers and degrades their signal-to-background ratio. Scanning techniques can solve this problem but are limited by reduced imaging speed and increased cost. We greatly reduce stray light by modulating the illumination intensity to different structures. We use a digital micromirror device in the illumination channel of a common widefield microscope and use real-time image processing to pattern the illumination. With the setup, we illuminate bright cell bodies with minimal light intensity, and illuminate in focus fiber-like structures with high light intensity to reveal weak signals. Thus, we minimize the background and enhance the visibility of fibers in the final image. This targeted illumination significantly improves fiber contrast while maintaining a fast-imaging speed and low cost. Using a targeted illumination setup in a widefield microscope, we demonstrate confocal quality imaging of complex neurons in live C. elegans and zebrafish larva, as well as in in vitro mice brain slice.