Temporal resolution doubling in fluorescence light-sheet microscopy via a hue-encoded shutter and regularization

Abstract

Studying dynamic biological processes, such as heart development and function in zebrafish embryos, often relies on multi-channel fluorescence labeling to distinguish multiple anatomical features, yet also demands high frame rates to capture rapid cell motions. Although a recently proposed method for imaging dynamic samples in transmission or reflection allows to conveniently switch between color imaging or boosting the frame rate by use of spectrally-encoded, temporally-modulated illumination sequences and a hue-encoded shutter (hue-encode shutter method, HESM), the technique is not applicable directly in fluorescence microscopy, where the emitted light spectrum is mostly independent of the excitation wavelength. In this paper, we extend HESM by using samples labeled with multiple fluorophores, whose emission signal can either be used to distinguish multiple anatomical features when imaged in multi-channel mode or, if the fluorophores are co-localized in a dynamic tissue, to increase the frame rate via HESM. We detail the necessary steps to implement this method in a two-color light-sheet microscope to image the beating heart of a zebrafish embryo. Specifically, we propose an adapted laser modulation scheme for illumination, we identify caveats in choosing a suitable multi-color fluorophore labeling strategy, and derive an ℓ1-regularized reconstruction technique that is sufficiently robust to handle the low signal-to-noise ratio and labeling inhomogeneities in the fluorescence images at hand. Using the case of a beating heart in a zebrafish embryo, we experimentally show an increase in the frame rate by a factor two while preserving the ability to image static features labeled in distinct channels, thereby demonstrating the applicability of HESM to fluorescence. With a suitable illumination setup and fluorescent labeling, the method could generalize to other applications where flexibility between multiple channel and high-speed fluorescence imaging is desirable. For fluorophores that are not co-localized, the imaging system is similar to a conventional light sheet microscope.