A study of focus control and photobleaching in a light-sheet microscope designed for long-term in vivo imaging of neuronal processes in Drosophila

In animals, daily activity rhythms are regulated by an endogenous circadian clock. To understand the underlying mechanisms, we study the fruit fly Drosophila melanogaster. In these flies, a specific group of neurons known as small ventral lateral neurons (s-LNvs) play a key role in generating the circadian rhythm [1]. The ultimate goal of this project is to use fluorescence microscopy to monitor processes occurring in s-LNv neurons throughout the day in live adult flies. Our laboratory has built a light-sheet microscope designed to image the s-LNv neurons in vivo in adult flies[2]. However, long-term acquisition is limited by focus drift, which degrades the image quality, and photobleaching, whereby the emission of fluorophores is lost due to prolonged light excitation. We present two strategies for controlling drift: a control loop that corrects the relative distance between the objectives that constitute the remote refocusing system and a sample drift correction based on phase cross-correlation [3]. We also systematically quantified photobleaching in live flies, through metrics that defined image quality over time. These improvements enabled imaging of the s-LNvs for five hours in a live fly, allowing observation of neuronal remodelling and intracellular mitochondrial movement. [1] MP Fernandez et al., PLoS Biol 6 (2008) e69. [2] F Tassara et al., bioRxiv (2024) 2024.11.06.622263. [3] M Guizar-Sicairos et al., Opt. Lett. 33 (2008) 156.