Rapid Lightsheet Volume Imaging For Developmental Biology

Abstract

Optical fluorescence microscopy is one of the key tool, that is indispensable for the study of complex and dynamic biological processes. Key issues associated with the existing fluorescence imaging systems are, photo-bleaching, long-time imaging and low-quality volume reconstruction. Continuous monitoring of large specimens for long duration requires fast volume imaging. This is essential for understanding processes occurring during developmental stages of multi-cellular organisms. One of the key obstacles for prolonged monitoring and data collection is photo-bleaching. To overcome the effect of bleaching, a single and multi- color Lightsheet Illuminated Volume Expedite (LIVE) imaging technique is developed that enables rapid screening of multiple tissues in an organism with an order-less photo-bleaching. Our approach based on LIVE imaging employs quantized step rotation of the specimen to record 2D angular data that reduces data collection time by a factor of more than 10 when compared to existing light sheet fluorescence microscopy. A co-planar multicolor light sheet is generated to excite spectrally-separated fluorescent probes that label the target tissues. Arduino-based control systems were employed to automatize and control the volume data acquisition process. To illustrate the advantages of our approach, we have noninvasively imaged Drosophila larvae (without removing chitinous cover) and Zebrafish embryo. Dynamic studies of multiple organs (muscle and yolk-sac) in Zebrafish over a prolonged duration of time (5 days) were carried out to understand muscle structuring and metabolism. Volume reconstruction, intensity plots and inter-dependence ratio analysis allowed us detailed visualization of organs during early development (Pharyngula period) in Zebrafish. The advantage of multicolor lightsheet illumination, fast volume imaging, simultaneous multiple organ imaging and order-less photo-bleaching makes LIVE imaging the system of choice for rapid monitoring and real-time assessment of macroscopic biological organisms at microscopic resolutions. We envision developed LIVE system as the next generation state-of-art-the-art light sheet fluorescence imaging