A customized light sheet microscope to measure spatio-temporal protein dynamics in small model organisms.

Matthias Rieckher,Ilias Kyparissidis-Kokkinidis,Nektarios Tavernarakis,Athanasios Zacharopoulos,Giannis Zacharakis,Jorge Ripoll,Georgios Kourmoulakis,Irene Georgakoudi

doi:10.1371/journal.pone.0127869

Abstract

We describe a customizable and cost-effective light sheet microscopy (LSM) platform for rapid three-dimensional imaging of protein dynamics in small model organisms. The system is designed for high acquisition speeds and enables extended time-lapse in vivo experiments when using fluorescently labeled specimens. We demonstrate the capability of the setup to monitor gene expression and protein localization during ageing and upon starvation stress in longitudinal studies in individual or small groups of adult Caenorhabditis elegans nematodes. The system is equipped to readily perform fluorescence recovery after photobleaching (FRAP), which allows monitoring protein recovery and distribution under low photobleaching conditions. Our imaging platform is designed to easily switch between light sheet microscopy and optical projection tomography (OPT) modalities. The setup permits monitoring of spatio-temporal expression and localization of ageing biomarkers of subcellular size and can be conveniently adapted to image a wide range of small model organisms and tissue samples.

Highlights

Biomedical research increasingly requires in vivo imaging of cellular molecular dynamics as they are unfolding over time in the context of the whole organism
In this work we present and describe in detail a customized, cost-effective light sheet microscopy (LSM), which tackles the challenge of 3 dimensional (3D) visualization of protein dynamics during ageing in C. elegans
A typical data set for a single light sheet microscopy recording consists of a stack of 80–120 images (1000x1000 pixels each) of the plane of illumination through the specimen taken in 0.007mm steps and stored as one collected file with a size of approximately 200 megabyte

Summary

Introduction

Biomedical research increasingly requires in vivo imaging of cellular molecular dynamics as they are unfolding over time in the context of the whole organism. The graphical user interface (GUI) controls all relevant parameters (camera, stage movement, and filters) and allows for convenient design of automatic data acquisition sequences for imaging experiments, including light sheet microscopy, OPT, extended time lapse and video-recorded live imaging. A typical data set for a single light sheet microscopy recording consists of a stack of 80–120 images (1000x1000 pixels each) of the plane of illumination through the specimen taken in 0.007mm steps and stored as one collected file with a size of approximately 200 megabyte.

Results

Conclusion