Microfluidic immobilization and subcellular imaging of developing Caenorhabditis elegans

Abstract

Living matter is highly dynamic. The cell itself is a collection of constituents whose material properties vary extensively through time and space. Developing an understanding of the physical principles that guide spatiotemporal cellular organization requires improved techniques for studying the material properties of subcellular structures in developing and aging organisms. This thesis describes the development of a novel microfluidic device to enable reversible compressive immobilization and imaging of individual Caenorhabditis elegans larvae, thus enabling the tracking of changes in the biophysical properties of subcellular structures throughout its lifetime. As a test case, the device is demonstrated to enable novel measurements within developing C. elegans larvae. We focus on using fluorescence recovery after photobleaching (FRAP), to study changes in the diffusivity of the proteins fibrillarin (FIB1) and nucleophosmin (NPM1) within intestinal nucleoli as a function of age. Our results suggest that the material properties of NPM1 remain stable throughout larval development, while FIB1 exhibits an age-related reduction in recovery, consistent with prior observations in other in vitro and in vivo systems.