Integrating Optical Trapping into Light Sheet Florescence Microscopy Build for Cytoskeleton Network Structural Analysis

Cytoskeletal networks pose a unique problem in regards to imaging. Cytoskeletal networks formed through the weaving of cytoskeletal filaments via molecular motors contract into dense asters such that traditional methods of microscopic imaging cannot reveal their structure. One of the most elegant solutions that prevents extreme photo-bleaching while allowing for detailed imaging of these hyper dense structures is Light Sheet Florescence Microscopy. Additionally, it is valuable to understand the rheological properties of cytoskeletal networks. Optical tweezers micro-rheology studies of such composites at lower densities demonstrate tunable properties such as viscoelasticity and force responses correlated with restructuring of the composite. A current gap in the literature of Cytoskeletal networks is a simultaneous analysis of physical structure and rheological properties of higher density asters. We hypothesize that the properties observed at low density will be amplified by the increased contraction. To this end, we are adding optical tweezers (OT) capability to a light sheet fluorescence microscope (LSFM) to perform micro-rheology measurements while simultaneously acquiring a high. resolution 3D image of the surrounding filamentous structure to tens of microns in each direction. By combining these two techniques into a single build, we will be able to analyze simultaneously the structure and material properties of both active and post-activity cytoskeletal asters. This talk seeks to explain our own work in building our Single Objective LSF Microscope and its Optical Trap capabilities, as well as the theory behind its functions and experimental benefits.