"IDBR: TYPE A" Development of a High Resolution Fluorescence Force Microscope

Emory University, Atlanta GA

Investigators

Abstract

An award is made to Emory University to develop a high resolution fluorescence force microscope (HRFFM) to generate molecularly specific force maps orders of magnitude higher in spatial, orientation, force, and time resolution compared with the current methods. Forces are central to living systems, but few tools exist to investigate them at the molecular level. Developing the basic tools and instrumentation to characterize and visualize the molecular interplay between physical forces and biochemical signals is paramount in gaining a global understanding of signaling pathways in living systems. The HRFFM instrument will address these needs by mapping forces that are orders of magnitude higher in spatial, orientation, force, and time resolution compared with the current methods for cellular force imaging. The research, education, and outreach efforts will be integrated though their emphasis on forces and optical microscopy. K-12 outreach will be organized with Students for Science, a program that brings microscopes to the classroom, thus challenging students to visualize and probe the "micro- world". The program will expand multidisciplinary education across multiple departments at Emory University though restructuring of two courses that will bring together students interested in the interface between the biological and physical sciences. Importantly, practical knowledge about the HRFFM instrument will be disseminated to the broad scientific community through multiple channels. Firstly, a force imaging Wikipedia style website providing a community resource and educational information on force imaging will be developed. Students working directly on the project will post and record instructional YouTube videos on topics including: force chip preparation and polarization imaging. Analysis programs, protocols and references will also be posted, creating an interactive community focused on molecular forces in cell biology. The HRFFM instrument will be showcased at workshops and seminars across academic institutions nationally, thus ensuring that the broader cell biology and biophysics communities are fully aware of the advent and utility of this new tool. The current gold standard for measuring cellular mechanics, traction force microscopy, is based on polymer surfaces that deform under tension. Unfortunately, the lack of molecular specificity, ~1 µm spatial resolution, ~1 s time resolution, and ~1 nN force sensitivity inherent to traction force microscopy obscures the molecular underpinnings of cellular mechanotransduction. For example, traction force data is reported in units of nN/µm2; three orders of magnitude less sensitive than the resolution required for measuring molecular forces. The HRFFM instrument will address these shortcomings and map forces with pN-range force resolution, ~ms time resolution, ~20 nm spatial resolution, chemical specificity, and 3D force vectors in living cells. Moreover, the HRFFM instrument will provide a common platform bridging the single molecule FRET and super-resolution imaging communities, as well as creating a unique connection between the fields of cell biology and single molecule biophysics. This award is being made jointly by two Programs: (1) Instrument Development for Biological Research, in the Division of Biological Infrastructure (Biological Sciences Directorate), and (2) Physics of Living Systems, in the Division of Physics (Math and Physical Science Directorate).