Dynamic nanoscale cellular imaging via time-resolved cryo-vitrification - Summary: Many cellular processes are triggered by ligands such as ions, small molecules, peptides/proteins (e.g., hormones, cytokines), and viruses. However, rapid cellular adaptations that follow stimulation often fall in the blind spot of microscopy: limitations of spatiotemporal resolution prevent detailed exploration of nanoscale cellular dynamics. Motivated by the need to address this technological barrier, we aim to develop a biophysical tool that will allow stimulating biological cells with various chemical or biological compounds and then freezing these cells for subsequent analysis by high-resolution microscopy. We refer to this technique by an acronym: Vitrification Experiment Resolved In Time After Stimulation (VERITAS). Broad utility of this new method in biological research is envisioned, including investigations concerning the dynamics of cellular membranes and membrane-protein interactions, the dynamics of protein-protein and protein-nucleic acid interactions, and the dynamics of post-translational modifications. In addition, the need for the proposed instrument is supported by a user survey that we conducted. Our approach is conceptually innovative because it will permit imaging of nanoscale cellular dynamics with millisecond temporal resolution and because it establishes a connection between time-resolved cryo-vitrification and super-resolved fluorescence imaging. The approach is methodologically innovative because it will feature millisecond-level control over the stimulation-to-vitrification time, and reliable chemical stimulation of cells. Moreover, we will develop new protocols to integrate cryo-vitrification and nanoscale optical microscopy, and to validate the technique against live optical microscopy measurements. In Aim 1, we will develop the time-resolved cryo-vitrification instrument by first achieving millisecond-level control over the stimulation-to-vitrification time and then developing a method for precise chemical stimulation of the sample. This work will complete our instrumentation development efforts and allow proceeding to the validation stage. In Aim 2, we will develop post-vitrification imaging and data analysis protocols and use these procedures to validate VERITAS using two representative biological systems. These studies will test the boundaries of utility of the method because they use different types of cells and different imaging methods. Based on our preliminary data, the project is feasible. Our efforts will establish the groundwork for future biological studies using the proposed time-resolved cryo-vitrification technique. We suggest that this is significant because time-resolved cryo-vitrification will provide detailed molecular and structural understanding of how cells respond to a variety of stimuli of relevance both to normal physiology and disease. Furthermore, the proposal includes detailed plans for technology dissemination via direct collaboration, open-source sharing, commercialization, and integration with local instrumentation facilities.
