Correlative light and electron microscopy (CLEM) is emerging as a powerful tool for nanoscale structural studies in both the biological and physical sciences. However, the challenges associated with obtaining precisely registered images of nanoscale neighborhoods within a sample on multiple microscopes severely limit the adoptability and throughput of CLEM. Additionally, the low throughput and lack of robust image analysis lead to CLEM being largely used as a qualitative approach with limited ability to assess structural heterogeneity or identify subtle changes associated with early stages of disease. By leveraging the high throughput of the Vutara single molecule localization microscope and taking advantage of structural fiducials / landmarks identifiable via both light and electron microscopy, we have developed indirect CLEM [iCLEM] as a low-cost, high throughput option with extensive quantitative capabilities.
This approach has enabled us to undertake systematic investigation of the structural underpinnings of electrical signal propagation in the heart, a critical process that coordinates the contraction of ~12 billion muscle cells during each heartbeat. Motivation for this work derives from that fact that disruption of this process leads to life-threatening disruptions in the heart’s rhythm (arrhythmias). Therefore, we used iCLEM to study the intercalated disk (ID), a specialized structure that affords electrical and mechanical coupling between muscle cells in the heart. Although, the ID is widely recognized as a complex, heterogeneous structure, the lack of quantitative structural data has forced computational models to omit it or simplify it to a homogenous cylinder. Using iCLEM, we have obtained the first-ever quantitative picture of the ID, enabling us to construct realistic computational models. Transmission electron microscopy (TEM) enabled us to quantify ID ultrastructure from micro- through nano-scales and thereby, construct populations of finite element models of IDs, capturing both intra- and inter- individual variability. We then populated these meshes with electrogenic proteins based on STORM-based Relative Localization Analysis (STORM-RLA) of single molecule localization data. By incorporating these data into computational models of electrical signal propagation, we are uncovering previously unappreciated structure-function relationships that determine the regularity of the heart’s rhythm. These predictions, along with functional imaging studies of electrical signal spread in the heart, are providing the basis for the development of novel classes of anti-arrhythmic drugs.
Rengasayee (Sai) Veeraraghavan, Ph.D.
Heather Struckman, BS, MS