To gain a comprehensive understanding of cellular structures and function, correlative light and electron microscopy has been widely utilized to facilitate the acquisition of both macroscopic view of the whole organism and the localization of single molecules or macromolecular complexes. However, current correlative microscopy solutions require extensive sample preparation before biological specimens can be investigated using EM, which potentially alters the relevant structures and induces image artifacts. Therefore, this Phase II project is aimed to develop and bring to market a universal continuous-flow environmental specimen holder system for the light microscope (LM) and the scanning and transmission electron microscope (SEM and TEM) with a biofunctionable sample substrate and a micron-precision location reference system for correlative microscopy to take advantage of multiple imaging modalities spanning a range of spatial scales and frequencies. This powerful and versatile correlative microscopy specimen holder platform will enable environmental LM and EM, allow accurate temperature regulation (from below 0 C to 100 C), and empower microfluidic experiments conducted inside the chamber, where biological samples are incubated or drug treated. It will also allow sample imaging across LM, SEM, and TEM platforms without additional manipulation. In Phase I we have successfully created a working prototype of the system. The proposed cross-correlated specimen holder platform offers the capability to transform any existing individual microscopes into a complete correlative microscopy system that permits the entire microscopy field to join forces to not only uncover many insights and research the most urgent advances in modern medicine, but more importantly to open new doors and offer opportunities to the entire field of life and health sciences.
The environmental cross-correlative light and electron microscopy characterization techniques that will be developed in this project will have a direct impact on the understanding of biological processes. This project targets the cross-correlative nanometer-scale imaging of biological structures in their native liquid environment with sample temperature control, as well as the ability to observe biological processes with high resolution as they are occurring. This new correlative approach, which represents a giant leap forward compared to current correlative microscopy techniques using cryogenic freezing, will be used to gain a comprehensive understanding of cellular structures and function.