Novel Nano- and Immuno-Probes for Multicolor Electron Microscopy of Neural Cells and Tissues - Project Summary Abstract: Our goal is to develop methods for combined molecular and ultrastructural imaging of neural cells and tissues. This goal is motivated by the critical need in neuroscience and neuropathology to understand the interplay between morphological and molecular phenotypes of neural networks in health and disease. Ultrastructure refers to cellular features that can be seen in electron microscopy (e.g., various membranes), while molecular specificity is typically achieved by fluorescence microscopy. A combination of these two techniques would provide orthogonal information on neural physiology. However, state-of-the-art approaches to combine fluorescence and electron imaging of cells and tissues have limitations. The major limitation of correlative light and electron microscopy (CLEM) methods is the requirement to use antibodies that are difficult to deliver into cells without permeabilization. The major limitation of heavy-metal-based tags is their reliance on the same contrast mechanism as ultrastructural imaging in EM, making the two difficult to distinguish. We will address the limitation of CLEM probes by developing a set of smaller immunofluorescent labels based on recombinant fragments of antibodies called single chain variable antibody fragments (scFv). We will also address the limitations of heavy-metal-based tags by developing multicolor nanoparticle and small-molecule probes (cathodophores) whose optical emission is induced directly by the electron beam in a process termed cathodoluminescence (CL). Thus, we will establish two generally applicable methods for multicolor EM: one via CLEM using scFv probes and the other via CL using cathodophores. The following three aims are proposed: Aim 1: To develop detergent-free multicolor immunoprobes that reveal molecular components via CLEM. Aim 2: To develop sub-10-nm lanthanide nanoparticle cathodophores that can be used as multicolor probes for localization of single proteins in electron microscopy. Aim 3: To establish small-molecule dyes as cathodoluminescent contrast agents in multicolor EM. Our approach is innovative because scFvs that we will engineer in Aim 1 are a new type of molecular probe that holds promise for multiplexed CLEM of neuronal tissue. In Aims 2 and 3, we are exploring a new light-electron-matter interaction in neurobiology research: optical emission induced by the electron beam – cathodoluminescence. Our extensive preliminary results confirm feasibility and lay the necessary foundation for the proposed work. Our technology-driven approach has the potential to be impactful in neurobiology and medicine, redefining the way we visualize and understand disease at the level of brain ultrastructure.
