Biochemical Microscopy for imaging across Molecular Scales

Cryo-Electron Tomography (cryo-ET) can map native vitrified cells and tissues at near atomic scale, but is limited by electron transparency to thin lamellae (~200 nm) that are prepared using focussed ion beam (FIB) milling while structural and chemical knowledge of surrounding regions is lost. Yet, the network of molecular interactions between cells is a critical component of understanding how cells communicate, divide, or are subjugated to pathogens. Mass spectrometry imaging (MSI) can be used to assess and localise molecules such as lipids, metabolites, peptides and proteins, while SEM (Scanning Electron Microscopy) can build a picture of the local morphology. Using these techniques together, we aim to exploit MSI to build up a 3D picture of the chemical and biological structure of tissue and cell environment.

Our initial focus is to exploit the 3D cryo preparative step for cryo-ET that uses plasma focussed ion beam scanning electron microscopy (pFIB-SEM) and the 3D nature of secondary ion mass spectrometry (SIMS) to enable precise label-free chemical targeting during lamella preparation. However, bridging the gap in size domains and getting structural and chemical information on the same areas of interest is a significant challenge. In this project we aim to explore how recent developments in these technologies could be used to bridge this gap and radically enhance each other through a series of proof-of-concept experiments, on cell and tissue samples, that will enable design of transformative technology in life sciences.  This conceptual design would deliver a completely new type of instrument, capable of exploring biological tissues in a way previously not possible – bringing together high-resolution SEM imaging with SIMS imaging to create a single tool for 3D cryogenic biochemical microscopy at subcellular resolution.

Broadening this concept further, we plan to consider the direct analysis of protein structure -through the integration of high resolution microscopy of cryo-ET to determine protein structure and native ambient mass spectrometry which is able to chemically map proteins within their natural environment. Furthermore, we plan to harness native ambient mass spectrometry to extract, separate, and capture protein complexes from tissue for subsequent super resolution mass spectrometry.

These developments will transform interpretations of complex cellular images by providing chemical information that will shine a light on cell functions, as well as structure. Fundamental processes, such as neuronal connectivity, dysregulation leading to cancer, and infection are chemically driven, but this can only be understood in the context of the molecular structures that drive cellular changes affecting function.