STEM-EELS tomography of biological materials

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Biological structures within a cell are difficult to assess at high resolution, which poses a critical limit to understanding a wide range of biological processes that are relevant to various human diseases. Light microscopy has made exceptional advancements in cell biology, enabling spatial and temporal resolution of biological molecules within single cells, however these techniques are limited in resolution and their ability to quantify protein density. Conventional electron microscopy can provide higher resolution of cell structures, but rely on the use of heavy metal stains to provide contrast. To circumvent these limitations, we plan to develop a 3D imaging technique to examine the elemental composition of unstained biological material in situ. We will develop and advance a technique known as scanning transmission electron microscopy in combination with electron tomography. This will allow us to obtain electron-energy loss spectroscopy (EELS) information of biological samples, that not only informs about the shape and structure of materials at exquisite resolution, but also reveals the chemical composition - allowing us to identify the nature of the molecules that we observe in addition to their shape and structure. This technique will combine the power and resolution of traditional electron microscopy with chemical information from the biological spectra, enabling detailed structural information without the use of conventional contrast enhancing stains. While common in material science research, several barriers have limited the use of STEM- EELS in revealing fine structural details of biological specimens, which we are now in a unique position to overcome to reveal unprecedented insight into the structure and composition of cells. We have previously demonstrated the feasibility of energy loss spectroscopy in resolving biological molecules using electron spectroscopic imaging by mapping phosphorus (abundant in nucleic acids) and nitrogen (present in all protein and nucleic acids) mapping. We will expand on this technique by collecting spectra at each pixel, and will obtain pixel specific spectra at multiple tilt angles to generate element-specific pixel resolution 3D information about our biological sample.