STEM-EELS tomography of biological materials

细胞内的生物结构很难以高分辨率进行评估，这对理解与各种人类疾病相关的广泛生物过程构成了关键限制。光学显微镜在细胞生物学方面取得了非凡的进步，使单细胞内生物分子的空间和时间分辨率成为可能，然而这些技术在分辨率和量化蛋白质密度的能力方面受到限制。传统的电子显微镜可以提供细胞结构的更高分辨率，但依赖于使用重金属染色剂来提供对比度。为了克服这些限制，我们计划开发一种3D成像技术来原位检查未染色生物材料的元素组成。我们将开发和推进一种称为扫描透射电子显微镜与电子断层扫描相结合的技术。这将使我们能够获得生物样品的电子能量损失光谱（EELS）信息，不仅可以以精细的分辨率了解材料的形状和结构，还可以揭示化学成分-使我们能够识别我们观察到的分子的性质以及它们的形状和结构。这种技术将联合收割机的功率和分辨率的传统电子显微镜与化学信息的生物光谱，使详细的结构信息，而无需使用传统的对比度增强染料。虽然在材料科学研究中很常见，但有几个障碍限制了STEM- EELS在揭示生物标本的精细结构细节方面的使用，我们现在处于一个独特的位置，可以克服这些障碍，以揭示对细胞结构和组成的前所未有的洞察力。我们以前已经证明了能量损失光谱的可行性，在解决生物分子使用电子光谱成像映射磷（丰富的核酸）和氮（存在于所有蛋白质和核酸）映射。我们将通过在每个像素处收集光谱来扩展这种技术，并将在多个倾斜角度处获得像素特定的光谱，以生成关于我们的生物样本的元素特定像素分辨率3D信息。

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.