Correlating Soft X-Ray Cryo-Tomography With Super-Resolution Cryo-Microscopy

在我的研究员期间，我将在带头发展相关软X射线冷冻层析成像(SXT)和荧光冷冻显微镜的课题组中从事相关低温超分辨显微镜的工作。在过去的十年里，拉贝尔集团(UCSF)开发了世界上第一台用于细胞生物学的软X射线冷冻显微镜，以及第一台用于相关研究的荧光低温显微镜(Cryo-FM)。使用这两种成像模式从同一细胞测量的数据的关联产生了细胞的整体视图，分子和细胞过程直接在其近天然的冷冻保存的超微结构环境中可视化。在SXT中，厚度可达15微米的低温完好细胞被‘水窗’X射线(2.3-4.4 nm)照射，含碳生物分子对这种X射线的吸收强度是水的10倍。这导致了细胞超微结构的定量高对比度图像，而没有对比度增强染色。现有的低温调频是受衍射限制的，因此，只能在比SXT所能达到的空间分辨率低得多的空间分辨率下对过程进行局部化。为了提高这种定位精度，必须开发一种超分辨率的低温调频系统。与其他亚衍射技术不同的是，结构照明显微镜(SIM)不需要具有特定光开关特性的荧光团，因此它代表了一种有吸引力的低温条件下的超分辨策略。与传统的光学显微镜相比，线性SIM可以实现两倍的横向分辨率提高。我在小组中的角色将是实施结构照明低温显微镜，并使用自行编写的图像处理算法和标准断层图像重建软件将其数据与SXT图像相关联。在第一次原理验证实验之后，我将应用新的相关超分辨率成像技术，共同培育一个研究计划，旨在了解核组织和染色质区划在小鼠嗅觉受体基因调控中所起的作用。SXT和Cryo-SIM的结合将代表着细胞成像方面的突破性进展，并将以亚衍射精度对冷冻保存的完整细胞的分子组织和超微结构进行前所未有的解释和分析。简而言之，我在拉拉贝尔小组的研究工作将使我获得最高水平的技术技能和科学见解，并对细胞生物学和显微镜领域具有巨大的潜在价值。我将开发的技术也适用于电子显微镜，这是一个科学实力很强的领域，如果低温SIM可用于相关的成像研究，将大大提高这一领域的科学实力。

During my Fellowship, I will work on correlative cryogenic super-resolution microscopy in the research group spearheading the development of correlative soft x-ray cryo-tomography (SXT) and fluorescence cryo-microscopy. During the past decade, the Larabell group (UCSF) has developed the world's first soft x-ray cryo-microscope for cell biology together with the first fluorescence cryo-microscope (cryo-FM) for correlative studies. The correlation of data - measured from the same cell using these two imaging modalities - generates a holistic view of the cell, with molecules and cellular processes being visualized directly within their near-native cryo-preserved ultrastructural context. In SXT, cryogenic intact cells of up to 15 micrometers in thickness are illuminated with 'water window' X-rays (2.3-4.4 nm), which are absorbed 10 times more strongly by carbon-containing biomolecules than by water. This leads to quantitative high-contrast images of the cellular ultrastructure without contrast-enhancing staining. The existing cryo-FM is diffraction-limited, and, therefore, can only localize processes at a spatial resolution significantly lower than that achievable with SXT. To improve this localization accuracy, a super-resolution version of the cryo-FM has to be developed. Since, unlike other sub-diffraction techniques, structured illumination microscopy (SIM) does not require fluorophores with specific photoswitchable properties, it represents an attractive super-resolution strategy for cryogenic conditions. Linear SIM can achieve a twofold lateral resolution improvement compared to conventional light microscopy. My role in the group will be to work on the implementation of a structured illumination cryo-microscope, and correlate its data with SXT images using self-written image processing algorithms and standard tomogram reconstruction software. After first proof-of-principle experiments, I will apply the novel correlative super-resolution imaging technique in a collaboration to cultivate a research program aimed at understanding the role played by nuclear organization and chromatin compartmentalization in gene regulation of mouse olfactory receptors. The combination of SXT and cryo-SIM will represent a ground-breaking advance in cell imaging, and will allow unprecedented interpretation and analyses of molecular organization and ultrastructural architecture of cryo-preserved intact cells at sub-diffraction accuracy. In short, my fellowship work in the Larabell group will give me technical skills and scientific insights at the highest level, and of great potential value to the fields of cell biology and microscopy. The technologies I will be developing are also applicable in electron microscopy, an area of scientific strength that would be advanced significantly by the availability of a cryo-SIM for correlative imaging studies.