D epartment of Biomedical Sciences, Heritage College of Osteopathic Medicine, Ohio University, Athens, OH, USA.
Advanced Photon Source, Argonne National Laboratory, Lemont, IL, USA.
Metallomics. 2023 Feb 16;15(2). doi: 10.1093/mtomcs/mfad006.
Synchrotron X-ray fluorescence microscopy (SXRF) presents a valuable opportunity to study the metallome of single cells because it simultaneously provides high-resolution subcellular distribution and quantitative cellular content of multiple elements. Different sample preparation techniques have been used to preserve cells for observations with SXRF, with a goal to maintain fidelity of the cellular metallome. In this case study, mouse pancreatic beta-cells have been preserved with optimized chemical fixation. We show that cell-to-cell variability is normal in the metallome of beta-cells due to heterogeneity and should be considered when interpreting SXRF data. In addition, we determined the impact of several immunofluorescence (IF) protocols on metal distribution and quantification in chemically fixed beta-cells and found that the metallome of beta-cells was not well preserved for quantitative analysis. However, zinc and iron qualitative analysis could be performed after IF with certain limitations. To help minimize metal loss using samples that require IF, we describe a novel IF protocol that can be used with chemically fixed cells after the completion of SXRF.
同步辐射 X 射线荧光显微镜（SXRF）为研究单个细胞的金属组提供了有价值的机会，因为它可以同时提供多个元素的高分辨率亚细胞分布和定量细胞含量。已经使用了不同的样品制备技术来保存用于 SXRF 观察的细胞，其目的是保持细胞金属组的保真度。在本案例研究中，使用优化的化学固定法保存了小鼠胰岛β细胞。我们表明，由于异质性，β细胞金属组中的细胞间变异性是正常的，在解释 SXRF 数据时应考虑到这一点。此外，我们确定了几种免疫荧光（IF）方案对化学固定β细胞中金属分布和定量的影响，发现β细胞的金属组对于定量分析不能很好地保存。然而，在 IF 之后可以进行锌和铁的定性分析，但有一定的局限性。为了帮助使用需要 IF 的样品最小化金属损失，我们描述了一种新的 IF 方案，该方案可在 SXRF 完成后用于化学固定的细胞。
