Molecular Biophysics, Center for Integrative Physiology and Molecular Medicine, Saarland University, 66421 Homburg, Germany.
Department of Physics, Saarland University, 66123 Saarbrücken, Germany.
Nano Lett. 2020 Nov 11;20(11):7948-7955. doi: 10.1021/acs.nanolett.0c02644. Epub 2020 Oct 9.
Quantum dots exhibit unique properties compared to other fluorophores, such as bright fluorescence and lack of photobleaching, resulting in their widespread utilization as fluorescent protein labels in the life sciences. However, their application is restricted to relative quantifications due to lacking knowledge about the labeling efficiency. We here present a strategy for determining the labeling efficiency of quantum dot labeling of HER2 in overexpressing breast cancer cells. Correlative light- and liquid-phase electron microscopy of whole cells was used to convert fluorescence intensities into the underlying molecular densities of the quantum dots. The labeling procedure with small affinity proteins was optimized yielding a maximal labeling efficiency of 83%, which was applicable to the high amount of ∼1.5 × 10 HER2 per cell. With the labeling efficiency known, it is now possible to derive the absolute protein expression levels in the plasma membrane and its variation within a cell and between cells.
量子点与其他荧光团相比具有独特的性质，例如明亮的荧光和无荧光漂白，因此它们被广泛用作生命科学中荧光蛋白标记物。然而，由于缺乏对标记效率的了解，它们的应用仅限于相对定量。我们在这里提出了一种确定过表达乳腺癌细胞中 HER2 量子点标记效率的策略。利用全细胞的相关光和液相电子显微镜，将荧光强度转换为量子点的潜在分子密度。通过优化小亲和力蛋白的标记程序，得到了 83%的最大标记效率，这适用于每个细胞约 1.5×10 的高 HER2 量。有了标记效率，现在就可以计算出质膜中的绝对蛋白表达水平及其在细胞内和细胞间的变化。
