Counting fluorescently labeled proteins in tissues in the spinning-disk microscope using single-molecule calibrations.
Counting fluorescently labeled proteins in tissues in the spinning-disk microscope using single-molecule calibrations.
复制标题
使用单分子校准在旋转圆盘显微镜中计数组织中的荧光标记蛋白质。
DOI：
10.1091/mbc.e21-12-0618
复制
发表时间：
2022-05-15
影响因子：
3.3
通讯作者：
Howard, Jonathon
中科院分区：
文献类型：
--
作者：
Liao, Maijia;Kuo, Yin-Wei;Howard, Jonathon
活细胞中分子数量和浓度的量化对于测试复杂生物现象的模型至关重要。计数细胞中的分子需要估计单个分子的荧光强度，这通常仅限于在细胞表面附近、孤立的细胞中或运动扩散的地方成像。为了克服这个困难，我们设计了一种旋转盘共聚焦显微镜的校准技术，通常用于组织成像，它使用单步漂白动力学来估计单荧光团强度。为了交叉检查我们的校准，我们将 SDC 显微镜中的荧光团亮度与全内反射和落射荧光显微镜中的荧光团亮度进行了比较。我们应用这种校准方法来量化生长微管末端彗星中末端结合蛋白 1 (EB1)-eGFP 的数量，并测量果蝇幼虫感觉神经元中 EB1-eGFP 的细胞质浓度。这些测量使我们能够估计 EB1-eGFP 与微管的解离常数以及 GTP-微管蛋白帽的大小。我们的结果显示了使用转盘共聚焦显微镜进行单分子成像的未开发潜力，并提供了一种直接的方法来计算组织中荧光团的绝对数量，可应用于广泛的生物系统和成像技术。
Quantification of molecular numbers and concentrations in living cells is critical for testing models of complex biological phenomena. Counting molecules in cells requires estimation of the fluorescence intensity of single molecules, which is generally limited to imaging near cell surfaces, in isolated cells, or where motions are diffusive. To circumvent this difficulty, we have devised a calibration technique for spinning–disk confocal microscopy, commonly used for imaging in tissues, that uses single–step bleaching kinetics to estimate the single–fluorophore intensity. To cross–check our calibrations, we compared the brightness of fluorophores in the SDC microscope to those in the total internal reflection and epifluorescence microscopes. We applied this calibration method to quantify the number of end–binding protein 1 (EB1)–eGFP in the comets of growing microtubule ends and to measure the cytoplasmic concentration of EB1–eGFP in sensory neurons in fly larvae. These measurements allowed us to estimate the dissociation constant of EB1–eGFP from the microtubules as well as the GTP–tubulin cap size. Our results show the unexplored potential of single–molecule imaging using spinning–disk confocal microscopy and provide a straightforward method to count the absolute number of fluorophores in tissues that can be applied to a wide range of biological systems and imaging techniques.