Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520.
Mol Biol Cell. 2022 May 15;33(6):ar48. doi: 10.1091/mbc.E21-12-0618. Epub 2022 Mar 24.
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.
定量检测活细胞内的分子数量和浓度对于测试复杂生物现象的模型至关重要。在细胞中计数分子需要估计单个分子的荧光强度，而这通常限于在细胞表面附近、在分离的细胞中或在扩散运动的情况下进行成像。为了克服这一困难，我们设计了一种用于旋转盘共聚焦显微镜的校准技术，该技术常用于组织成像，可以使用单步漂白动力学来估计单荧光团的强度。为了核对我们的校准，我们将 SDC 显微镜中的荧光团的亮度与全内反射和荧光显微镜中的荧光团的亮度进行了比较。我们将这种校准方法应用于定量测量生长微管末端彗星中结合蛋白 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。这些测量结果使我们能够从微管上估计 EB1-eGFP 的离解常数以及 GTP-微管蛋白帽的大小。我们的结果显示了使用旋转盘共聚焦显微镜进行单分子成像的未被开发的潜力，并提供了一种简单的方法来计算组织中荧光团的绝对数量，该方法可应用于广泛的生物系统和成像技术。
