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首页> 外文会议>Single molecule spectroscopy and superresolution imaging VII >Design and development of BODIPY-based photoswitchable fluorophores to visualize cell signaling with multispectral super resolution microscopy
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Design and development of BODIPY-based photoswitchable fluorophores to visualize cell signaling with multispectral super resolution microscopy

机译：基于BODIPY的光开关荧光团的设计和开发，可通过多光谱超分辨率显微镜观察细胞信号

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摘要

Super resolution microscopy (SRM) has overcome the historic spatial resolution limit of light microscopy, enabling fluorescence visualization of cellular structures and multi-protein complexes at the nanometer scale. Using single-molecule localization microscopy, the precise location of a stochastically activated population of photoswitchable fluorophores is determined during the collection of many images to form a single image with resolution of ～10-20 nm, an order of magnitude improvement over conventional microscopy. However, the spectral resolution of current SRM techniques are limited by existing fluorophores with only up to four colors imaged simultaneously, limiting the number of intracellular components that can be studied in a single sample. In the current work, a library of novel BODIPY-based fluorophores was synthesized using a solid phase synthetic platform with the goal of creating a set of photoswitchable fluorophores that can be excited by 5 distinct laser lines but emit throughout the spectral range (450-850 nm) enabling multispectral super resolution microscopy (MSSRM). The photoswitching properties of all new fluorophores were quantified for the following key photoswitching characteristics: (1) the number of photons per on cycle (2) the number of on cycles (switching events), (3) the percentage of time the fluorophore spends in the fluorescent on and off states, and (4) the susceptibility of the fluorophore to photobleaching (time of last event). To ensure the accuracy of our photoswitching measurements, our methodology to detect and quantitate the photoswitching properties of individual fluorophore molecules was validated by comparing measured photoswitching properties of three commercial dyes to published results. We also identified two efficient methods to positionally isolate fluorophores on coverglass for screening of the BODIPY-based library.
机译：超分辨率显微镜（SRM）克服了光学显微镜的历史空间分辨率限制，可在纳米级对细胞结构和多种蛋白质复合物进行荧光可视化。使用单分子定位显微镜，可以在收集许多图像以形成分辨率约为10-20 nm的单幅图像时确定随机激活的可光转换荧光团的精确位置，这比传统显微镜提高了一个数量级。但是，当前的SRM技术的光谱分辨率受到现有荧光团的限制，同时最多只能同时成像四种颜色，从而限制了可以在单个样品中研究的细胞内组分的数量。在当前工作中，使用固相合成平台合成了一个基于BODIPY的新型荧光团库，其目的是创建一组可光切换的荧光团，它们可以被5条不同的激光线激发，但在整个光谱范围内发射（450-850）纳米），实现多光谱超分辨率显微镜（MSSRM）。针对以下关键的光开关特性，对所有新的荧光团的光开关特性进行了量化：（1）每个接通周期的光子数量（2）接通周期的数量（开关事件），（3）荧光团花费的时间百分比（4）荧光团对光漂白的敏感性（最后事件的时间）。为了确保我们的光开关测量的准确性，通过将三种商业染料的测得的光开关特性与公开的结果进行比较，验证了我们检测和定量单个荧光团分子的光开关特性的方法。我们还确定了两种有效的方法来在盖玻片上定位荧光团，以筛选基于BODIPY的文库。

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