Bittel Amy M, Davis Ashley M, Wang Lei, Nederlof Michel A, Escobedo Jorge O, Strongin Robert M, Gibbs Summer L
Biomedical Engineering Department, Oregon Health & Science University, Portland, OR, 97201, USA.
Quantitative Imaging, Pittsburgh, PA, 15238, USA.
Sci Rep. 2018 Mar 15;8(1):4590. doi: 10.1038/s41598-018-22892-8.
Multicolor microscopy tools necessary to localize and visualize the complexity of subcellular systems are limited by current fluorophore technology. While commercial fluorophores cover spectral space from the ultraviolet to the near infrared region and are optimized for conventional bandpass based fluorescence microscopy, they are not ideal for highly multiplexed fluorescence microscopy as they tend to have short Stokes shifts, restricting the number of fluorophores that can be detected in a single sample to four to five. Herein, we synthesized a library of 95 novel boron-dipyrromethene (BODIPY)-based fluorophores and screened their photophysical, optical and spectral properties for their utility in multicolor microscopy. A subset of our BODIPY-based fluorophores yielded varied length Stokes shifts probes, which were used to create a five-color image using a single excitation with confocal laser scanning microscopy for the first time. Combining these novel fluorophores with conventional fluorophores could facilitate imaging in up to nine to ten colors using linear unmixing based microscopy approaches.
用于定位和可视化亚细胞系统复杂性的多色显微镜工具受到当前荧光团技术的限制。虽然商业荧光团覆盖了从紫外到近红外区域的光谱空间，并针对基于传统带通的荧光显微镜进行了优化，但它们并不适用于高度多重荧光显微镜，因为它们往往具有较短的斯托克斯位移，将单个样品中可检测到的荧光团数量限制为四到五个。在此，我们合成了一个包含95种新型硼二吡咯亚甲基（BODIPY）基荧光团的文库，并筛选了它们的光物理、光学和光谱特性，以评估其在多色显微镜中的实用性。我们基于BODIPY的荧光团子集产生了不同长度斯托克斯位移的探针，首次使用共聚焦激光扫描显微镜通过单次激发创建了五色图像。将这些新型荧光团与传统荧光团相结合，可以使用基于线性解混的显微镜方法实现多达九到十种颜色的成像。
