Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Institute for Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario M5S 3G9, Canada.
Rev Sci Instrum. 2020 Dec 1;91(12):123703. doi: 10.1063/1.5143319.
New innovations in single-molecule localization microscopy (SMLM) have revolutionized optical imaging, enabling the characterization of biological structures and interactions with unprecedented detail and resolution. However, multi-color or hyperspectral SMLM can pose particular challenges which affect image quality and data interpretation, such as unequal photophysical performance of fluorophores and non-linear image registration issues, which arise as two emission channels travel along different optical paths to reach the detector. In addition, using evanescent-wave based approaches (Total Internal Reflection Fluorescence: TIRF) where beam shape, decay depth, and power density are important, different illumination wavelengths can lead to unequal imaging depth across multiple channels on the same sample. A potential useful approach would be to use a single excitation wavelength to perform hyperspectral localization imaging. We report herein on the use of a variable angle tunable thin-film filter to spectrally isolate far-red emitting fluorophores. This solution was integrated into a commercial microscope platform using an open-source hardware design, enabling the rapid acquisition of SMLM images arising from fluorescence emission captured within ∼15 nm to 20 nm spectral windows (or detection bands). By characterizing intensity distributions, average intensities, and localization frequency through a range of spectral windows, we investigated several far-red emitting fluorophores and identified an optimal fluorophore pair for two-color SMLM using this method. Fluorophore crosstalk between the different spectral windows was assessed by examining the effect of varying the photon output thresholds on the localization frequency and fraction of data recovered. The utility of this approach was demonstrated by hyper-spectral super-resolution imaging of the interaction between the mitochondrial protein, TOM20, and the peroxisomal protein, PMP70.
单分子定位显微镜（SMLM）的新创新彻底改变了光学成像，使生物结构和相互作用的特征能够以空前的细节和分辨率进行。然而，多色或超光谱 SMLM 可能会带来特定的挑战，这些挑战会影响图像质量和数据解释，例如荧光团的光物理性能不均一和非线性图像配准问题，这两个问题会随着两个发射通道沿着不同的光路到达探测器而出现。此外，在光束形状、衰减深度和功率密度等重要的基于消逝波的方法（全内反射荧光：TIRF）中，不同的照明波长会导致同一样品上多个通道的成像深度不均一。一种潜在的有用方法是使用单一激发波长进行超光谱定位成像。我们在此报告使用可变角度可调谐薄膜滤光片来光谱分离远红发射荧光团。该解决方案使用开源硬件设计集成到商业显微镜平台中，能够快速获取源自约 15nm 到 20nm 光谱窗口（或检测带）内荧光发射的 SMLM 图像。通过在一系列光谱窗口中表征强度分布、平均强度和定位频率，我们研究了几种远红发射荧光团，并使用该方法确定了用于双色 SMLM 的最佳荧光团对。通过检查改变光子输出阈值对定位频率和恢复数据分数的影响，评估了不同光谱窗口之间荧光团串扰的情况。通过对线粒体蛋白 TOM20 与过氧化物酶体蛋白 PMP70 之间相互作用的超光谱超分辨率成像，证明了该方法的实用性。
