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Three-dimensional single molecule localization close to the coverslip: a comparison of methods exploiting supercritical angle fluorescence
Biomedical Optics Express ( IF 3.4 ) Pub Date : 2021-01-12 , DOI: 10.1364/boe.413018
Philipp Zelger 1 , Lisa Bodner 1 , Martin Offterdinger 2 , Lukas Velas 3 , Gerhard J Schütz 3 , Alexander Jesacher 1
Affiliation  

The precise spatial localization of single molecules in three dimensions is an important basis for single molecule localization microscopy (SMLM) and tracking. At distances up to a few hundred nanometers from the coverslip, evanescent wave coupling into the glass, also known as supercritical angle fluorescence (SAF), can strongly improve the axial precision, thus facilitating almost isotropic localization performance. Specific detection systems, introduced as Supercritical angle localization microscopy (SALM) or Direct optical nanoscopy with axially localized detection (DONALD), have been developed to exploit SAF in modified two-channel imaging schemes. Recently, our group has shown that off-focus microscopy, i.e., imaging at an intentional slight defocus, can perform equally well, but uses only a single detection arm. Here we compare SALM, off-focus imaging and the most commonly used 3D SMLM techniques, namely cylindrical lens and biplane imaging, regarding 3D localization in close proximity to the coverslip. We show that all methods gain from SAF, which leaves a high detection NA as the only major key requirement to unlock the SAF benefit. We find parameter settings for cylindrical lens and biplane imaging for highest z-precision. Further, we compare the methods in view of robustness to aberrations, fixed dipole emission and double-emitter events. We show that biplane imaging provides the best overall performance and support our findings by DNA-PAINT experiments on DNA-nanoruler samples. Our study sheds light on the effects of SAF for SMLM and is helpful for researchers who plan to employ localization-based 3D nanoscopy close to the coverslip.

中文翻译:

靠近盖玻片的三维单分子定位:利用超临界角荧光的方法比较

单个分子在三维空间的精确定位是单分子定位显微镜(SMLM)和跟踪的重要基础。在距盖玻片数百纳米的距离处,衰减波耦合到玻璃中,也称为超临界角荧光 (SAF),可以大大提高轴向精度,从而促进几乎各向同性的定位性能。特定检测系统,作为超临界角定位显微镜(SALM) 或具有轴向定位检测的直接光学纳米显微镜引入(DONALD),已开发用于在修改后的双通道成像方案中利用 SAF。最近,我们的小组已经表明,离焦显微镜,即在有意的轻微散焦处成像,可以同样好地执行,但仅使用单个检测臂。在这里,我们比较了 SALM、离焦成像和最常用的 3D SMLM 技术,即柱面透镜和双平面成像,关于靠近盖玻片的 3D 定位。我们展示了所有方法都从 SAF 中获益,这使得高检测 NA 作为解锁 SAF 优势的唯一主要关键要求。我们找到了柱面透镜和双平面成像的参数设置,以获得最高的 z 精度。此外,我们根据对像差、固定偶极子发射和双发射器事件的鲁棒性来比较这些方法。我们表明双平面成像提供了最佳的整体性能,并通过对 DNA 纳米样品的 DNA-PAINT 实验支持我们的发现。我们的研究阐明了 SAF 对 SMLM 的影响,有助于计划在盖玻片附近使用基于定位的 3D 纳米镜的研究人员。
更新日期:2021-02-01
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