Mlodzianoski Michael J, Schreiner John M, Callahan Steven P, Smolková Katarina, Dlasková Andrea, Santorová Jitka, Ježek Petr, Bewersdorf Joerg
Department of Cell Biology, Yale School of Medicine, 333 Cedar Street, New Haven, CT 06510, USA.
Opt Express. 2011 Aug 1;19(16):15009-19. doi: 10.1364/OE.19.015009.
The recent development of diffraction-unlimited far-field fluorescence microscopy has overcome the classical resolution limit of ~250 nm of conventional light microscopy by about a factor of ten. The improved resolution, however, reveals not only biological structures at an unprecedented resolution, but is also susceptible to sample drift on a much finer scale than previously relevant. Without correction, sample drift leads to smeared images with decreased resolution, and in the worst case to misinterpretation of the imaged structures. This poses a problem especially for techniques such as Fluorescence Photoactivation Localization Microscopy (FPALM/PALM) or Stochastic Optical Reconstruction Microscopy (STORM), which often require minutes recording time. Here we discuss an approach that corrects for three-dimensional (3D) drift in images of fixed samples without the requirement for fiduciary markers or instrument modifications. Drift is determined by calculating the spatial cross-correlation function between subsets of localized particles imaged at different times. Correction down to ~5 nm precision is achieved despite the fact that different molecules are imaged in each frame. We demonstrate the performance of our drift correction algorithm with different simulated structures and analyze its dependence on particle density and localization precision. By imaging mitochondria with Biplane FPALM we show our algorithm's feasibility in a practical application.
无衍射远场荧光显微镜的最新进展已将传统光学显微镜约250纳米的经典分辨率极限提高了约十倍。然而，分辨率的提高不仅以前所未有的分辨率揭示了生物结构，而且在比以前更精细的尺度上也容易受到样品漂移的影响。如果不进行校正，样品漂移会导致图像模糊、分辨率降低，在最坏的情况下会导致对成像结构的错误解读。这对于诸如荧光光激活定位显微镜（FPALM/PALM）或随机光学重建显微镜（STORM）等技术来说尤其成问题，因为这些技术通常需要数分钟的记录时间。在此，我们讨论一种方法，该方法可校正固定样品图像中的三维（3D）漂移，而无需使用 fiducial标记或对仪器进行修改。通过计算在不同时间成像的局部粒子子集之间的空间互相关函数来确定漂移。尽管每帧中成像的是不同分子，但仍可实现低至约5纳米精度的校正。我们用不同的模拟结构展示了我们的漂移校正算法的性能，并分析了其对粒子密度和定位精度的依赖性。通过双平面FPALM对线粒体进行成像，我们展示了我们算法在实际应用中的可行性。
