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Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

University of Maine

Imaging volumes as thick as whole cells at three-dimensional (3D) super-resolution is required to reveal unknown features of cellular organization. We report a light microscope that generates images with translationally invariant 30 x 30 x 75 nm resolution over a depth of several micrometers. This method, named biplane (BP) FPALM, combines a double-plane detection scheme with fluorescence photoactivation localization microscopy (FPALM) enabling 3D sub-diffraction resolution without compromising speed or sensitivity.

Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples.

Three-dimensional (3D) particle localization at the nanometer scale plays a central role in 3D particle tracking and 3D localization-based super-resolution microscopy. Here we introduce a localization algorithm that is independent of theoretical models and therefore generally applicable to a large number of experimental realizations. Applying this algorithm and a convertible experimental setup we compare the performance of the two major 3D techniques based on astigmatic distortions and on multiplane detection. In both methods we obtain experimental 3D localization accuracies in agreement with theoretical predictions and characterize the depth dependence of the localization accuracy in detail.

Experimental characterization of 3D localization techniques for particle-tracking and super-resolution microscopy.

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.

Article	Year
Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples. Nature methods\| PubMed ID: 18469823	2008
Experimental characterization of 3D localization techniques for particle-tracking and super-resolution microscopy. Optics express\| PubMed ID: 19434159	2009
Sample drift correction in 3D fluorescence photoactivation localization microscopy. Optics express\| PubMed ID: 21934862	2011

Michael J. Mlodzianoski

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