Abstract
We present an open source, real-time data analysis and rendering tool for super-resolution imaging techniques that are based on single molecule detection and localization (e.g. stochastic optical reconstruction microscopy - STORM and photoactivation localization microscopy – PALM). The recent availability of commercial STORM and PALM microscopes has made these techniques accessible to a wide range of researchers. However, the availability of high speed data analysis and rendering software lags behind, requiring researchers to develop their own analysis platforms or rely on commercial ones. We implemented GraspJ (GPU-Run Analysis for STORM and PALM), an ImageJ plug-in with a convenient user interface, that allows high accuracy localization of single molecules as well as processing and rendering of high resolution images in real-time. GraspJ includes several features such as drift correction, multi-color, 3D analysis/rendering, and is compatible with a large range of data acquisition software. In addition, it allows easy interfacing with other image processing tools available with ImageJ. Overall we believe that GraspJ will be a valuable tool for the super-resolution imaging field.
Highlights
In the past decade, a number of techniques have been developed that overcome the diffraction limit in fluorescence microscopy (Hell, 2007, Bates et al, 2008)
The speed achieved by QuickPALM falls short of real-time analysis when high fluorophore densities and fast acquisition speeds are needed (e.g. 60 Hz or higher speeds used for live cell imaging (Jones et al, 2011))
When used for real-time analysis, QuickPALM is only compatible with one data acquisition software, which is a drawback for incorporating the data analysis with pre-existing acquisition software. rapidSTORM is another open source program that uses a more accurate algorithm for determining molecule positions and that can achieve high speed analysis of super-resolution data (Wolter et al, 2010)
Summary
A number of techniques have been developed that overcome the diffraction limit in fluorescence microscopy (Hell, 2007, Bates et al, 2008) Among such techniques, those that are based on single molecule photoactivation and localization (e.g. STORM, PALM, fPALM) depend heavily on data processing and analysis to produce high resolution images. RapidSTORM is another open source program that uses a more accurate algorithm (nonlinear least squares fitting of Gaussian PSFs) for determining molecule positions and that can achieve high speed analysis of super-resolution data (Wolter et al, 2010). There is still need for open source, easy to use, high speed and high accuracy software packages that include essential data processing features and are compatible with a wide range of data acquisition software Such analysis tools will make super-resolution microscopy more accessible to researchers
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