Abstract
Three-dimensional structured illumination microscopy (3D-SIM) is a versatile and accessible method for super-resolution fluorescence imaging, but generating high-quality data is challenging, particularly for non-specialist users. We present SIMcheck, a suite of ImageJ plugins enabling users to identify and avoid common problems with 3D-SIM data, and assess resolution and data quality through objective control parameters. Additionally, SIMcheck provides advanced calibration tools and utilities for common image processing tasks. This open-source software is applicable to all commercial and custom platforms, and will promote routine application of super-resolution SIM imaging in cell biology.
Highlights
The last decade has witnessed the spread of super-resolution light microscopy techniques that surpass the long-standing diffraction limit of optical resolution[1,2,3]
We describe SIMcheck, a user-friendly and simple solution to this challenge, which lowers the activation energy required for less experienced users and helps specialists and non-specialists to capture excellent quality 3D-SIM data and interpret it correctly
Raw and reconstructed data from any commercial SIM platform can be analysed with several options, and produce a variety of quality check outputs, a log text containing important metrics, and an output table with assessment of the data quality based on key statistics
Summary
The last decade has witnessed the spread of super-resolution light microscopy techniques that surpass the long-standing diffraction limit of optical resolution[1,2,3]. Linear two- or three-dimensional structured illumination microscopy (SIM, 3D-SIM) provides multicolour optical sectioning with double the conventional resolution in the lateral[5,6] and axial[7,8] dimensions allowing fast 3-dimensional imaging of fixed and live specimens[9,10,11,12]. SIMcheck consists of a suite of plugins for the widely-used open-source application ImageJ17 It provides tools for assessment of the resolution, image quality and identification of sources of errors and artifacts in SIM imaging, as well as simplifying calibration and image handling steps. Performs tests for stability and alignment of phases (bead lawn recommended) pattern frequency (n) Phase step size (radians)
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