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SIMcheck

SIMcheck is a package of ImageJ tools for assessing the quality and reliability of Structured Illumination Microscopy (SIM) data.

Copyright Graeme Ball and Lothar Schermelleh, Micron Oxford, Department of Biochemistry, University of Oxford. License GPL unless stated otherwise in a given file (in particular, SIMcheck uses modified versions of ImageJ's Slicer plugin and Paul Baggethun's Radial Profile Plot plugin).

Installation

ImageJ updater

The simplest way to install SIMcheck and keep it up to date on your system is using the ImageJ updater. SIMcheck is available via an ImageJ update site so you only need to activate it from the list of sites. See "How to follow an update site" for more details

Manual install

The jar file for the latest release can also be downloaded manually for cases where the ImageJ updater is not available. Older versions of SIMcheck can be obtained by navigating the SIMcheck update site.

Building from source

The project uses the maven build and dependency management tool, so to build it, run the following command (.jar file appears in target/):

mvn package

Note that the parallel fftj dependency must be copied to the local lib/ directory before building (no maven artifact for this is available).

In addition, SIMcheck is also released via Sonatype to ease its use by other projects:

<dependency>
  <groupId>uk.ac.ox.micron</groupId>
  <artifactId>SIMcheck</artifactId>
  <version></version>
</dependency>

Features

0: SIMcheck main dialog

  • dialog to choose and set up all checks with standard parameters
  • cropping utility for raw and reconstructed data
  • results: images will appear and key statistics will be logged
  • help button: link to instructions, help, documentation

1: Pre-processing, Raw Data Checks

Check Statistic(s) Comments
Intensity Profiles bleaching, flicker, angle intensity
Motion / Illum Var None: angle differences colored
Fourier Projections None: check pattern / spots OK
Modulation Contrast feature MCNR acceptable? Wiener estimate

2: Post-reconstruction Checks

Check Statistic(s) Comments
Intensity Histogram +ve/-ve ratio acceptable? top/bottom 0.01%
SA Mismatch stdDev of miniumum vs. mean OTF mismatch
Fourier Plots None: symmetry+profile OK?
Mod Contrast Map None: inspect MCNR of features green=saturated

3: Calibration Checks

Check Statistic(s) Comments
Illum. Phase Steps phase step & range stable? +k0, linespacing
Pattern Focus None: check for "zipper" pattern

4: Utilities

  • Format Converter for SIM data
  • Raw SIM Data to Pseudo-Widefield conversion
  • Threshold and 16-bit conversion (i.e. "discard negatives")
  • Stack FFT, performs 2D FFT on each slice

PROJECT STRUCTURE

  • README.md - This file
  • LICENSE.txt - Project's license
  • CHANGES.txt - History of versions and changes
  • pom.xml - maven Project Object Model describing dependencies, build etc.
  • src/main/java/ - Application sources (in SIMcheck/ package)
  • src/main/resources/ - Application resources (IJ menu config, html help text)
  • src/test/java/ - Test sources
  • src/test/resources/ - Test resources
  • target/ - output SIMcheck_.jar file
  • target/classes/ - build output .class files
  • target/test-classes/ - classes produced by tests
  • lib/ - local dependencies not available via maven (i.e. parallel FFTJ)

Style Notes (1.0)

  • no run-time dependencies other than ImageJ1 (Parallel FFTJ optional)
  • simple, modular structure - each check is a standalone plugin
  • plugin exec methods take input images and return ResultSet (no GUI calls within exec when easily avoidable)
  • ImageJ1-like preference for pre- java 5 features and reliance on float primitive type for most calculations

TODO

  • 1.2: post-release updates, bugfixes & refactoring

    • MCN: report per. angle MCNR & warn about saturated pixels
    • si2wf: add option to select 1 angle
    • raw FPJ: add target overlay? (hide by default)
    • progress bar for FPJ plugin (& others?)
    • MCN, show saturated pixels in raw data? (add to LUT?)
    • Rec Fourier: improve 3D FFT dependency (maven dep: IJ2? JTransforms?)
    • more crop utility tests, move to separate utility plugin
    • turn FTR profile into multi-color and/or plot
    • improve "Fourier Transform Phases" info / log output
    • angle labels etc. should be overlaid, not drawn

  • 1.3: PSF and OTF symmetry, extent, shape & order separation

  • 1.4: additional numerical stats, including resolution estimate

    • stat for motion & illumination variation
    • SIR Fourier: resolution estimate? stat for artifacts?
    • multi-frame: test / document stats for current time-point only?

  • 1.5: ImageJ2 reimplementation / headless running

    • get rid of IJ.run calls & intermediate results from window list
    • documentation: macro examples / document batch running

  • 1.6: check name updates & test suite

    • Rec Fourier: rename ortho to axial?
    • better name for spherical aberration mismatch?
    • expand sample / test data set
    • optional tests using failsafe plugin? (check data & pass/fail)

  • 2.0: integrated swing GUI control

SIM Reconstruction Problems & Remedies

(Kai Wicker)

  • Camera background: subtraction

  • optical distortions & uneven gain: flat-field correction

  • illumination intensity fluctuations: normalisation (after background subtraction)

  • sample drift between images: drift correction by means of cross-correlation

  • unknown grating period: find grating vector using component cross-correlations

  • fluctuations in grating phase: optimization of mixing matrix

  • fluctuation of order strengths between images

  • sample drift between focal slices: drift correction through cross-correlation

  • unknown zero grating phase: global phase via cross-correlation

  • drift between rotational orientations: drift correction through cross-correlation

  • unknown order strengths: compare different separated components