SIMcheck

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

• More information can be found on the Micron Oxford Website
• Further help is available here

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

If for some reason the ImageJ updater is not available, the jar file for the latest release can still be downloaded manually from the update site. If so, the file extension needs to be manually adjusted adn dependencies will need to be installed somehow.

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

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 | shows 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

Style Notes (1.0)

• no run-time dependencies other than ImageJ1
• 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
  • Rec Fourier & Stack FFT utility: tidy options, use RadioButtons
  • 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