GDSC Single Molecule Light Microscopy (SMLM) ImageJ Plugins¶

GDSC SMLM plugins provide various tools for single molecule localisation analysis.

The tools are easily installed into ImageJ. The library works for Java 1.8+.

The code is hosted on GitHub.

Citation¶

GDSC SMLM: Single-molecule localisation microscopy software for ImageJ [version 1; peer review: 3 approved].
Etheridge TJ, Carr AM and Herbert AD.
Wellcome Open Res 2022, 7:241

Overview¶

Super-resolution microscopy can be used to obtain a higher level of resolution from an image than conventional light microscopy.

In conventional light microscopy the subject is illuminated and all the light interacting with the subject is captured simultaneously into an image. The resolution is limited since the optics of the microscope cannot focus the light perfectly.

Super-resolution microscopy uses small molecules that can be activated to emit light. If a single molecule is activated its position can be localised using software analysis. Using thousands of localisations it is possible to reconstruct a virtual image of the subject. This increases the image resolution to the precision of the localisation method. The improvement depends on the imaging conditions but is often 5-10 times higher.

Table 1 shows the increased resolution of single molecule localisation microscopy. The difference is most noticeable where structures meet since the single molecules have a cleaner signal when activated individually than the combined signal of all the molecules together.

Table 1 Example images of standard and single molecule localisation microscopy¶
(A) Standard image	(B) Super-resolution image
(C) Standard image 8x	(D) Super-resolution image 8x

The GDSC Single Molecule Light Microscopy (SMLM) plugins provide various tools for single molecule localisation analysis.

Fit an image, or series of images, using a 2D Guassian Point Spread Function (PSF)
Save results to a table, a file, an image and/or to memory
Trace or cluster localisations through time to identify molecules
Correct drift in long time course images
Estimate fluorophore dark-time and blinking rate
Create localisation density images
Create custom PSFs from calibration images
Create simulated data using a Gaussian or custom PSF with configurable molecule populations and diffusion
Calibrate the gain and read noise of your microscope camera
Estimate noise in an image
Estimate resolution using Fourier Ring Correlation (FRC)

The following plugins are available:

Fitting Plugins¶

Simple Fit	Performs fitting on an image to generate a table of molecule localisations and an image reconstruction. Provides a step through guide for initial configuration of microscope parameters
Peak Fit	Performs fitting on an image to generate a list of molecule localisations.
Template Manager	Allows management of templates for the fitting configuration.
Fit Configuration	Allows setting the fitting parameters and saving them to file.
Peak Fit (Series)	Performs fitting on folder of images to generate a list of molecule localisations.
Spot Finder	Identifies candidate maxima in an image.
Spot Finder (Series)	Identifies candidate maxima in a folder of images.
Spot Finder (Preview)	Shows an interactive view of the candidates that will be identified in an image.
Fit Maxima	Performs PSF fitting on candidate maxima.
Gaussian Fit	Allows interactive identification of maxima using filters and 2D Gaussian fitting.
Fail Count Manager	Provides analysis of the fail count settings used in the `Peak Fit` plugin.
Spot Fit Tool	Allows interactive fitting of clicked spots on an image.

Results Plugins¶

Results Manager	Allows conversion of the localisation results into different formats, e.g. files or images.
Batch Load Results	Allows multiple files to be loaded into memory.
Batch Save Results	Allows multiple results sets to be saved to file.
Summarise Results	Displays a summary of the results held in memory.
Clear Memory Results	Removes all results held in memory.
Clear Memory Results (Multi)	Removes selected results held in memory.
Rename Results	Allows the results sets to be renamed.
Resequence Results	Allows the frame number of results to be rebuilt assuming a repeating pattern of data and non-data frames.
Calibrate Results	Allows results held in memory to be calibrated.
Update Results Bounds	Allows the bounds of the results to be updated.
Convert Results	Allows results held in memory to be converted to different units.
Show Results Header	Shows the header information from any support localisation results file format.
Overlay Results	Draws an overlay on an image using all the localisations from a results dataset.
Load Localisations	Loads localisation data from a delimited text file in a user-specified format.
Save Localisations	Saves localisation data to a delimited text file in a user-specified format.
Trace Exporter	Export traced datasets to file.
Track Population Importer	Imports track categories from file and assigns them to an existing track dataset.
Filter Results	Filters a list of localisations using signal strength, coordinate shift and PSF width.
Crop Results	Filters a set of localisations using a 2D bounding rectangle.
ROI Crop Results	Filters a set of localisations using any `ImageJ` region of interest (ROI).
Free Filter Results	Filters a list of localisations using a custom filter specified using a text description. Multiple filters can be combined with AND/OR operators.
Filter Molecules	Filters a set of molecules using various criteria.
Split Results	Splits a set of localisation results using a 2D mask.
Translate Results	Allows the localisations in a dataset to be translated.
3D Results Viewer	Show an interactive 3D view of the localisations in a dataset.
Results Match Calculator	Calculate the match statistics between two results sets.
Trace Match Calculator	Calculate the match statistics between two sets of traced molecules.
Classification Match Calculator	Calculate the match between two classifications of the same localisations.
Spot Inspector	Extracts the fitted spots from an image into a stack ordered by the user-selected score.
Yeast Mask	Creates a mask of a fission yeast cell (S. pombe).
Depth Mask	Creates a 3D mask using three 2D masks to define the shape.
Nucleus Mask	Creates a 3D mask using spheres to model a nucleus of a cell.

Analysis Plugins¶

Drift Calculator	Corrects stage drift using: sub-image alignment; fiducial markers within an image; reference stack alignment; or a drift file.
Trace Molecules	Traces molecules through time using time and distance thresholds (using a type of single-linkage clustering).
Cluster Molecules	Clusters molecules through time using time and distance thresholds (using centroid-linkage clustering).
Dynamic Trace Molecules	Traces localisations through time and collates them into traces using a probability model to connect localisations.
Trace Molecules (Multi)	Allows the `Trace Molecules`, `Cluster Molecules` and `Dynamic Trace Molecules` plugins to be run with multiple input datasets.
Trace Diffusion	Trace molecules through consecutive frames. Mean-squared displacement analysis is performed on the traces to calculate a diffusion coefficient.
Trace Diffusion (Multi)	Allows the `Trace Diffusion` plugin to be run with multiple input datasets.
Trace Length Analysis	Analyses the track length of traced data.
Track Population Analysis	Analyses the local diffusion of tracks within a window and classifies tracks into sub-populations based on diffusion metrics.
Residence Time Analysis	Analyses the residence time of stationary (bound) molecules.
OPTICS	Runs the OPTICS algorithm to perform interactive density-based clustering of localisation data.
DBSCAN	Runs the DBSCAN algorithm to perform interactive density-based clustering of localisation data.
Draw Clusters	Draws collections of localisations with the same ID on an image, for example the output from `Trace Molecules` or `Cluster Molecules`.
Density Image	Calculates the local density around localisations and displays an image coloured using the density score.
Dark Time Analysis	Determines the maximum dark time for a fluorophore from localisation data.
Blink Estimator	Estimate the blinking rate of fluorophores in a results set.
Time Correlated (TC-PALM) Analysis	Performs time correlated photo-activated light microscopy (TC-PALM) analysis.
Neighbour Analysis	Saves all localisations paired with their neighbour (if present) to file.
Filter Analysis	Performs filtering on a set of categorised localisation results and computes match statistics for each filter.
Create Filters	Used to prepare a large set of filters for use in the `Filter Analysis (File)` plugin.
Filter Analysis (File)	Performs filtering on a set of categorised localisation results and computes match statistics for each filter defined in the input file.
Spot Analysis	Allows analysis of the signal and on/off times for fixed fluorophore spots in an image stack.
Spot Analysis (Add)	This plugin provides a named plugin command for the `Add` button of the `Spot Analysis` plugin.
Crosstalk Activation Analysis	Perform crosstalk activation analysis of photo-switchable probes under pulsed light activation.
Pulse Activation Analysis	Perform multi-channel super-resolution imaging by means of photo-switchable probes and pulsed light activation.
Density Estimator	Estimates the local density using NxN squares around localisations.
Fourier Image Resolution	Analyses the resolution of an image using Fourier Ring Correlation.
FIRE Q Estimation	Estimate the spurious correction factor Q for use in `Fourier Image Resolution` (FIRE) analysis.

PC PALM Plugins¶

PC-PALM Molecules	Prepare a set of localisations for Pair Correlation analysis.
PC-PALM Analysis	Produce Pair Correlation curve for a set of localisations selected from a super-resolution image.
PC-PALM Spatial Analysis	Performs spatial analysis to plot the molecule density around each localisation as a function of distance from the localisation.
PC-PALM Save Results	Saves all the PC-PALM results held in memory to a results folder.
PC-PALM Load Results	Load all the PC-PALM results from a results folder to memory.
PC-PALM Fitting	Combine multiple Pair Correlation curves and fit them using models for random or clustered distributions.
PC-PALM Clusters	Find clusters of molecules using a partial centroid-linkage hierarchical clustering algorithm.

Model Plugins¶

PSF Creator	Extracts the PSF from a Z-stack image of markers, e.g. quantum dots or fluorescent beads.
PSF Drift	Computes the drift in a PSF image.
PSF Combiner	Combines several PSF images into a single average PSF.
PSF HWHM	Diplays the half-width at half-maxima (HWHM) curve for a PSF image.
Cubic Spline Manager	Allows management of the cubic spline models of point spread functions (PSFs).
Astigmatism Model Manager	Allows management of 2D Gaussian astigmatism models for a 2D astigmatic PSF.
Create Data	Creates an image by simulating single molecule localisations using a model of photoactivated diffusing fluorophore complexes. The PSF can be simulated or real using an input PSF image
Create Simple Data	Creates an image by simulating single molecule localisations at a specified density.
Create Track Data	Creates an image by simulating single molecule localisations diffusing in tracks that do not overlap in time.
Create Benchmark Data	Creates an image by simulating single molecule localisations in a fixed location.
Fit Benchmark Data	Fit the image created by `Create Benchmark Data` and compute statistics on the accuracy and precision of fitting.
Benchmark Analysis	Compute statistics on the accuracy and precision of fitting using different methods. Statistics are only computed for all the localisations that were fit successfully by each method.
Create Spot Data	Creates a sparse image by simulating zero or one localisation per frame.
Load Benchmark Data	Load benchmark data using an open image and a localisations text file.
Filter Spot Data	Filter the image created by `Create Simple Data` or `Create Spot Data` and compute statistics on the accuracy and precision of identifying spot candidates.
Filter Spot Data (Batch)	Run a batch of filters through the analysis of `Filter Spot Data`.
Fit Spot Data	Fits all the candidate spots identified by the `Filter Spot Data` plugin.
Benchmark Filter Analysis	Run different filtering methods on a set of benchmark fitting results outputting performance statistics on the success of the filter.
Benchmark Filter Parameters	Run different filter control parameters on a set of benchmark fitting results outputting performance statistics on the success of the filter.
Iterate Filter Analysis	Iterates the `Benchmark Filter Analysis` and `Benchmark Filter Parameters` plugin to optimise performance on a set of benchmark fitting results.
Score Filter	Scores a filter against a set of benchmark fitting results.
Doublet Analysis	Fits candidates in a benchmark localisation image as a single or double spot (doublet) and scores the results.
Doublet Filter Analysis	Filters all the fit results produced by the `Doublet Analysis` plugin using the specified filter settings and scores the results.
Smart Spot Ranking	Compare methods for classifying spot candidates.

Calibration Plugins¶

PSF Calculator	Allows calculation of the Point Spread Function (PSF) size given the microscope imaging parameters.
PSF Estimator	Estimates the PSF by performing fitting on a sample image.
Mean-Variance Test & Mean-Variance Test (EM-CCD)	Opens a folder of images and computes a Mean-Variance test to determine the gain and read noise of the microscope camera. Gain can be calculated for standard or Electron Multiplying (EM) cameras
EM-Gain Analysis	Analysis a white light image from an EM-CCD camera and fits a model to obtain the bias, EM-gain, read noise and average photons per pixel.
EM-Gain PMF	Displays a plot of the probability mass function (PMF) of the expected value of a pixel on an EM-CCD camera given an average number of photons.
sCMOS Analysis	Analyse the per pixel offset, variance and gain from a sCMOS camera.
Camera Model Analysis	Model the on-chip amplification from an EM-CCD camera, CCD or sCMOS camera.
Camera Model Fisher Information Analysis	Model the Fisher information from an EM-CCD camera, CCD or sCMOS camera.
Camera Model Manager	Allows management of per-pixel camera models for the fitting configuration.
Diffusion Rate Test	Simulate molecule diffusion and fit a graph of mean-squared displacement to determine the diffusion coefficient.

Tool Plugins¶

In addition to the principle plugins for localisation fitting and analysis there are several utility plugins provided.

Smooth Image	Performs smoothing on an image (identical to that performed when identifying maxima).
Binary Display	Switches an image to binary (white/black) to allow quick visualisation of localisations.
Reset Display	Resets a binary image back to the standard display.
Pixel Filter	Perform filtering to remove hot pixels from an image.
Noise Estimator	Estimate the noise in an image using various methods.
Background Estimator	Estimate the background in a region of an image.
Median Filter	Compute the median of an image, on a per-pixel basis, using a rolling window at set intervals.
Image Background	Produces a background intensity image and a mask from a sample in vivo image. This can be used to simulate realistic fluorophore distributions.
Overlay Image	Allow an image to be added as an overlay with a transparent background.
Image Kernel Filter	Convolve an image with a kernel from another image.
TIFF Series Viewer	Opens a TIFF image as a read-only virtual stack image. Images can be extremely large.

Toolset Plugins¶

These plugins provide quick access to the various SMLM plugins via ImageJ toolsets and a customisable SMLM Tools window.

Install SMLM Toolset	Adds an `ImageJ` Toolset containing common commands that can be used from the `ImageJ` toolbar.
Show SMLM Tools	Display a `SMLM Tools` window with buttons to run plugins.
Create SMLM Tools Config	Create a configuration file allowing the `SMLM Tools` panel to be customised.
SMLM Macro Extensions	Register functions that can be used in the `ImageJ` macro language.

All of the plugins can be incorporated into ImageJ macros to allow automation of image analysis workflows.

Contents¶

Index¶

Issues¶

Please file a bug report on GitHub.