GDSC Single Molecule Light Microscopy (SMLM) ImageJ Plugins

Build Status Coverage Status Documentation Status License: GPL v3

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.

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
example_image_standard_jpg
(B) Super-resolution image
example_image_superres_jpg
(C) Standard image 8x
example_image_standard_8x_jpg
(D) Super-resolution image 8x
example_image_superres_8x_jpg

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.

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 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.

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 candidate spots identified by the Filter Spot Data plugin 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.