Single-molecule Plugins

Single-molecule Light Microscopy uses photo-activatable molecules that can be switched to a fluorescent state. If the number of active molecules at a single time is low then they can be imaged as small distanct spots. Software can be used to find the centres of the spots and then an image reconstructed from the positions of thousands of molecules. See the Super-Resolution example below:

Standard image 8x Super-resolution image 8x

Available Plugins

The GDSC Single Molecule Light Microscopy (SMLM) plugins provide various tools for single molecule localisation analysis. This includes PALM, STORM and other single molecule microscopy methods. Read the SMLM User Manual for full details.

Install using ImageJ2/Fiji

The SMLM plugins are distributed using an ImageJ2/Fiji update site. To install the plugins using Fiji (an ImageJ distribution) just follow the instructions How_to_follow_a_3rd_party_update_site and add the GDSC SMLM update site. Fiji will automatically check for a new version during start-up and install it if desired.

All the plugins will appear under the 'Plugins > GDSC SMLM' menu.

Install using ImageJ version 1

If you do not want to use ImageJ2/Fiji to get the plugins then you can download the latest Jar files from the update site and put them in your ImageJ plugins folder. The jars can be found here:

You will also need to install the additional Apache Commons Math library. This is already included in Fiji but for ImageJ 1 you can get the jar file here:

Source Code

The source code for the SMLM plugins can be found on GitHub.

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
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
Batch Fit Runs the Peak Fit analysis on an image for each configuration option in the batch
Spot Finder Identifies candidate maxima in an image
Spot Finder (Series) Identifies candidate maxima in a folder of images
Fit Maxima Performs PSF fitting on candidate maxima
Gaussian Fit Allows interactive identification of maxima and 2D Gaussian fitting

Results Plugins

Result Manager Allows conversion of the localisation results into different formats, e.g. files or images
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
Show Results Header Shows the header information from any support localisation results file format
Filter Results Filters a list of localisations using signal strength, coordinate shift and PSF width
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
Split Results Splits a set of localisation results using a 2D mask
Results Match Calculator Calculate the match statistics between two results sets
Trace Match Calculator Calculate the match statistics between two sets of traced molecules
Spot Inspector Extracts the fitted spots from an image into a stack ordered by the user-selected score

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)
Custer Molecules Clusters molecules through time using time and distance thresholds (using centroid-linkage clustering)
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 flourophore from localisation data
Blink Estimator Estimate the blinking rate of fluorophores in a results set as per the paper by [Annibale, et al (2011) PLoS ONE 6 e22678]
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
Fourier Image Resolution Analyses the resolution of an image using Fourier Ring Correlation
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. Performs pair-correlation analysis in the frequency domain as per the paper by [Sengupta, et al (2013) Nature Protocols 8 345-354] to produce a g(r) correlation curve.
PC-PALM Spatial Analysis Performs spatial analysis as per the paper by [Puchnar, et al (2013) PNAS 110 16015-16020]. This methods plots 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 Combiner Combines several PSF images into a single average 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 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
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
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
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
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
Load Localisations Loads the XYZ location file from Create Data into memory for analysis

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 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 as per the paper by [Ulbrich & Isacoff (2007) Nature Methods 4 319-321]
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
Diffusion Rate Test Simulate molecule diffusion and fit a graph of mean-squared displacement to determine the diffusion coefficient
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

Tool Plugins

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
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
Median Filter Compute the median of an image, on a per-pixel basis, using a rolling window at set intervals.
Overlay Image Allow an image to be added as an overlay with a transparent background

Full details of the SMLM plugins can be found in the Single Molecule Light Microscopy User Manual.

The software is in active development. For further details on the software please contact Alex Herbert.