Nature Methods

Fiji: an open-source platform for biological-image analysis

Johannes Schindelin, Ignacio Arganda-Carreras, Erwin Frise, Verena Kaynig, Mark Longair, Tobias

Pietzsch, Stephan Preibisch, Curtis Rueden, Stephan Saalfeld, Benjamin Schmid, Jean-Yves Tinevez,

Daniel James White, Volker Hartenstein, Kevin Eliceiri, Pavel Tomancak & Albert Cardona

Supplementary Figure 1 Organization of Fiji plugin folder

Supplementary Figure 2 Scripting in Fiji

Supplementary Figure 3 ImgLib code examples

Supplementary Table 1 Overview of Fiji plugins

Note: Supplementary Videos 1–4 are available on the Nature Methods website.

Nature Methods: doi:10.1038/nmeth.2019

Supplementary Figures

Command Launcher

Supplementary Figure 1: Fiji commands The Fiji application packs several hundred commands,organized by category. Each command executes a plugin. Above, a snapshot of the Registrationcategory. The separation of hundreds of commands into categories relevant to biological researchersgreatly aids new users in the discovery of such commands. Most Fiji plugins appear as commandsunder the Plugins Menu; others are inserted into the other menus inherited from ImageJ. For best easeof use, a Command Finder command (below), launched via the keybinding l (lowercase L), providesthe means to find and launch any command with a real-time search that responds to keystrokes. Wherea plugin appears in the menu is specified in the plugins.config file packed in the plugin’s jar file. Anyplugins that are not part of Fiji but which adhere to this ImageJ convention will integrate transparentlywith the Fiji menus.

Nature Methods: doi:10.1038/nmeth.2019

Supplementary Figure 2: Scripting in Fiji. An example of a simple Jython script that achieves rela-tively simple task of swapping the channels (using ImgLib lines 15 and 16) but is able to apply it to adirectory of images using file manipulation commands inherent to the scripting language.

Nature Methods: doi:10.1038/nmeth.2019

Supplementary Figure 3: Examples of ImgLib code. (a) Screenshot of the Script Editor with ImgLibcode that computes Difference of Gaussian (DoG) for float type input images with any number of dimen-stions. (b) Screenshot of the Script Editor with ImgLib code that computes Maximally Stable ExtremalRegions (MSER) for float type input images with any number of dimenstions.

Nature Methods: doi:10.1038/nmeth.2019

PluginsAnalysis3D ObjectsCounter

Quantification of connected components in 3D

Analyze Skele-ton

Calculate statistics on skeletonized objects in bi-nary images

Colocalization Calculate Pearson and Manders coefficients andmake scatter plots for colocalization analysis

Coloc 2 Calculate Pearson, Manders and other colocal-ization coefficients and make scatter plots forcolocalization analysis

Delaunay/Voronoi Provide a triangulation or Voronoi diagram for agiven set of points

Directionality Quantify direction in images with oriented tex-tures

Exact EuclideanDistance

A linear-time exact algorithm for multi-dimensional Distance Transform

FlowJ Optical flow analysisIsoData Classi-fier

Histogram-based multi-level thresholding

Local Thickness Quantification of linear structures’ thickness intwo or three dimensions

PIV Analyser Particle Image Velocimetry, an optical flow algo-rithm

Shape IndexMap

Local surface shape classification

Shape-basedAveraging

Make averages of multiple images preservingshapes

Skeletonize 3D Skeletonize 3D objects in binary image stacksSurfaceness Study the morphology of volumetric data con-

taining surface-like structuresTemporal ColorCoder

Color-coded maximum intensity projection oftime series

TopoJ Analysis of surface topologyTubeness Enhance tube-like structures in volumetric data

Image transformation3D Morphology Morphological operations on 3D binary imagesAnisotropic Dif-fusion

Edge-preserving blurring

Bilateral Filter An edge-preserving Gaussian blurColour Decon-volution

Identification of fluorophores by color

Differentials Differential operatorsDynamicReslice

Make a kymograph along a line of interest, up-dated interactively

Extended DepthField

Stack focusing, i.e. make in-focus images fromstacks where each (x,y) coordinate is in focus inonly one or two z slices

Nature Methods: doi:10.1038/nmeth.2019

Gray Morphol-ogy

Morphological operations on grayscale images

Radial Reslice Transform to polar coordinatesTransformJ Arbitrary affine transformations

Image ArithmeticCalculator Plus Advanced arithmetic operations on two imagesImage Expres-sion Parser

Application of free-form terms (e.g. A + 2 ∗ B −gauss(A, 0.5), where A denotes an image).

Image enhancingCLAHE Contrast Limited Adaptive Histogram Equaliza-

tion method for enhancing the local contrast ofan image.

Frangi Vessel-ness

Frangi’s filter to enhance linear structures innoisy images

Kalman StackFilter

Kalman’s temporal denoising method

Kuwahara Filter Kuwahara’s denoising filterRetinex Perception-based contrast and color enhancingROF Denoiser Image denoising based on the paper by Rudin,

Osher and Fatemi

TrackingManual Track-ing

Manual tracking of multiple objects in time series

MTrack2 Tracking of multiple objects in time seriesToAST Automated tracking of sporozoids in time series

SegmentationAuto Threshold Reusable histogram-based global and local

threshold calculationColour Thresh-old

Threshold segmentation based on colors, e.g.selecting ranges of hues and ranges of satura-tion

FeatureJ Classical differential image featuresGraphCut Graph-based segmentationLasso and BlowTool

interactive tools adapting the selection to thepixel intensities

Level Sets Segmentation based on Level Sets (Active Con-tours)

Linear Kuwa-hara

Enhancement of linear structures based on theKuwahara filter

Robust Adap-tive ThresholdSelection

Local gradient-based threshold selection

SIOX Segmen-tation

Color segmentation based on example segmen-tations

Nature Methods: doi:10.1038/nmeth.2019

Simple NeuriteTracer

Semi-interactive segmentation of fiber-like struc-tures, as well as analysis of the resulting data.

Snakuscule Circular active contoursStatistical Re-gion Merging

Automatic Region Merging based on a statisticaltest of the intensity differences

TrainableSegmenta-tion/WekaSegmentation

Advanced machine learning technique to extracta segmentation model from example segmenta-tions

RegistrationbUnwarpJ Automatic bijctive elastic registration of 2D im-

agesAlign images bya common line

Rigid registration according to line selections intwo images marking the same structure

Moving LeastSquares

Landmark-based registration, interpolating thetransformation as rigidly as possible

Register VirtualStack Slices

Register unaligned slices of one stack, with min-imal memory requirements

Rigid Registra-tion

Automatic and manual rigid registration of two 3Dimages

SPIM Registra-tion

Register multiple 3D images of the same sample,recorded from different angles

StackReg Automatic 3D alignment of two image stacksTurboReg Automatic 2D alignment of two imagesVIB Protocol The Virtual Insect Protocol aims to facilitate

anatomical studies from multiple samples

StitchingMosaicJ Semi-automatic stitching of 2D image mosaicsStitching 2D/3D Automatic stitching of 3D image mosaics

AnnotationArrow Tool Draw and adjust arrowsSeries Labeler Versatile time stamperStack Manipula-tion

Reorder, recombine, interleave stacks and otherstack-specific operations

Sync Win Track current mouse coordinates in other images

Visualization3D/4D Viewer Using Java3D for hardware-accelerated 3D/4D

display of imagesDichromcay Simulating color blindnessInteractive 3DSurface Plot

Interactively display a 2D image as a height field

Multi Kymo-graph

Kymographs (intensity over time plots) fromstacks and line selections

Panoramaequirectangularview

Visualize panorama images in a pannable view

Nature Methods: doi:10.1038/nmeth.2019

Record Screen Make a movie from a window, e.g. for screen-casts

Replace Red byMagenta

A tool to fix color blind-unfriendly red/green im-ages

Video Editing Basic video editing tools for editing screencastsView 5D Display up to 5D volumetric datasets, offering

convenient data inspection, basic particle track-ing, and more

Volume Viewer Volume rendering without requiring hardware ac-celeration

Input/outputAmiraReader/Writer

Load and save 3D images in the Amira file format

AnalyzeReader/Writer

Load and save files in the Analyze file format

Animated GIFReader/Writer

Load and save image stacks as animated GIFfiles

Bio-Formats Read/write support for 100 file formats com-monly used by microscope vendors’ software

BioradReader/Writer

Load and save images in the format written byBiorad’s software

DF3Reader/Writer

Load and save files in the Povray texture format

DM3 Reader Import files produced by the Gatan Digital Micro-graph

IPLab Reader Import files in the IPLab formatExport EPS Write images as .eps files for publicationLSMReader/Toolbox

Special-purpose I/O for the file format used byZeiss

Nrrd Reader Reader for the Nearly Raw Raster Data formatPDF Reader Render a PDF document into an image stackPDF Writer Make a .pdf file from one or more imagesReconstructReader

Import Reconstruct projects as TrakEM2 projects

SVG Reader Render SVG into a raster image

ScriptingBeanshell, Clo-jure, Javascript,JRuby, Jython

Support for scripting and interactive evaluation ofcommands

Scripting Exam-ples

Fiji comes with a large number of examples thatcan be adjusted to one’s own needs

Java as a”script” lan-guage

For rapid prototyping, Fiji can treat simple Javaclasses as if they were scripts

Script Editor A versatile editor helping to write scripts

Working with Fiji

Nature Methods: doi:10.1038/nmeth.2019

Automatic classversion transla-tor

Make it possible to run plugins compiled for Java1.6 on older Java versions (e.g. on MacOSX ver-sions prior to 10.6)

Bug Submitter Provide an easy and convenient way to submit abug report

Context help Open the Fiji Wiki page corresponding to a givenmenu item

Fiji Updater Effortless and user-friendly way to stay up-to-date

IJ Robot Automate mouse clicks and keyboard pressesMultiple ImageProcessor

Batch process images in a directory output to an-other directory

Upload SampleImage

Allow users to upload large sample images foruse by the Fiji developers

InteroperabilityMiji Provide a user-friendly way to use Fiji from within

Matlab

MiscellaneousImageJ/ImageJA A custom ImageJ version that frequently con-

tains cutting edge features which are then inte-grated into ImageJ

QuickPALM Software support for Photo-Activated Light Mi-croscopy (superresolution microscopy)

RandomJ Generate artificial noise adhering to given pa-rameters

Thread killer Stop runaway processesTrakEM2 Work with large mosaics, e.g. obtained via EM

(segmentation, stitching, registration and muchmore)

Developing FijiEclipse Net-beans IntelliJsupport

Fiji has support for popular development environ-ments

Tutorial Maker A very fast way to make tutorials in Fiji and up-load them to the Fiji Wiki

Table 1: Overview of Fiji plugins The table list all plugins currently provided by the Fiji Updater systemtogether with a short description. Plugins developed specifically for Fiji are highlighted in blue (77 of111 total). The remaining plugins are inherited or selected from ImageJ.

Nature Methods: doi:10.1038/nmeth.2019