About this Manual

Introduction

This manual is designed to be used in conjunction with a series of demonstration images to show users how to perform some basic and complicated analysis using the Fiji distribution of ImageJ. It is not an exhaustive list of techniques and methods by any stretch. It does however touch on many of the key components that most users will need in carrying out image analysis using Fiji.

Additionally, there is an advanced section on recording and writing macros for advanced and automated analysis.

The techniques presented in this manual, while biology based, can be applied to any type of image analysis. A blob is a blob, and they can all be segmented.

This manual has evolved over six editions (multiple sub-editions in number 6) gaining extra techniques, extra demo images and the section on programming. The current 6th Edition has extra detail added to the basic module sections and had more programming elements added to the advanced module section showing how to loop for regions, output useful log data and creating custom dialog boxes. A Small update was made, generating the 6.1 edition that changed screen shots to Windows 10 and includes extra features that had been added to Fiji. 6.2 and 6.3 added section on useful tools such as kymograms and histogram analysis. 6.4 added AI tools for cell counting and segmentation. 6.5 

Provided Resources

There is a selection of demo images and plugins available to be used in conjunction with this manual. They can be downloaded from the full project page here http://bit.ly/3Y3ujrN

The project page contains links to both a print quality, searchable PDF version of this manual and the resources file containing the demo images and plugins that need to be manually installed.

A Note on Image Analysis

This manual will demonstrate how to perform specific types of image analysis. The rationale behind the choices made in each method (where applicable) will be explained. Knowing which filters, methods, techniques etc. to use for a given analysis is something that cannot be easily taught. It comes from experience and a full understanding of the underlying mathematics of image analysis.

Distribution/Licence

All contents of this manual and the corresponding resources are licensed CC-BY 4.0 by the authors and contributors, unless mentioned otherwise. 

For any questions or support please contact Cameron Nowell (cameron.nowell@monash.edu).

Authors

This manual was written by Cameron J. Nowell over the course of many years (beginning in 2010) initially while at the Centre for Advanced Microscopy at the Ludwig Institute for Cancer Research, further expanded while working at the Centre for Dynamic Imaging at the Walter and Eliza Hall Institute of Medical Research with the current and previous (4th, 5th and 6th) editions at the Monash Institute for Pharmaceutical Sciences, Monash University.

While written by Cameron the content of this manual has evolved and been enhanced by the numerous suggestions of those people that have attended the many workshops that this manual has been the basis of. Without those suggestions it wouldn’t be what it is today.

Requirements

This manual relies on the users having the Fiji distribution of ImageJ installed. Fiji can be obtained from here

http://fiji.sc/

The base install is all that is required for most of this manual. But to fully carry out all the examples contained within the following plugins need to be installed. These can be added from the update sites part of the Fiji updater.

•  3D ImageJ Suite

• BAR

•  BIG-EPFL

•  CLIJ (CLIJ, CLIJ2 and assistants)

•  CSBDeep

•  IJPB-plugins

• Ilastik

• ImageScience

• Labkit

•  Morphology

• StarDist

Initial Setup

To be able to have results that match the screen shots in this manual, some of the default settings within Fiji need to be changed.

1.  Set the colours of foreground, background and selection to white, black and yellow respectively. Go to Edit → Options → Colours and configure the dialog box as follows

2.  Set the binary image options so that all images are assumed to have a black background (this stops most of the binary images that are generated looking black in white parts and vice versa). Go to Process → Binary → Options and set the dialog box as follows.