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CZI Workshop I

August 20th, 2021

Ellen Dobson

ellen.dobson@wisc.edu

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Center for Open Bioimage Analysis

openbioimageanalysis.org/

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Introduction to ImageJ

Getting Started with the ImageJ User Application

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What is ImageJ?

Open source tool for scientific analysis
End user application, a tool for image science
Reusable set of libraries & shared framework for image analysis
Multiple ‘flavors’ of ImageJ…

ImageJ

ImageJ2

Fiji

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What is ImageJ?

Open source tool for scientific analysis
End user application, a tool for image science
Reusable set of libraries & shared framework for image analysis
Multiple ‘flavors’ of ImageJ…

ImageJ

ImageJ2

Fiji

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What is ImageJ?

stable version of ImageJ(1.x) which has been under continuous development since 1997
developed in 1997 as a cross-platform version of NIH Image
ImageJ2 includes the latest version of ImageJ(1.x) and a legacy layer for backwards compatibility

transparently converts between IJ1 and IJ2 data structures as needed

https://imagej.nih.gov/ij/

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Fiji is just ImageJ

Is an image processing package—a "batteries-included" distribution of ImageJ2
FOR USERS:

easy to install
has an automatic update function
bundles a lot of plugins
offers comprehensive documentation.

FOR DEVELOPERS:

open source project hosted in a Git version control repository
access to the source code of all internals, libraries and plugins
eases the development and scripting of plugins.

https://imagej.net/Fiji

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What can Fiji do?

Tracking

Segmentation

Big Data

Stitching

and more...

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Learn how to fish...

https://imagej.net/

GitHub repository:

https://github.com/imagej/imagej.github.io

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Teach me how to fish...

https://forum.image.sc/

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Teach me how to fish...

https://forum.image.sc/

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Let’s open/run Fiji...

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CTRL + L: Search Bar

Edit ▶ Options ▶ Search Bar... ▶ Pressing L focuses the search bar

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The Main Window

Tip: click on the status bar
Tip: right / double-click on Tools

Getting Started page of the ImageJ wiki

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Staying Up-To-Date

Updater page of the ImageJ wiki

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Staying Up-To-Date

Following an Update Site

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Opening Data…

Drag and Drop

File ▶ Open…

File ▶ Import

▶ Bio-Formats

Importing Image Files page of the ImageJ wiki

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Image Window

Zoom factor (use +/- keys)

Image Type

Image Size in physical units (and pixels)

Dataset positions

File ▶ Open Samples ▶ Mitosis (26MB, 5D Stack)

ImageJ User Guide: Image

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Bit Depth & Pixel Types

8-bit = 256 levels
12-bit = 4096 levels
16-bit = 65536 levels

Qualitative/”Just looking”

Quantitative/Measurements

ImageJ User Guide: Image Types & Formats

https://www.olympus-lifescience.com/en/microscope-resource/

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Lookup Tables (LUTs)

Image ▶ Lookup Tables
Image ▶ Colors ▶ Display LUTs

ImageJ User Guide: Lookup Tables

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Helpful LUTs

Grays

HiLo

Royal

ImageJ User Guide: Lookup Tables

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Get to Know Your Data…

File ▶ Open Samples ▶ Boats
Analyze ▶ Histogram

ImageJ User Guide: Analyze

What would cause this histogram?

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Profile Plots

File ▶ Open Samples ▶ Blobs (Shift + B)
Draw a line using the Line tool
Analyze ▶ Plot Profile

Image ▶ Stacks ▶ Plot Z-Axis Profile...

ImageJ User Guide: Plot Profile

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2D Visualization

File ▶ Open Samples ▶ Mitosis (26MB, 5D Stack)
Image ▶ Lookup Tables ▶ Magenta
Image ▶ Color ▶ Channels Tool (Shift + Z)
Image ▶ Properties… (Shift + P)

ImageJ User Guide: Stacks

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2D Visualization: Brightness & Contrast

ImageJ User Guide: Brightness/Contrast...

Defining grey value range to be visualized

File ▶ Open Samples ▶ Boats
Image ▶ Adjust ▶ Brightness/Contrast… (Shift + Command + C)

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Thresholding

ImageJ User Guide: Thresholding

Isolate grey values of interest

File ▶ Open Samples ▶ Blobs (Shift + B)

Image ▶ Adjust ▶ Threshold…

Which method is best?

Image ▶ Adjust ▶ Auto Threshold, Try All

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Regions of Interest (ROI)

ImageJ User Guide: Selections

File ▶ Open Samples ▶ Clown (14K)
Freehand selection tool
Circle the clown nose

Analyze ▶ Measure (Ctrl + M)

Can you draw the same exact circle on a new clown??

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Regions of Interest (ROI)

ImageJ User Guide: ROI Manager

Select a nose
Press T or Analyze ▶ Tools ▶ ROI Manager (Ctrl + T)
Select other clown image
Click the ROI in manager or Edit ▶ Selection ▶ Restore Selection

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Plugins...

What is a plugin?

A special-purpose software component that extends functionality

There are four tiers of plugins:

Core ImageJ plugins (1000+)
Core Fiji plugins (1000+)
Plugins installable from Update Site.
Additional manually-installed plugins available from various online sources

Plugins page of the ImageJ wiki

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Colocalization in Fiji...

Fiji Plugin = Coloc 2

pixel intensity correlation over space methods of Pearson, Manders, Costes, Li and more
scatterplots, analysis, automatic thresholding and statistical significance testing

Coloc2 page of the ImageJ wiki