Image Analysis with Fiji

What is ImageJ?

A tool for scientific image analysis

  • Open source
    • You wouldn't write:
      Added 500mg of unknown chemical.
    • So don't put your data in a black box!
  • Rich ecosystem: thousands of plugins

Plugins ▶ Utilities ▶ Make Fiji Package

Fiji Is Just ImageJ

Learn how to fish

Learn how to fish

Teach me how to fish!

Ctrl + L: Search Bar

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

Ctrl + L: find commands

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

The main window

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

Staying up-to-date

Memory management

Edit ▶ Options ▶ Memory & Threads

Plugins ▶ Utilities ▶ Monitor Memory

Opening data

Drag and Drop
File ▶ Open...

File ▶ Import ▶ Bio-Formats

Pixel types

Pixel type pitfalls

  • Know the limitations of your data
    • File ▶ New ▶ Image... (32-bit, ramp, 20x20)
    • Process ▶ Math ▶ Multiply... : 100,000,000
    • Probe values
    • Process ▶ Math ▶ Add... : 1
    • Probe values
  • Can you find any problems?

Get to know your data

  • File ▶ Open Samples ▶ Boats
  • Analyze ▶ Histogram
  • Compare histograms:
    File ▶ Open Samples ▶ Blobs

What would cause this histogram? ➙

Profile Plots

Qualitative observation → Quantitative data

  • Open Blobs (Shift + B)
  • Use any Line tool
  • Analyze ▶ Plot Profile

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

Image processing principles

What does this image tell us about the volume of this pipette?

Image formats are not created equal!

Edit ▶ Options ▶ Appearance...

File ▶ Open Samples ▶ Adelsons Squares

File ▶ Open Samples ▶ Comparing Lengths

File ▶ Open Samples ▶ Straight Lines

Purves, D., Lotto, R. B., & Nundy, S. (2002). Why we see what we do. American Scientist, 90(3), 236-243.

1x

1/4x

No stripes?

1/12x

WTF?

Addressing Color Blindness ...

Many more details: Basics of Quantitative Image Analysis

Thresholding

Isolate values of interest

  • Open Blobs (Shift + B)
  • Image ▶ Adjust ▶ Threshold... (Shift + T)

Which method is best?

Image ▶ Adjust ▶ Auto Threshold, Try All

Regions of Interest (ROIs)


  • 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?

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

2D Visualization

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

3D Visualization

Image registration

Unify coordinates of 2+ images

  • File ▶ Open Samples ▶ Centipede Drawing
  • File ▶ Open Samples ▶ Centipede Mivart
  • Draw lines between equivalent points
  • Plugins ▶ Registration ▶ Align Image by line ROI

Segmentation

Identify blobs of interest

  • File ▶ Open Samples ▶ Blobs (25k)
  • Image ▶ Duplicate...
  • Image ▶ Adjust ▶ Auto Threshold
  • Process ▶ Binary ▶ Dilate (x2)
  • Process ▶ Binary ▶ Watershed
  • Analyze ▶ Analyze Particles...

Macros: Never forget again

Plugins ▶ Macros ▶ Record...

Further Reading