University Division/Office

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Introduction to scientific

image processing in FIJI

Lunch Seminar Series

Moritz Kirschmann Moritz.kirschmann@zmb.uzh.ch

Center for Microscopy and Image Analysis

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Table of Contents

1) What is Fiji and what can it do?

2) Open/import image data

3) Basics of the user interface and meta data

4) Changing color mode

5) Changing contrast

6) Inspecting stacks in orthogonal views

7) Z projections of stacks

8) 3D projections of stacks

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1. What is Fiji

- A distribution of ImageJ including many useful plug-ins for life sciences

- Open source

- Written in Java

- Under GNU General Public License

- Runs on Windows, MacOS, Linux

- Under daily development by a large community

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What can FIJI do?

- Reads many data formats (TIF, JPG, PNG, …), including proprietary

formats (CZI, LIF, DM3, DM4, STK, ICS, IMS, …)

- Image contrast and look-up-table modifications

- Modification of metadata such as pixel sizes

- Maximum intensity projections of image stacks

- Segmentation

- Counting objects

- Measuring intensities, length, areas, shape parameters and more

- Reconstruct objects from serial section

- Visualize in 3D

- […]

- everything is scriptable!

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Open/import image data

Single files:

Drag and drop your image file onto the FIJI window.

- simple file formats (PNG, TIF) open in a window directly.

- complex file formats (LIF, CZI, IMS, ICS…) will prompt a dialog box “Bio-

formats import options”.

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Don´t panic!

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Choose: view stack with: Hyperstack and leave Stack order: Default

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For files containing multiple stacks/time series (typically CZI, LIF) you can choose

which of these you want to open. Activating the Open all series checkbox opens

everything.

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This stitching option simply puts the tiles of an image mosaic together assuming no

overlap. There are more sophisicated ways of stitching in FIJI.

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Color mode: Composite shows all channels at the same time and assigns default colors. If

you want set your colors choose Color mode: Custom.

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Autoscale adapts the contrast of the images (of bit depth >8). The highest intensity in each

channel is set to maximum screen brightness.

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Metadata can also be shown later if necessary.

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If your data is bigger than the available RAM of your computer

you must activate Use virtual stack.

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Open/import image data

You have two possibilities to open a series of image files which you want to open

together as a stack:

A) Drag and drop the folder containing the images onto the FIJI window.

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Open/import image data

B) choose from the menu

File > Import > Image sequence

File name contains can

be used to filter by file

name.

For example entering

“Kartoffel” will only open

images with “Kartoffel” in

their names.

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Open/import image data

B) choose from the menu

File > Import > Image sequence

FIJI estimates how much

RAM will be used to open

this stack. If this is more

than what FIJI has available

an error is given. In this

case activate Use virtual

stack.

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Basics of the user interface

B) choose from the menu

File > Import > Image sequence

placing the mouse over a

pixel gives intensity values.

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Basics of the user interface

B) choose from the menu

File > Import > Image sequence

Name of file, of sub

experiment and zoom factor

(at 100 % not shown).

Keyboard shortcuts:

- Arrow keys left and right

scroll in C

- ALT and arrow keys left

and right scroll in Z

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Basics of the user interface

B) choose from the menu

File > Import > Image sequence

Metadata overview:

- current channel and slice

number.

- field of view in calibrated

units and pixels

- bit depth

- size

Color and frame around

image are displayed in the

color of the selected

channel.

Detailed metadata can be

viewed via menu Image >

Show info …

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Basics of the user interface

To change pixelsizes

choose from the menu

Image > Properties…

Pixelsize in x, y and z

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Basics of the user interface

Attention:

Activating Global applies

these settings to all open

images

To change pixelsizes

choose from the menu

Image > Properties…

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Changing color mode

Open Channels tool

from Image > Color > Channels tool

Composite shows all

channels at once.

Color and grayscale only

one channel at a time.

Keyboard shortcuts:

- Ctrl shift Z opens

Channels tool

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Changing color mode

Open Channels tool

from Image > Color > Channels toolEach channel can be

hidden one by one.

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Changing contrast

Open Channels tool

from Image > Adjust> Brightness / Contrast

Note:

On images with more than 8

bits changes in brightness

and contrast do not change

the intensity values of the

pixels. Only the display

settings are adjusted.

Keyboard shortcuts:

- Ctrl shift C opens B&C

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Orthogonal views of a stack

Open Channels tool

from Image > Stacks > Orthogonal views

Selection tools define the line of the virtual

slices by clicking.

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Z projections

Open Channels tool

from Image > Stacks > Z Project

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3D projections

Open Channels tool

from Image > Stacks > 3D Project…

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Further info

Download Fiji at http://www.fiji.sc

If you don’t remember where in the menus and submenus a command is hidden

press l for a list of all commands!

All menus explained in depth:

http://imagej.net/docs/guide/

Manuals and guides:

http://fiji.sc/User_Guides

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Which other features do you need to make FIJI

useful for you?

Stitch image tiles measuring distances, areas and volumes

batch convert file format invert contrast

overlay scale bar change color crop

blur create a montage of a stack

correct for uneven background crop in 3D

export as video display power spectrum find edges

synchronize windows analyze gels

record and run macros of repetitive tasks colocalization analysis

reslice stack register time series to compensate drift

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Thank you!