University Division/Office
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Introduction to scientific
image processing in FIJI
Lunch Seminar Series
Moritz Kirschmann [email protected]
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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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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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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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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