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Sam'S Fiji Imagej Basic Quantification Manual: Save An Image As Text (Ie Numbers)

This document provides a manual for basic quantification and image analysis in FIJI/ImageJ. It describes various tools and techniques for: 1) Counting objects manually using point and multipoint selection tools; 2) Line and region measurements of intensity; and 3) Semi-automatic region creation using thresholding, the wand tool, and Analyze Particles. It also discusses accuracy considerations and provides exercises applying these techniques.

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139 views9 pages

Sam'S Fiji Imagej Basic Quantification Manual: Save An Image As Text (Ie Numbers)

This document provides a manual for basic quantification and image analysis in FIJI/ImageJ. It describes various tools and techniques for: 1) Counting objects manually using point and multipoint selection tools; 2) Line and region measurements of intensity; and 3) Semi-automatic region creation using thresholding, the wand tool, and Analyze Particles. It also discusses accuracy considerations and provides exercises applying these techniques.

Uploaded by

priftif
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Sam's FIJI ImageJ basic quantification manual

Save an image as text (ie numbers)


Open any image - eg blobs, ctrl+shift+b or
File/SaveAS/TextImage

File/OpenSamples/Blobs.gif

Look at the resulting .txt file with a text editor with wrap text turned off.
Or Analyze/Tools/saveXYcoordinates

Count objects manually


Point or multipoint tool - right click on icon to switch between these.

Double left-click on icon to get options for the option selected.


This approach is often slightly faster and more accurate than counting in your head, provides a record and
the potential for other measurements.

Multi-point
Click on several objects
See the count on the image (if label points is selected)
Measure (ctrl-m, Analyze/Measure) gives point measurements for all points

Single-point
The Auto-measure option records the accumulating number and other measurements
Mark to see where you have clicked - puts on foreground colour. Need to be an RGB image to see colour.
Add to ROI manager add the position of each point individual to the ROI manager list

Either
Marker size can be adjusted
Save an image including the markings by flatten (Image/Overlay/Flattenor ctrl-shift-F) then save as
PNG etc

Count dots with find maxima


Process/FindMaxima..

CountFoci_foci.tifis quite a good example for this


Noise tolerance determines how much brighter than the surrounding the spot needs to be to be picked as a
local maxima.
Preview to see how many and where they are found (the markers are those under multi-point selection) and
a count is displayed
Exclude edge maxima prevent half objects/artifacts at the edge of the image being included
Various output types - try them "Single point" gives an image with the maxima as single pixel objects
"Maxima Within Tolerance" shows all the pixels within the tolerance value for each maxima
"Segmented particles" watersheds the image around the maxima
"Point Selection" produces an image with a multipoint selection overlayed
Try it on a thresholded image, especially segmented particles

Line measurements
Select a line
Analyze/PlotProfile(ctrl-K) to measure intensity across a line
Double-left click on the line icon to change width. Wide lines are averaged across the orthogonal pixels.
Same if you draw a rectangle.

Region measurements
Draw a region (geometric, freehand etc)
Edit/Selection/Specifyallows input of specific sizes
Measure (ctrl-m,Analyze/Measure)to produce numbers
Analyze/setMeasurementsselects what to measure
Help presents a very good description of the options, my favourites . . .

Area - in pixels or micron^2 if the image is calibrated.


Mean, Mode, median grey values. Uncalibrated unless you calibrated them (eg to OD). Standard deviation
is the SD of the gray values within the selection or object.
Integrated density is the sum of the pixel values in the ROI.
Centroid and center of mass both give X Y coordinates, center of mass takes into account the differing
intensity across an object.
Shape descriptors - Circularity, 4*area/perimeter^2. 1=a circle, 0=very much not a circle. Other shape
descriptors involve fitting the ROI to a shape, eg elipse to give an aspect ratio (major/minor axis).
Feret's diameter - length and angle of the two most distant points around a selection. Useful for assessing
the length and orientation of objects.
Stack position and Display label are useful as these include the name and slice number of the image in the
results.
Invert Y coordinates mean 0,0 is the bottom left of an image (rather than top left based on mn matrix
notation), which makes it more like the standard cartesian coordinates of familiar graphs.

ROI manager
Allows Multiple regions, transfer or region . . .
press t

Semi-Automatic region creation with the Wand tool

Double left click on the icon to input a tolerance - how much the pixel intensity can vary by and the
selection to continue to flood out.
8-connected mean it looks at all 8 neighbouring pixel values, which generally makes sense
Click on an object to make a selection
shift-click for multiple regions
or use t to add to ROI manager list

Threshold-based regions
Image/Adjust/Thresholdor ctrl-shift+T
Select an intensity range that best separates objects from background.

Try with blobs and simple_threshold.tiff


Auto threshold methods choose for you. Image/Adjust/AutoThresholdtries them all.
createselectionmakes a single selection/region around all thresholded pixels. This can be added to
ROI manager as one region.

Analyze particles
Analyze/Analyze Particles . . .
A very useful tool for dealing with multiple objects. Allows exclusion
by size and circularity and offers a range of outputs.

Try range of options for "show:"

Add to manager sends region to ROI manager allowing transfer and more.

Useful methods
Process an image, make regions via Analyze particles from the processed image, apply regions to original
image.
Use one channel to make regions to measure another - eg nuclei from dapi stain.

Exercises
How good can you get regions and count.tif?
Count Foci - how many foci, how many foci per cell?

Binary operations
Press apply on the threshold box or Process/Binary/MakeBinary
Mouse-over to see the two intensity values. Histogram - looks pretty boring, black and white, no grey.

Binary logic
Process/ImageCalculator..

Try AND and OR on two different binary images of the same size
Use LinesForBlobs.tif to measure the height of blobs across the x axis.

Binary operations
Process/Binary/...

Erode,Dilate,Open,Close,Outline,Fillholes,Skeletonize

Try these on mask.tif and fingerprint_binary.tif

Watershed to separate objects

Accuracy of the numbers


Spatial
You need to make sure your images are accurately calibrated
Check or input the values at Images/Properties
(or Analyze/Set Scale)
Add a scale bar with Analyze/Tools/Scale Bar

Intensity
Relative measurements are generally made. Absolutely intensities are more challenging.
Background correction is required - measure it or subtract it from the images before measurements.

Limits to accuracy of the numbers

Aim to be accurate with sufficient precision. Error is inevitable. Systematic error is bad. Even worse if you
are precisely systematically inaccurate because you are more likely to believe your experiment.

Potential confounds producing inaccuracy


(may be differentially between your A and B samples)
Changing the acquisition settings
Saturation or clipping intensities in some samples
Bad processing - "auto"
(hopefully you never do any of these!)

Illumination artifacts is the field perfectly even?


Day-to-day variation laser power (helps to warm them up), bulb age, alignment of the optics
(especially laser coupling), dirt
Bleaching over t and z
Antibodies incomplete permeabilization, non-linearity. FPs dont have these problems.
Fluorophore saturation (confocal)
Bleedthrough, other fluorophore interactions (eg quenching)
Imaging efficiency over z
Image aberrations eg chromatic: colocalization accuracy, spherical: intensity over z (do all your
samples have the same aberrations?), intensity and resolution loss
Reagent instability batch variation between antibodies, mount, coverslips

A standard sample can help ease your mind.

Stack measurements
Measure bleach and background regions on the FRAP time series - FRAP_neuron.tif
Draw a few ROIs, In ROI manager, More/MultiMeasure
Define region by subtraction - threshold and erode.

Gel measurements
Open your gel image or File/OpenSamples/Gel
Draw a region around the first lane, ctrl-1 or Analyze/Gels/SelectFirstLane
Move the region to the second lane, ctrl-2 or Analyze/Gels/SelectNextLane
Repeat for all lanes
ctrl-3 or Analyze/Gels/Plotlanes
Use the line tool to manual demarcate the regions of the graph accounting for each band
Use the wand tool to measure the area under the curve representing the intensity of the band
Think about linearity - fluorescence based ones ok, ECL non-linear
Analyze/Calibrateallows a protein curve to be added

Background of the gel may need to be taken into account.

3D measurements
Are projections ok to quantify?

Yeast example
Use the DIC images to make a mask
Make it great using all the tricks you now know
Measure the total background corrected GFP expression per yeast cell.

Try your images . . .


Use all the clever tricks from this week and last to do your best with our real data

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