Counting and measuring using image analysis software

Counting and measuring using image analysis software
Counting and measuring
Counting using Image J
Open file in Tiff format, and convert to 8 bit if
necessary (Image, Type, 8-bit)
To threshold the image, go to Image, Adjust,
Threshold
Use Otsu as the type of threshold method
(Otsu thresholding calculates the area to
select for thresholding based on the intensity
histogram)
Make the objects B&W
To threshold using Metamorph see the Thresholding section of the Metamorph guide; Page 7
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Counting and measuring
If some of your objects are touching each other, go
to Process, Binary, Make Binary
Ok
Then go to Process, Binary, Watershed
You can also separate objects manually
Make sure the Draw Tools are visible by clicking the
>> icon and selecting Draw Tools
Use the colour picker tool and click on the
background
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Select the pencil tool, and double click the icon to
adjust the line width
Counting and measuring
Draw lines through adjacent objects to separate
them
NB. You can also use the draw tools to colour in, or
erase objects you don’t want to count, just make
them the same colour as the background
When you are finished, make sure the background is
black and the objects are white.
If this is not the case you can select tick the Dark
Background option in the Threshold box
To count the number of objects, click on Analyse,
Analyse Particles
Adjust the parameters of the objects you want to
count. Make sure the Summarize box is ticked
A summary window will appear. The Count column
tells you the number of objects
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Measuring using Image J
Counting and measuring
Start by opening the Region of Interest Manager by
going to Analyse, Tools, ROI Manager
Or click the ROI Manager icon
Then you need to select something to measure, so
pick one of the selection tools and draw a region of
interest
In the ROI Manager window click Add, and your
selection will appear in the list
NB. You can rename your selections to help you
identify them
When measuring intensity it is best to use 16-bit images, and make sure your channels are split, otherwise
you will just record an average intensity across all the different channels.
If you are measuring area or length you may want to set the scale of your image, so your measurements are
in microns instead of pixels. See Set the Scale section.
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Counting and measuring
You can draw multiple regions of interest using the different drawing tools, just remember to Add each one
to the ROI Manager. To delete a selection click Delete
You then need to decide what measurements you
want to record.
Go to Analyse, Set Measurements
And pick what you want in the Set Measurements
window
This is the average
intensity within the
region selected
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Counting and measuring
In the ROI Manager window, select one of your
regions of interest and click Measure
A Results window will appear. This contains the measurements you requested.
The Label column displays the name of the image and the name of the region of interest
The Area and Perimeter are
measured in pixels, unless you
have set the scale of your image
The Mean column is the average
intensity within the region
selected (Mean Gray Value)
Min and Max are the minimum
and maximum intensity values
within the region selected
To save these results you will need to copy and paste them into Excel
To transfer a region of interest to another image, select the image you want to transfer to, and then select
the region from the ROI Manager. You can then make the same measurements on this image by clicking
Measure
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Counting and measuring
The measurements from this new image are distinguishable from your previous ones, as the Label tells you
which image has been used
To select multiple areas and group them as one region of interest, hold down the shift key when making
selections. Once you have finished selecting things, add the selection to the ROI Manager as a single item
Now when you hit Measure, the area will be a sum of all the selections, and the Mean will be the average
intensity across all the selections
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Counting and measuring
Quick measurements
To make a quick measurement on just one image
you don’t have to use the ROI Manager. You can just
use the selection tools, draw your selection, and
click Analyse, Measure
See instructions above on how to change what measurements are displayed (Analyse, Set Measurements)
To remove a selection go to Edit, Selection, Select
None
Counting and measuring using Metamorph
• To count the number of cells see the Cell Scoring section of the Metamorph guide; Page 12
Cell scoring uses a maximum of 2 colours, one of which needs to be DAPI nuclei staining.
See also the Nuclei counting section of the Metamorph guide; Page 15, to count just nuclei.
• To count using two or more wavelengths, see the Multi-Wavelength cell scoring section of the
Metamorph guide; Page 13
• To measure distance or area in Metamorph you will need to calibrate your images. See the
Calibrating Images section of the Metamorph guide; Page 7
• To make measurements from different regions of interest see the Region Measurements section of
the Metamorph guide; Page 8
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