Measuring cell morphology Cell area, length, width and shape features

The analyse particles menu in ImageJ can be used to extract some basic features from your cells
These include area, perimeter, staining intensity. See Measuring protocols.
If you have multiple regions interest to measure this can be done either in ImageJ using the Multi
Measure tool or in Metamorph using the Region Measurements tool. See Measure Multiple Regions
protocol.
Metamorph can be used for more complex Cellular analysis.
Robust measurement of multiple cellular features using Integrated Morphometry
Analysis (IMA):
Requires: 16 bit, background subtracted, noise reduced/ filtered images. Software is
never as good as the human eye at detecting edges so the better your images the better
your results.
To use IMA you first of all need the threshold your cells (see Thresholding protocol)
Select the IMA tab

The IMA menu will appear. Make sure the image you want measured is selected
In the source window.
If you want to use a mask for segmentation (recommended) check the box

Click on the measurements tab. There are a lot of possibilities.

Pick what you need.
In the Select measurements
dialogue you can refine the
measurements which are made,
there are cascading drop down
menus so the specific
measurements you need can be
made.

You can use the comparison and limits boxes to filter data, stating if
the area or intensity need to be smaller, larger or between defined
values.
Select Preferences tab: choose the options required
1

Click on Measure. The selected area in the image to be measured will be green, excluded area will
be blue
In the Object Data dialogue the
measurements for the selected parameters
can be viewed
These can be exported to Excel by opening a
Data log.
The parameters exported can be selected by
choosing configure log (see logging data
protocol)

A summary of the data which has been measured can be viewed in the Summary Tab
The summary measurements can be
exported by opening a Summary Log
The parameters exported can be selected
by choosing configure log
Ensure these are being sent to a different
excel sheet than the Raw data

If the settings arent selecting what you want refine the parameters in the measure tab.
If there is a problem you can start again using Reset Current.
When the parameters are ok you can save the state. This
can be reloaded for the next set of images
The Histogram Tab in the IMA can be used to view graphs of the data if required
2

Cell scoring/ Multi Wavelength cell scoring

- Useful for measureing nuclear cytoplasmic staining
Requires 16 bit tifs of tissue or cells stained with multiple wavelengths, ONE OF WHICH
MUST BE A COMPLETE NUCLEI STAIN SUCH AS DAPI. Tifs should be background subtracted,
and may be calibrated if size or area is required.
Use: To count cells with multiple stains, eg- total cells in a section and those which are c-fos
positive.
Choose : cell scoring for 2 wavelengths in or multiple wavelength cell scoring for 3 or more.
Open your images and pick the cell scoring app: (can also be found in the Menu bar in Apps)

Select the image with the nuclear stain as Wavelength 1 and set the parameters
for measuring the nucleus
Measure the minimum and maximum width of your nuclei using the line tool
and add a slightly lower value into the Min width boxes and a slightly higher
value into the Max width box.
Measure the background intensity and the intensity of the staining you want to
measure. Put the difference between these values into the Intensity above local
background box.
To see what is selected in your image for measurement with these parameters
press Preview
The preview image will have the nuclei selected in red.
If you are happy with what is selected you can now set the
parameters for the second staining in a similar way. Ensure you
state if your staining is nuclear, cytoplasmic or both so the
algorithm knows where to expect your staining to be.
If you are not happy with what has been selected change the
parameters slights and repeat the process until what is selected
is acceptable.
The preview image for the second staining will highlight this in
green.
Both the red and the green selections are a mask and can be
removed by clicking the mask button
3

Before making the measurement you will need to set

up a data log to record your data.
Go to the log menu in the main window.
Click open data log and open summary log.
Click apply in the Cell scoring Window.

Once the data logs are opened you can configure what
information you would like to save from the measurements
(Configure Data Log) and from the Summary of all the
measured points in your image (Configure Summary Log)
Data Log:
Summary Log:
Tick the boxes for the data you want in both logs

If you know you will need to measure

multiple images you can save the settings
you have set for measuring using the Save
settings button. These can then be
reloaded later using the Load settings
button.

Once you have set the measurement parameters and also decided which data you want you can go
ahead and make your measurements. To do this press the Apply button in the cell scoring dialogue.

Metamorph will now calculate your cells scores. It

will give an image showing positive (green) cells
and negative (red) cells. The interpretation of this
will depend on the scientific question you are
asking.
This image can be saved by right clicking it,
selecting copy to clipboard and pasting into Paint.
The table shows the scores per cell as well as a
summary. This can be logged using the log data
button

Multi wavelength Cell Scoring.

This allows measurements of two or more stains in a cell to be made and works exactly the same
way as the cell scoring application
Select the number of wavelengths .A tab will
appear for each one. You can change the name.

For each wavelength select the source image

Choose a colour for each- red and green are good
as the overlap yellow is very easy to see
Proceed as for cell scoring to define settings for
each wavelength.
Open excel and configure the logs
Open logs as for cell scoring
When configuring logs the summary log gives the
simple read out of number of cells and number
or % staining overlap for each channel compared
to DAPI and each other.
Click apply
You will get a result image and a data log. Save
both.