Cell Morphology Analysis.pdf

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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

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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

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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)

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

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

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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

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.

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.

The table shows the scores per cell as well as a

summary. This can be logged using the log data

button

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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.