Posted by
Gabriel Landini on
Apr 16, 2011; 9:45am
URL: http://imagej.273.s1.nabble.com/effect-of-magnification-on-area-measurements-tp3684968p3684969.html
On Friday 15 Apr 2011, Cheuk Tam <
[hidden email]> wrote:
> I am new to ImageJ and am using it to measure the area of DAB-stained
> cells. I tried measuring the area of the same cell at different
> magnifications (and set the scale to the respective stage micrometer
> images), but I found the area measurements yielded different numbers. The
> 40X objective had an area that was about 4-fold smaller than the 10X
> objective image. Is this because the resolution and the number of pixels
> in each image are different?
Are you sure that your calibration is correct? I would look at this first.
Second I would measure a single object that is easily defined and has s simple
shape - let's say 1 nucleus or the same erythrocyte (do you get the right
diameter at all magnifications?).
If you still got the wrong numbers, you probably have done wrong the
calibration of the objectives or made a mistake with the photos thinking that
you used a different objective. Or you did not apply the right calibration to
the image. Or, the thresholds you apply to the different images are selecting
different things.
It also matters what you are measuring and what shape and how close your
objects are.
Euclidean shapes should return a similar area size across maginifications plus
minus some error associated to the digitisation and inter-pixel distance.
The numbers in the stage micrometer are a good test because they are well
contrasted.
If what you are measuring is not Euclidean, maybe at low magnification many
objects merge into one, or empty holes appear filled at low magnification.
Without any other details or images (please upload them to some site for us to
see) it is not possible say much more.
Cheers
G