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Setting scale and subtracting background

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Setting scale and subtracting background

becky_d_04
Hi all,

I am completely new to ImageJ and need to use the program for some IHC analyses. My first question regarded how to set the scale of my image. I know from my microscope that my images are 0.6297 um per pixel, therefore, 1.588 pixels per um. I already have a 100 um scale bar already placed onto my images. Do I just need to draw a line under my scale bar and then enter the known distance (100 um) and 1.588 into the "distance in pixels" box under Analyse;Set Scale? Does this measurement then just stay put during all my analyses or do I need to enter this information in every time?

The second question regards subtracting background fluorescence. I was wondering if the following method would be acceptable?

1) Analyse;Measure (I intend to measure my entire object)

2) Take multiple measurements (between 2-7 from what I've read) from my negative control using small boxes (of the same size!) which represent the background fluorescence in that image

3) Average these measurements together and then subtract this value from the mean gray value/integrated density of my measured object in step one.

Apologies if I'm babbling. I am completely new to this, and would appreciate any help I could get!

Thanking you all kindly in advance :)