Scaling for analysis of average size (.czi to .tiff)

Image Analysis
#1

Hi,

I am doubting if the scaling that I use for measuring areas is indeed correct and if it is the best way to do.
I first drew a line from the left to the right corner in my .czi file and measured the distance in µm with the zen blue software.
To write the macro, I opened one of the tiff files (czi converted to tiff) drew the same line and set the µm distance that I got from the czi image.
Each image consists of several tiles (4x4) stitched together taken with a 5x objective.
Is this a good way to do the scaling? If so, can I check somewhere in the summary table if indeed areas are expressed in µm2 rather than pixels or inches?

This is my macro:

run("Set Scale...", "distance=5000.4052 known=6455.941 unit=inch");
run("8-bit");
setAutoThreshold("Default dark no-reset");
//run("Threshold...");
setThreshold(11, 255);
//setThreshold(11, 255);
setOption("BlackBackground", true);
run("Convert to Mask");
run("Set Measurements...", "area mean standard min median limit display redirect=None decimal=2");
run("Analyze Particles...", "size=100-Infinity circularity=0.01-1.00 display exclude clear include summarize add");

The summary table gives me these numbers for the image attached
ARPE19IFNy-4dpi-Scene-01-B1-B01-Stitching-01-Image Export-01_c1.tif 265 1877700.887 7085.664 9.311 250.426 255
which would mean the average size of the plaques in this image is 7085 µm2?

Thank you for your help!

Marlies

ARPE19IFNy-4dpi-Scene-01-B1-B01-Stitching-01-Image Export-01_c1.tif (6.6 MB)

cziwithscale
cziwithscale1920×1080 378 KB

#2

Hi Marlies,
Measuring the distance in the ZEN software will only work if the ZEN software has been calibrated. In your screen shot it shows in the status bar a scale of 1 px/px. It is a long time since I used the ZEN software, but this suggests to me that this is not properly calibrated - I would expect something like x px/um. It is also unusual that it would be exactly 1 pixel per um.
The best way of checking would be to take an image of an object with a known distance (e.g. a calibration slide) and then measure the length of the known object on the image (if you don’t have a calibration guide, but have a good idea how big the structures that you image are, you could use this as a rough guide).
Also, when you set the scale in ImageJ as you have done (run(“Set Scale…”, “distance=5000.4052 known=6455.941 unit=inch”)) , you actually say that 5000 pixels are equal to 6455 inches, which would mean that 1 pixel equals 6455/5000 inches, which clearly doesn’t make sense. It also means that your measurements are in inches as this is the unit you defined. Depending on your camera, etc I would expect that the calibration is somewhere around 1-2 um/pixel for a 5x objective.
There are various tutorials describing the spatial calibration process, e.g. Spatial Calibration - ImageJ.
Hope this helps,
Volko

2 Likes
#4

Hi @Volko
The ZEN software gives these dimensions 1,29µm/pixel could thus be correct?

image
image1920×1080 374 KB

Using this command would work for all images (taken with same objective of course)?

run("Set Scale...", "distance=100 known=129 unit=µm global");

Entire macro

title = getTitle();
run("8-bit");
setAutoThreshold("Default dark no-reset");
//run("Threshold..."); 
setThreshold(11, 255);
//setThreshold(11, 255);
run("Convert to Mask");
run("Set Scale...", "distance=100 known=129 unit=µm global");
run("Set Measurements...", "area mean standard min median display redirect=None decimal=3");
run("Analyze Particles...", "size=150-Infinity circularity=0.01-1.00 display exclude clear include summarize add");
#5

This sounds about right and is in the range of values I would expect for a 5x objective. Once you have applied this calibration to your images, you can use the line selection tool to measure a cell/structure of interest and see whether the length measurements in the ImageJ status bar look appropriate.
Good luck,
Volko

1 Like