Rescale intensities - Load Images module

Sorry if this questions are too obvious, I tried to find the answer to them in this forum but I didn’t found any of them!
I am still just a beginner!

I am using the module Load Images to analyze a time serial images.

In the pop-up help window it’s written
"Check this setting to rescale the image intensity so that saturated values are rescaled to 1.0 by dividing all pixels in the image by the maximum possible intensity value."

1- What means “maximum POSSIBLE intensity value?
2- There are a set of " maximum possible intensity value”, one for each image, or it takes in to account all the images together to find the maximum of the maximums?
3- If not, which is the best option for compare images of the same time serial? check the rescale intensities or “uncheck” that?
4- I tried both and when I visualize the image; I am not able to see any difference between this two options? Is it because to show the image, the Cell profiler reduces them to 8-bits?
5- Once you have load the images, if you use the MeasureImageIntensity module, which data takes uses for that?

Thank you,

Núria

Hi Nuria,

The issue can best be understood in light of the example provided in the same help that you quoted: “For instance, a microscope might acquire images using a 12-bit A/D converter which outputs intensity values between zero and 4095, but stores the values in a field that can take values up to 65535.” The inclusion of this setting was spurred by this forum post, in which the image has a different dynamic range than would be expected from the image format. Keep this in mind as I answer below.

For the above example, the maximum possible value is 65535 since the images are 16-bit.

This setting only uses the value of the current image, not all of them together. Since it is typical that the images are all the same format (e.g., 16-bit), the maximum will be the same for all of them anyway.

I’m not sure what you mean by “time serial” here. If the acquired images are from the same microscope hardware/software and in the same format, it should really be the same comparison whether it is checked or unchecked. What you don’t want to do is check it for one run of the pipeline, uncheck for another run, and attempt to compare the two results as if they were the same. What matters is being consistent.

In your case, it is most likely that the dynamic range matches the image format, so unchecking/checking the setting makes no difference.

Any assay which requires a per-image intensity measurement could use this data. For example, you could write the result directly to a spreadsheet if that’s your final readout, or you could use a downstream module such as CalculateMath if you need to do additional operations on this data prior to the final readout.

Regards,
-Mark