How do I get the original pixel values of the image?

[Meghana]

Hi,

Thank you for this wonderful software. It's been of great help to me. 

I was wondering how the fluorescence intensity values are calculated, seeing as the original tiff images have pixel values ranging from 1000-4000's, while the values stored in signal2 (or signal1) lies between 0 and  0.2. I understand that there's some normalizing that's carried out. Could you please point out where in the source code I can find the logic for carrying out the normalization?

Thanks in advance!

Cheers,

Meghana

[CJW Lab]

Dear Meghana,

Thanks for using Oufti. There was indeed normalization. I believe most of the normalization is similar to what was done in function normim (https://github.com/JacobsWagnerLab/Oufti/blob/master/oufti_windows/source/divisionFunctions/normim.m). As you see, all pixels were normalized between 0 to 1 based on the range of the pixels of that particular frame. The reason of this normalization is to use improve the computation efficiency. For example, a 16-bit image has a highest value of 2^16-1 = 65535. If you sum up all the pixels, the sum could go up to 2^38 (assuming a 2048x2048 image), which overflows a 32-bit integer. Other mathematical operations could make this worse. 

Hope this answers your question.