Would like to flat-field correct images that were obtained on a spinning disk confocal.
I collected some images using our confocal, and would like to correct for uneven illumination using a procedure similar to what is described here:
I obtained my dark images and collected bright images for the 4 channels that I’m using, and then took the z-projected average and median, respectively. I then try to apply the following formula to calculate my corrected image:
resulting image = (acquired image - background) / (flatfield image - background)
My question is: should I be checking the “32-bit (float) result” option in the calculator setting?
All my images are 16-bit, but I’ve found that if I don’t check 32-bit in the final calculation, the resulting image is just dark. If I do check 32-bit, the intensity values in the resulting images are now on the order of ~0.1 instead of ~1000. The image itself looks flat-fielded, but it’s unclear to me how the values are changing?
Thanks for any help!