Fluorescent Imaging Overlay

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In response to several questions, here is the outline of an algorithm for overlaying your fluorescent image on top of the bright field image.

· Load the fluorescent onion image.

· Apply flat field correction.

· Adjust the contrast/histogram, if needed.

· Apply a global threshold to generate a mask. (useful function: im2bw)

· Multiply corrected fluorescent onion image by mask.

· Convert to a color image in RGB. (Example, for a red image: colorMaskedImage(:,:,1) = maskedImage; colorMaskedImage(:,:,2) = zeros(480,640); colorMaskedImage(:,:,3) = zeros(480,640);)

· Load the bright field onion image.

· Adjust contrast/histogram, if needed.

· Multiply by the complement of the mask (1 – mask).

· Convert to RGB grayscale image. (You can use the function im2RGB. There is a predefined color map called “gray” in Matlab.)

It is helpful to display the image after each step so you can debug any errors in your code.