Hi Ahn,

Yes, I wrote and tested this code with RGB images in mind ( (255,0,0) is just a simple red color), I think yours may be grayscale, like something from a fluorescence-based technique. Try using a single number instead as the message suggests, like 255.
The snippet does some kind of edge detection, and the extra part is the upscaling of the segmentation image to the resolution of the microscopy image. The edges are going to be 2-pixel wide on the image this way, while doing an edge detection first on the segmentation image and upscaling the result afterwards would result in very thick outlines, also with different width along the vertical and horizontal axes.

Best regards,
Gergely