A couple thoughts:
- Are you using a very slow scan speed for the Bleach event? This is the line after “Iterations” in the Bleach control window. I find it hard to bleach using a fast scan speed, but the default is the Scan Speed for the image collection. I typically use bleach speed 2 or 3 while the Scan Speed in the Aquisition Mode window is 6-9.
- Is the area you are bleaching connected to a pool of soluble fluorophore? If so, it might be replenishing faster than your imaging settings. You can test this a couple ways. (A) try to catch the bleach: Use a very small area and choose Zoom Bleach to make the time between the bleach event and the first image as small as possible. When Zoom Bleach is unchecked, the mirrors still scan the entire image, not just the bleach region, so it takes longer. Zoom Bleach has the scanners move only in the area of the region. (b) Use a very large bleach area and measure the intensity of an area far away. This will only work if the contiguous solution is a relatively small volume such that the large bleach area is a substantial portion of the population of fluorescent molecules.