Confocal, laser-scanning microscopy was applied to acquire coenzyme F-420-based autofluorescence images of middle sections of sludge granules during start-up of a thermophilic reactor that were seeded with mesophilically-grown microorganisms of granular sludge. Digital images were analyzed to calculate weighted averages of autofluorescence. The values were related (r(2)=0.97) to specific methanogenic activities of granular sludge as the granules developed to steady state.