Patent ID: 8445867

Claim:
A method of photobleaching and intermittency localization microscopy (PhILM), the method comprising steps: a) processing a movie of fluorophores transitioning between fluorescent and dark states by sequentially subtracting frame (n+1) from frame n to get frame n′, frame n from frame (n−1) to get frame (n−1)′, etc. to obtain a “backwards-subtracted movie” comprising frames 1′, 2 ′, . . . , (n−1)′, n′, (n+1)′, etc.; b) detecting in the backwards-subtracted movie dark and bright spots, wherein bright spots correspond to transitions from fluorescing-states-to-dark-states (“photobleaching” events), and dark spots correspond to transitions from dark-states-to-fluorescing-states (“blinking” events) and storing corresponding frame numbers and positions of these photobleaching or blinking events; c) localizing positions of the fluorophore transition events with sub-pixel resolution; and d) creating a high resolution image by plotting the positions of the localized fluorophore transition events in a final image.