Increasing microscopy resolution with photobleaching and intensity cumulant analysis.

Super-resolution fluorescence microscopy and its applications for analysis of biological structures are evolving rapidly field. A number of approaches aimed at overcoming the fundamental limit imposed by diffraction have been proposed in recent years. Here we present a modification of super-resolution optical fluctuation imaging (SOFI), a technique based on spatio-temporal evaluation of the optical signal from independently fluctuating emitters. Instead of rapid, reversible photoswitching, photobleaching is used to produce irreversible transitions between emitting and nonemitting states of the fluorochrome molecules. Simulated images are used to demonstrate that, in the absence of noise, the proposed SOFI modification increases the efficiency of transfer of high spatial frequencies in a fluorescence microscope. Correspondingly, a decrease of the point spread function (PSF) width is obtained. Moreover, the modified SOFI algorithm is capable of resolving point emitters in the presence of simulated noise. Using real biological images we demonstrate that an increase of resolution is obtained in 2D optical sections through densely packed chromatin in cell nuclei and lamin layer at the nuclear envelope. Finally, the approach is extended to 3D wide-field microscopy, allowing reduction of out-of-focus image blurring.