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Visualization of organelles and molecules at nanometer resolution is revolutionizing the biological sciences. However, such technology is still limited for many cell biologists. We present here a novel approach using photobleaching microscopy with non-linear processing (PiMP) for sub-diffraction imaging. Bleaching of fluorophores both within the single-molecule regime and beyond allows visualization of stochastic representations of sub-populations of fluorophores by imaging the same region over time. Our method is based on enhancing the probable positions of the fluorophores underlying the images. The random nature of the bleached fluorophores is assessed by calculating the deviation of the local actual bleached fluorescence intensity to the average bleach expectation as given by the overall decay of intensity. Subtracting measured from estimated decay images yields differential images. Non-linear enhancement of maxima in these diffraction-limited differential images approximates the positions of the underlying structure. Summing many such processed differential images yields a super-resolution PiMP image. PiMP allows multi-color, three-dimensional sub-diffraction imaging of cells and tissues using common fluorophores and can be implemented on standard wide-field or confocal systems.

Sub-diffraction imaging on standard microscopes through Photobleaching Microscopy with non-linear Processing