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Cluster Analysis of Super Resolution Fluorescence Images

Direct Stochastic Optical Reconstruction Microscopy (dSTORM) is a novel, high resolution technique to produce fluorescence images. It uses conventional organic fluorophores which are fluorescent molecules that emit light when excited in either a spontaneous or induced way. Common examples of fluorophores are labeled antibodies, used as markers to indicate locations of certain proteins. The fluorophores attached to certain proteins are activated which then ”blink” intermittently. Due to the stochastic nature of the fluorophores, only a subset of them are activated at once. Then their positions are precisely determined by fitting Gaussian to each and localizing them, which means taking an image. After localization, they are deactivated to prepare activation of another subset of the fluorophores. Repetitive activation, localization, and deactivation – more than few thousand times – and reconstruction of all the images into one by overlapping them yield 10-fold better resolution compared to conventional fluorescence microscopy [3]. Also, with dSTORM, it is possible to generate 3D or multi-coloured images.