Localization fluorescence microscopy using quantum dot blinking

Blinking of fluorescent quantum dots can be used to discriminate them, which allows their successive localization. One method of separating non-moving blinking objects from a time series of images is by using independent component analysis (ICA). Here we investigate with the help of simulations, how well 5 particles within the radius of one point spread function can be separated and localized and how the precision of object position depends on various imaging parameters. As proposed previously (Lidke et al., Opt. Expr. 13, 2005) the vision is to take the step from localizing to painting an image, which we called "Pointillism". In contrast to methods based on identification of subframes showing only single particles, which practically limits the method to distance measurements between two targets, separation methods such as ICA can separate several overlapping particle images.