Balanced SOFI

Super-resolution optical fluctuation imaging (SOFI) achieves 3D super-resolution by computing temporal cumulants or spatio-temporal cross-cumulants of stochastically blinking fluorophores. In contrast to localization microscopy, SOFI is compatible with weakly emitting fluorophores and a wider range of blinking conditions. Balanced SOFI analyses several cumulant orders for extracting molecular parameter maps, such as the bright and dark state lifetimes, the concentration and the brightness distributions of fluorophores within biological samples. In combination with a deconvolution of the cumulant images, the estimated parameter maps proved useful to balance the image contrast and to linearize the brightness and blinking response. Thereby, the image quality and the resolution were improved significantly.