Spectrally Resolved Super-Resolution Microscopy

Abstract

Recent advances in super-resolution fluorescence microscopy methods have led to ∼10 spatial resolution and thus profound impacts on biology. However, previous super-resolution methods provide no spectral information, thus throwing away a potentially rich dimension of information, and making multicolor imaging difficult. We recently developed a method, named Spectrally Resolved - Stochastic Optical Reconstruction Microscopy (SR-STORM), to synchronously obtain the fluorescence spectra and positions of millions of single molecules in densely labeled cell samples in minutes, and hence the concept and realization of spectrally resolved, “true-color” super-resolution fluorescence microscopy. This allowed us to unambiguously identify single molecules of different dyes that overlapped heavily in spectrum. Crosstalk-free 3D super-resolution microscopy was thus achieved for four dyes that were only 10 nm apart in emission spectrum; excellent resolution was obtained for every channel, and the 3D localizations of all molecules were automatically aligned within one imaging path. Moreover, our technique is suitable for live-cell imaging, and using fluorophores spectrally responsive to local environments, we enter a new regime in which local physicochemical parameters, including pH and polarity, may be probed at nanometer resolution and single-molecule sensitivity.