Multicolor Super-Resolution Fluorescence Imaging via Multi-Parameter Fluorophore Detection

Understanding the complexity of the cellular environment will benefit from the ability to unambiguously resolve multiple cellular components, simultaneously and with nanometer‐scale spatial resolution. Multicolor super‐resolution fluorescence microscopy techniques have been developed to achieve this goal, yet challenges remain in terms of the number of targets that can be simultaneously imaged and the crosstalk between color channels. Herein, we demonstrate multicolor stochastic optical reconstruction microscopy (STORM) based on a multi‐parameter detection strategy, which uses both the fluorescence activation wavelength and the emission color to discriminate between photo‐activatable fluorescent probes. First, we obtained two‐color super‐resolution images using the near‐infrared cyanine dye Alexa 750 in conjunction with a red cyanine dye Alexa 647, and quantified color crosstalk levels and image registration accuracy. Combinatorial pairing of these two switchable dyes with fluorophores which enhance photo‐activation enabled multi‐parameter detection of six different probes. Using this approach, we obtained six‐color super‐resolution fluorescence images of a model sample. The combination of multiple fluorescence detection parameters for improved fluorophore discrimination promises to substantially enhance our ability to visualize multiple cellular targets with sub‐diffraction‐limit resolution. No dye‐lemma: By measuring two or more spectral parameters independently, many fluorescent probes may be identified unambiguously. Applying this approach to super‐resolution fluorescence microscopy expands the palette of detectable colors substantially, enabling six‐color imaging with sub‐diffraction limit spatial resolution (see picture).