Robust and fast multicolor Single Molecule Localization Microscopy using spectral separation and demixing

Single Molecule Localization Microscopy (SMLM) is a straightforward approach to reach sub-50 nm resolution using techniques such as Stochastic Optical Reconstruction Microscopy (STORM) or DNA-Point Accumulation for Imaging in Nanoscale Topography (PAINT), and to resolve the arrangement of cellular components in their native environment. However, SMLM acquisitions are slow, particularly for multicolor experiments where channels are usually acquired in sequence. In this work, we evaluate two approaches to speed-up multicolor SMLM using a module splitting the fluorescence emission toward two cameras: simultaneous 2-color PAINT (S2C-PAINT) that images spectrally-separated red and far-red imager strands on each camera, and spectral demixing STORM (SD-STORM) that uses spectrally-close far-red fluorophores imaged on both cameras before assigning each localization to a channel by demixing. For each approach, we carefully evaluate the crosstalk between channels using three types of samples: DNA origami nanorulers of different sizes, single-target labeled cells, or cells labeled for multiple targets. We then devise experiments to assess how crosstalk can potentially affect the detection of biologically-relevant subdiffraction patterns. Finally, we show how these approaches can be combined with astigmatism to obtain three-dimensional data, and how SD-STORM can be extended three-color imaging, making spectral separation and demixing attractive options for robust and versatile multicolor SMLM investigations.