Expanding the Spectral Resolution of Single-Molecule Localization Microscopy with Bodipy-Based Photoswitchable Fluorophores

Single-molecule localization microscopy (SMLM) has become an important tool for studying molecular biology. Through the use of photoswitchable fluorophores, SMLM can accurately localize individual molecules with 10-20 nm resolution, an order of magnitude better than conventional fluorescence microscopy. While SMLM succeeds in locating individual molecules, it is limited to 4-color emission based imaging due to the standard bandpass filter technology used to generate multicolor images, restricting the number of molecular entities that can be simultaneously localized in a single sample. The development of multi-spectral superresolution microscopy (MSSRM) improves the spectral resolution of SMLM enabling up to 20 color imaging in a single sample. However, MSSRM is currently restricted by the spectrally limited conventional fluorophores with adequate photoswitching for SMLM. Herein, we designed, synthesized and validated a series of novel BODIPY-based fluorophores with appropriate photoswitching for SMLM that span the visible spectrum permitting high-resolution, multi-color images using our MSSRM. The BODIPY-based probes were selected from a 110 member library of compounds synthesized through modification of a core BODIPY FL scaffold with diverse aromatic rings using a solid phase synthetic platform. All BODIPY-based probes were characterized for absorption and emission properties as well as key photoswitching properties to facilitate selection of the ideal probes for SMLM and MSSRM. Through labeling known cellular structures with selected BODIPY-based probes, we demonstrated that multiple fluorophores could be accurately resolved when excited by the same laser line due to varied length Stokes shifts. The developed BODIPY-based probes for SMLM will be beneficial in advancing understanding of cellular spatial organization by enabling the localization of an increased number of molecular compounds simultaneously.