Synthesis and characterization of bodipy-based fluorophores for multicolor microscopy Public Deposited

Multicolor microscopy tools necessary to localize and visualize the complexity of subcellular systems are limited by current fluorophore technology. While commercial fluorophores cover spectral space from the ultraviolet to the near infrared region and are optimized for conventional bandpass based fluorescence microscopy, they are not ideal for multicolor microscopy as they tend to have short Stokes shifts, restricting the number of fluorophores that can be detected in a single sample to four or five. Herein, we synthesized a library of 95 novel BODIPY (boron-dipyrromethene)-based fluorophores and screened their photophysical properties for conventional and superresolution multicolor microscopy applications. A subset of our BODIPY-based fluorophores with varied length Stokes shifts were utilized with confocal laser scanning microscopy and linear unmixing to create a five color image using a single excitation laser line for the first time. Using these novel fluorophores in combination with commercial fluorophores would enable a nine to ten color image using linear unmixing. We also demonstrated the applicability of our novel BODIPY-based fluorophores for single-molecule localization superresolution microscopy (SMLM). Using our developed screening methodology for efficient characterization of fluorophore photoswitching properties, we identified high-quality photoswitchable BODIPY-based fluorophores from our library that fill voids in commercially available fluorophore spectral space, facilitating multicolor superresolution microscopy (SRM) using conventional SMLM instrumentation. Overall, the fluorophores generated from our BODIPY-based library will enable both conventional and SRM multicolor imaging applications alone or in conjunction with commercially available fluorophores, increasing the available color palette for imaging.

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