Boron-alkylated BODIPY molecules initially have a low fluorescence quantum efficiency (fluorescence off state) and upon irradiation with approximately 500 nm wavelength light, the fluorescence quantum efficiency of the photoactivated product increases (fluorescence on state). This ‘off’ and ‘on’ switching of fluorescence states is advantageous for single molecule localization microscopy (SMLM).
Paclitaxel was attached to the boron-alkylated photoactivatable BODIPY via a 4-(N-hydroxysuccinamidylcarbonyl)styryl linker to image microtubules, which have a diameter that is smaller than the diffraction limit of light. This paclitaxel-BODIPY probe resulted in a three-fold increase in fluorescence upon photoactivation.
Different 488-nm laser irradiances were analyzed to find a suitable irradiance to photoactivate the paclitaxel-BODIPY molecule and achieve a sufficient signal-to-noise ratio for SMLM. Single molecules were stochastically activated with approximately 160 W/cm2 laser irradiance, which is comparably lower than the laser irradiances used for conventional SMLM probes. Using the signal detected from photoactivated probes, single molecules were localized with a median precision of 14 nm.
Super-resolution images of in vitro microtubules were generated using the paclitaxel-BODIPY probe with an approximately 40-nm FWHM, which is an order of magnitude lower than that of the diffraction-limited image. Microtubules were also imaged in fixed and live cells. The imaging buffers that were used for these experiments did not contain harsh additives, which are often necessary for photoswitching with conventional SMLM probes and prohibit live cell imaging.
Wijesooriya, C.S.; Peterson, J.A.; Shrestha, P.; Gehrmann, E.J.; Winter, A.H.; Smith, E.A. “A Photoactivatable BODIPY Probe for Localization-Based Super-Resolution Cellular Imaging.” Angewandte Chemie International Edition. 2018,57,12685