Abstract
Cells are dynamic and complex systems where a vast network of biomolecules continuously interact to sustain life. Capturing the full picture of these interactions requires molecular tools that can image biomolecules with high spatial and temporal resolution, minimal damage to the biological environment, and the ability to multiplex. Recent advancements in microscopy techniques allow researchers to surpass the 200 nanometer (nm) diffraction limit of traditional fluorescence microscopy, such as photoactivated localization microscopy (PALM). This technique requires compounds capable of switching from a nonemissive state to an emissive form under irradiation at an appropriate activation wavelength (λAc). However, the activation of these molecules often requires ultraviolet or violet irradiation, which can cause significant photodamage to live specimens. This leads to the compromising of data integrity, thus limiting the widespread use of these probes and imaging modalities. In addition, comprehensive libraries of synthetic dyes with the appropriate photochemical, photophysical, and structural properties for multiplexed super-resolution imaging in live cells are not yet available.
The current work therefore aims to address these critical limitations by describing the design and validation of next-generation photoactivatable fluorophores (PAFs) for live-cell super-resolution imaging. First, a spectrally resolved palette of BODIPY-based PAFs was developed for multiplexed single-molecule tracking using advanced imaging modalities, including single-particle tracking and spectroscopic single-molecule localization microscopy (sSMLM). Second, novel green-light-activatable fluorophores based on the same BODIPY scaffold were synthesized to minimize photodamage during live-cell imaging. Finally, molecular handles were incorporated into this new library of green-light PAFs to enable covalent binding to specific intracellular proteins via self-labeling protein tags or to nuclear DNA, allowing selective targeting of subcellular structures.