Conventional BODIPY Conjugates for Live-Cell Super-Resolution Microscopy and Single-Molecule Tracking

Area of Science:

• Biophysics
• Cell Biology
• Microscopy

Background:

• Single molecule localization microscopy (SMLM) offers super-resolution imaging beyond the diffraction limit.
• BODIPY dyes are versatile fluorophores, but their use in SMLM requires specific properties like transient dark-to-bright state transitions.
• Existing SMLM protocols may not be readily applicable to common BODIPY conjugates.

Purpose of the Study:

• To present a straightforward and adaptable protocol for SMLM using conventional BODIPY conjugates.
• To enable super-resolution imaging and single-molecule tracking of biomolecules in living yeast and mammalian cells.
• To demonstrate the utility of this protocol for resolving nanoscale cellular structures and dynamics.

Main Methods:

• Development of a protocol for SMLM utilizing the transient formation of red-shifted BODIPY ground-state dimers (DII).
• Application of the protocol to image lipid droplets, fatty acids, and lysosomes in living cells.
• Demonstration of multi-color imaging capabilities by combining BODIPY dyes with other fluorescent probes.
• Tracking of single BODIPY-DII states to extract spatio-temporal information.

Main Results:

• Successful acquisition of super-resolution images and tracking of single BODIPY-DII states in living yeast and mammalian cells.
• Nanoscopic resolution of intracellular structures like lipid droplets and lysosomes.
• Visualization of differential spatial distribution and mobility of BODIPY-fatty acids and neutral lipids in yeast under varying metabolic conditions.
• Demonstration of multi-color SMLM with BODIPY dyes.

Conclusions:

• The presented protocol provides a versatile and simple method for SMLM with a wide range of commercially available BODIPY conjugates.
• This approach significantly expands the applicability of SMLM for studying biological processes at the nanoscale.
• The protocol facilitates the investigation of cellular dynamics and structures with high spatial and temporal precision.