Superresolution microscopy on the basis of engineered dark states

Area of Science:

• Biophysics
• Optical Microscopy
• Molecular Imaging

Background:

• Superresolution fluorescence microscopy enables imaging beyond the diffraction limit.
• Photoswitchable and photoactivatable fluorophores are key to single-molecule localization microscopy (SMLM).
• Existing SMLM techniques rely on specific fluorophore properties for controlled blinking.

Purpose of the Study:

• To demonstrate a universal approach for superresolution imaging using various synthetic fluorophores.
• To enhance the duration of fluorophore dark states for improved localization precision.
• To achieve nanoscale resolution in biological samples.

Main Methods:

• Controlled manipulation of fluorophore emission properties by creating long-lived dark states.
• Oxygen removal to extend triplet state lifetime to milliseconds.
• Electron transfer reactions to generate radical ion states, further increasing dark state duration.
• Imaging of single molecules, actin filaments, and microtubules in fixed cells.

Main Results:

• Demonstrated superresolution imaging with practically any single-molecule compatible synthetic fluorophore.
• Successfully created dark states with millisecond-to-longer lifetimes.
• Achieved an imaging resolution of approximately 50 nm in fixed biological samples.
• Simulations confirmed the efficacy of the dark state manipulation for high-resolution imaging.

Conclusions:

• The developed method provides a versatile platform for superresolution fluorescence microscopy.
• Controlling fluorophore dark states is a broadly applicable strategy for achieving nanoscale resolution.
• This technique expands the toolkit for visualizing sub-diffraction limit structures in cells.