Direct stochastic optical reconstruction microscopy with standard fluorescent probes

Area of Science:

• Biophysics
• Microscopy
• Cell Biology

Background:

• Direct stochastic optical reconstruction microscopy (dSTORM) provides super-resolution imaging using conventional fluorescent probes.
• Achieves lateral resolution of approximately 20 nm, surpassing conventional fluorescence microscopy limits.

Purpose of the Study:

• To present a detailed protocol for dSTORM imaging in fixed and live cells.
• To optimize the control of fluorophore states for enhanced imaging quality.

Main Methods:

• Utilizes a wide-field fluorescence microscope with standard fluorescent probes.
• Involves transferring fluorophores to a reversible OFF state and reactivating sparse subsets for localization.
• Employs photoinduced control of fluorophore ON/OFF states and discusses labeling strategies and acquisition parameters.

Main Results:

• Demonstrates a step-by-step protocol applicable to various cellular imaging scenarios.
• Highlights the importance of fine-tuning the ratio of fluorophores in ON and OFF states.
• Achieves temporal separation of unresolved structures through repetitive activation and localization cycles.

Conclusions:

• The presented dSTORM protocol offers a robust method for high-resolution cellular imaging with standard probes.
• Optimized control over fluorophore states is crucial for achieving high spatial and temporal resolution.
• Data acquisition and processing can be completed rapidly, making dSTORM an efficient super-resolution technique.