Fast, three-dimensional super-resolution imaging of live cells

Area of Science:

• Cellular Biology
• Microscopy
• Biophysics

Background:

• Live-cell imaging is crucial for understanding dynamic cellular processes.
• Existing methods often lack the spatiotemporal resolution to visualize ultrastructural details in real-time.
• Stochastic optical reconstruction microscopy (STORM) offers potential for super-resolution imaging.

Purpose of the Study:

• To develop and demonstrate high spatiotemporal resolution live-cell super-resolution fluorescence imaging using STORM.
• To visualize dynamic cellular structures, such as clathrin-coated pits and transferrin cargo, in living cells.
• To establish 2D and 3D imaging capabilities with improved resolution and speed.

Main Methods:

• Utilized stochastic optical reconstruction microscopy (STORM) for super-resolution imaging.
• Employed photoswitchable dyes for labeling proteins directly or via SNAP tags.
• Applied clathrin-coated pits and transferrin cargo as model systems for live-cell imaging.
• Achieved 2D and 3D imaging with high spatial and temporal resolutions.

Main Results:

• Achieved 2D STORM imaging in live cells with spatial resolution of ~25 nm and temporal resolution as fast as 0.5 s.
• Demonstrated live-cell 3D STORM imaging with spatial resolution of ~30 nm (lateral) and ~50 nm (axial) at 1-2 s time resolutions.
• Successfully performed two-color 3D super-resolution imaging in live cells using distinct photoswitchable dyes.

Conclusions:

• STORM enables high spatiotemporal resolution imaging of live cellular ultrastructures.
• The developed imaging capabilities provide a new window into dynamic cellular processes at the ultrastructural level.
• This technique advances the study of molecular mechanisms within living cells.