Long time-lapse nanoscopy with spontaneously blinking membrane probes

Area of Science:

• Cell Biology
• Microscopy
• Biophysics

Background:

• Live-cell super-resolution microscopy faces challenges with labeling density, dye brightness, photostability, and toxicity.
• Long time-lapse imaging of cellular dynamics at the nanoscale is crucial for understanding biological processes.

Purpose of the Study:

• To introduce novel high-density, environment-sensitive (HIDE) membrane probes for enhanced live-cell nanoscopy.
• To enable long time-lapse imaging of cellular structures and organelles with high spatiotemporal resolution.

Main Methods:

• Development of HIDE probes based on the silicon-rhodamine dye HMSiR.
• Application of HIDE probes in live-cell super-resolution microscopy for time-lapse imaging.
• Analysis of 2D and 3D dynamics of mitochondria, plasma membrane, filopodia, and endoplasmic reticulum.

Main Results:

• HIDE probes allow for significantly longer nanoscopy movies (tens of minutes) compared to protein-based labeling (tens of seconds).
• Successfully visualized the dynamics of mitochondria, plasma membrane, filopodia, and endoplasmic reticulum in living cells.
• Demonstrated the capability for live-cell, two-color super-resolution imaging using HIDE probes.

Conclusions:

• HIDE probes represent a significant advancement for live-cell super-resolution microscopy.
• These probes overcome key limitations, facilitating detailed visualization of dynamic cellular processes.
• Expanded utility of nanoscopy for studying complex biological structures and events in real-time.