Surface-Functionalized Halo-Tag Gold Nanoprobes for Live-Cell Long-Term Super-Resolution Imaging of Endoplasmic...

Area of Science:

• Cell Biology
• Biophysics
• Nanotechnology

Background:

• Super-resolution fluorescence microscopy is crucial for observing endoplasmic reticulum (ER) dynamics.
• Current limitations include the lack of probes with sufficient brightness, photostability, and labeling stability for long-term ER imaging.

Purpose of the Study:

• To develop and characterize a novel nanofluorescent probe for enhanced super-resolution imaging of live-cell ER dynamics.
• To overcome challenges associated with probe delivery and stable ER labeling.

Main Methods:

• Synthesis of a surface-functionalized Halo-tag gold nanofluorescent probe (GNP-Atto565-fR8-CA).
• Incorporation of multiple Atto565 dye molecules for increased brightness.
• Modification with a cell-penetrating peptide (fR8) for improved cytoplasmic delivery.
• Utilizing Halo-tag for specific and stable labeling of ER proteins.
• Super-resolution microscopy techniques, including Structured Illumination Microscopy (SIM), for imaging.

Main Results:

• The GNP-Atto565-fR8-CA probe demonstrated superior brightness, photostability, and biocompatibility.
• Efficient cytoplasmic delivery of the probe was achieved, avoiding vesicular entrapment.
• Specific and stable labeling of the ER was confirmed through Halo-tag interaction.
• Successful long-term super-resolution imaging of ER dynamics and ER-organelle interactions was demonstrated.

Conclusions:

• The developed GNP-Atto565-fR8-CA probe is a practical and effective tool for studying live-cell ER ultrastructure and dynamics.
• This probe significantly advances the capabilities for long-term super-resolution imaging of the endoplasmic reticulum.
• The findings provide a new avenue for investigating dynamic cellular processes involving the ER.