Self-Healing Dye-Conjugated Nanobody Probe for Super-Resolution Imaging with Enhanced Photon Budget

Area of Science:

• Biophysics
• Molecular Imaging
• Nanotechnology

Background:

• Super-resolution microscopy demands probes with high photon output and minimal protein perturbation.
• Existing probes often face limitations in brightness and photostability.

Purpose of the Study:

• To develop a novel nanobody probe for enhanced super-resolution imaging.
• To improve photon budget and targeting affinity for GFP-tagged proteins.

Main Methods:

• Site-specific conjugation of a photostabilizer-fluorophore to an anti-GFP nanobody.
• Utilizing intramolecular triplet energy transfer to suppress triplet-state populations.
• Characterizing probe performance via molecular brightness and single-molecule photophysics measurements.

Main Results:

• The self-healing dye-conjugated nanobody demonstrated a 38-83% increase in molecular brightness.
• Achieved 6-fold and 42-fold increases in cumulative photon output under ambient and oxygen-scavenging conditions.
• Improved localization precision in single-molecule localization microscopy of GFP-tagged Nup96 in cells.

Conclusions:

• The developed probe offers a robust, ready-to-use solution for high-performance super-resolution imaging.
• Site-specific conjugation and photostabilizer integration enhance probe capabilities.
• This nanobody probe advances GFP-based super-resolution microscopy applications.