Enzyme self-label-bound ATTO700 in single-molecule and super-resolution microscopy

Area of Science:

• Biophysics
• Cell Biology
• Optical Microscopy

Background:

• Förster Resonance Energy Transfer (FRET) is a powerful technique for measuring molecular interactions in cells.
• SNAP-tag and Halo-tag protein labeling systems offer versatile tools for biological research.
• Near-infrared (NIR) dyes are advantageous for in vivo imaging due to reduced light scattering and autofluorescence.

Purpose of the Study:

• To evaluate the suitability of the near-infrared dye ATTO700 as an acceptor in FRET pairs for SNAP- and Halo-tag protein labeling.
• To assess the performance of ATTO700 in FRET measurements using both ensemble and single-molecule techniques.
• To demonstrate the application of this labeling strategy for live-cell microscopy, including super-resolution imaging.

Main Methods:

• Protein labeling using SNAP- and Halo-tag systems with ATTO700 as the acceptor.
• Förster Resonance Energy Transfer (FRET) measurements via ensemble (bulk) and single-molecule spectroscopy.
• Live-cell fluorescence microscopy, including super-resolution stimulated emission depletion (STED) nanoscopy.

Main Results:

• ATTO700 effectively functions as a near-infrared acceptor in FRET pairs with SNAP- and Halo-tagged proteins.
• Ensemble and single-molecule FRET measurements confirm efficient energy transfer.
• Successful imaging of cell surface proteins in live cells using STED nanoscopy demonstrates the practicality of the system.

Conclusions:

• Near-infrared ATTO700 is a viable and effective dye for SNAP- and Halo-tag based FRET studies.
• This labeling approach enables advanced optical microscopy techniques, such as STED nanoscopy, for high-resolution live-cell imaging.
• The developed method provides a valuable tool for investigating protein interactions and dynamics at the cell surface.