Targeting and imaging single biomolecules in living cells by complementation-activated light microscopy with...

Area of Science:

• Biophysics
• Cell Biology
• Microscopy

Background:

• Single-molecule (SM) microscopy offers deep insights into cellular biomolecular mechanisms.
• Selective detection of individual biomolecules within their native cellular environment presents significant challenges.

Purpose of the Study:

• To introduce a straightforward methodology for specific targeting and nanometer-accurate imaging of single biomolecules in living cells.
• To present complementation-activated light microscopy (CALM) as a novel technique for high-precision biomolecular imaging.

Main Methods:

• Proteins are genetically fused to dark split-fluorescent proteins (split-FPs).
• Activation into bright fluorescent proteins (FPs) occurs upon complementation with synthetic peptides.
• CALM enables imaging of diffusion dynamics and SM tracking of targeted proteins with nanometer precision.

Main Results:

• CALM allows imaging of diffusion dynamics for selected extracellular and intracellular proteins with nanometer precision.
• Single-molecule tracking is feasible using complementary fluorophores and quantum dots.
• Site-specific labeling is validated through coincidence SM detection and single-pair Förster resonance energy transfer.

Conclusions:

• CALM is a simple method combining genetic and synthetic probes for high-accuracy SM imaging in living cells.
• The technique is effective regardless of protein expression levels and at high probe concentrations.
• CALM facilitates advanced studies of biomolecular mechanisms at the single-molecule level within native cellular contexts.