Mapping molecules in scanning far-field fluorescence nanoscopy.

Area of Science:

• Biophysics
• Microscopy Techniques
• Cell Biology

Background:

• Traditional fluorescence microscopy struggles to accurately quantify molecule distribution due to variable emitter brightness and low molecule counts per pixel at high resolutions.
• Existing methods rely on dividing total fluorescence by single emitter brightness, which is often unreliable in complex biological samples.

Purpose of the Study:

• To develop a novel fluorescence microscopy approach for precise molecule counting and distribution mapping.
• To overcome the limitations of conventional methods in determining the number and brightness of emitters per unit volume.
• To apply this method for high-resolution 3D nanoscale mapping of cellular structures.

Main Methods:

• Utilized the principle that single molecules emit photons one at a time.
• Analyzed simultaneous photon arrival times during confocal imaging to determine the number and local brightness of molecules (up to 20 per confocal volume).
• Employed stimulated emission depletion (STED) microscopy for subsequent subdiffraction resolution mapping of molecule distribution.

Main Results:

• Successfully established a method to quantify the number and local brightness of multiple molecules within a diffraction-limited volume.
• Achieved subdiffraction resolution mapping of molecular distributions.
• Applied the technique to map the 3D nanoscale organization of internalized transferrin receptors in HEK293 cells.

Conclusions:

• The developed photon timing analysis method provides a robust solution for accurate molecule counting in fluorescence microscopy.
• This technique enhances the capability to study nanoscale molecular organization with improved resolution and reliability.
• Demonstrated the utility of the method in visualizing complex cellular structures like internalized transferrin receptors.