Counting fluorescently labeled proteins in tissues in the spinning-disk microscope using single-molecule calibrations

Area of Science:

• Cellular and Molecular Biology
• Biophysics
• Microscopy Techniques

Background:

• Accurate quantification of molecular numbers and concentrations in cells is essential for understanding biological processes.
• Traditional single-molecule imaging is often limited to specific cellular regions or conditions.
• Existing methods struggle with quantifying molecules in dense tissues or within living organisms.

Purpose of the Study:

• To develop a novel calibration technique for spinning-disk confocal microscopy (SDC) to enable accurate single-molecule quantification.
• To overcome the limitations of current methods for measuring molecular numbers in living cells and tissues.
• To apply the new calibration method to quantify specific proteins and cellular structures in vivo.

Main Methods:

• Devised a calibration technique using single-step photobleaching kinetics to estimate single-fluorophore intensity in SDC microscopy.
• Cross-validated calibration by comparing fluorophore brightness across SDC, total internal reflection, and epifluorescence microscopy.
• Applied the calibrated SDC method to quantify end-binding protein 1 (EB1)-eGFP in microtubule comet structures and neuronal cytoplasm.

Main Results:

• Successfully estimated single-fluorophore intensity using single-step bleaching kinetics in SDC microscopy.
• Quantified EB1-eGFP molecules at growing microtubule ends and measured its cytoplasmic concentration in fly larval neurons.
• Determined the dissociation constant of EB1-eGFP from microtubules and estimated the GTP-tubulin cap size.

Conclusions:

• Spinning-disk confocal microscopy, when calibrated with this method, offers significant potential for single-molecule imaging in tissues.
• The developed technique provides a straightforward approach for absolute fluorophore counting applicable to diverse biological systems and imaging modalities.
• This method enhances our ability to study molecular dynamics and concentrations within complex living tissues.