Toward Absolute Molecular Numbers in DNA-PAINT.

Area of Science:

• Optical Microscopy
• Nanotechnology
• Biophysics

Background:

• Single-molecule localization microscopy (SMLM) offers high resolution but requires calibration for accurate molecular counting.
• Existing methods for SMLM, especially DNA-PAINT, face challenges with heterogeneous samples and varying target densities.
• Calibration-dependent approaches are sensitive to blinking behavior and sample properties, limiting their biological application.

Purpose of the Study:

• To introduce localization-based fluorescence correlation spectroscopy (lbFCS) as a novel method for absolute molecular counting in SMLM.
• To demonstrate lbFCS as the first absolute molecular counting approach for DNA-PAINT microscopy.
• To enable molecular quantification independent of localization cluster density in SMLM.

Main Methods:

• Development and application of localization-based fluorescence correlation spectroscopy (lbFCS).
• Utilizing DNA-points accumulation for imaging in nanoscale topography (DNA-PAINT) microscopy.
• Systematic proof-of-principle study using DNA origami structures as model targets.

Main Results:

• lbFCS is presented as the first absolute molecular counting method for DNA-PAINT and SMLM.
• The method successfully quantifies target molecules irrespective of localization cluster density.
• Proof-of-principle studies on DNA origami structures validated the accuracy and robustness of lbFCS.

Conclusions:

• lbFCS overcomes limitations of previous DNA-PAINT counting methods.
• This technique provides absolute molecular quantification independent of sample-specific factors.
• lbFCS holds potential for quantitative analysis of challenging biological targets with heterogeneous structures.