Description

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Molecular Counting is a technique that enables quantitative mapping of the distribution of molecules using fluorescence microscopy. The approach is based on Counting by Photon Statistics (CoPS)¹, which involves a statistical analysis of detected photon coincidences to estimate the number of independent fluorescent emitters in the observation volume.

Mapping molecules quantitatively based on CoPS can be carried out using a MicroTime 200 time-resolved confocal microscope equipped with four SPAD detectors and a four channel HydraHarp 400. The application also requires additional optical elements and proprietary analysis software provided by Haisen Ta / PicoQuant.

The counting method as presented here currently works best for bright and stable red emitting fluorophores in fixed samples with low background emission.

¹ Ta, H., Kiel, A., Wahl, M. & Herten, D.-P. Experimental approach to extend therange for counting fluorescent molecules based on photon-antibunching.Phys.Chem. Chem. Phys.12,10295–10300 (2010)

Proof of principle experiment

In proof-of-concept studies, DNA origamis with varying emitter numbers (from 1 to 30) per origami were quantified. The plots below show the results obtained with DNA origami emitting in the red spectral range. The black bars indicate the expected distribution pattern of emitter number per origami (assuming a binomial distribution), while pink bars show the experimentally obtained numbers. The red curve is a Gaussian curve fitted to the experimental data. As can be clearly seen, the approach works well for up to 10 emitters per origami. At higher emitter numbers per cluster, the CoPS approach leads to an overestimation of this number.

Downloads

• Scientific presentation on molecular counting by CoPS
• Molecular counting by CoPS - Poster