Ms. Caroline Berlage Profile

Caroline Berlage

at PicoQuant GmbH

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Proceedings Article | 4 March 2019 Presentation

Mapping molecules quantitatively in confocal fluorescence microscopy (Conference Presentation)

Marcelle Koenig, Caroline Berlage, Paja Reisch, Felix Koberling, Uwe Ortmann, Haisen Ta, Rainer Erdmann

Proceedings Volume 10884, 1088409 (2019) https://doi.org/10.1117/12.2507391

KEYWORDS: Confocal microscopy, Luminescence, Microscopy, Molecules, Statistical analysis, Associative arrays, Single photon, Systems modeling, Tantalum, Molecular lasers

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Single-molecule fluorescence microscopy has been established in life science as an essential tool for studying the characteristics and dynamics of individual fluorescent emitters both in vitro as well as in vivo. Acquiring quantitative information from the confocal observation volume is still a challenging task and knowing the absolute number or concentration of proteins in, e.g., cellular structures can significantly improve our understanding of cell biology, which is an important step towards quantitative microscopy. In this talk, a new quantitative analytical tool called Counting by Photon Statistics (CoPS) will be presented. The approach relies on a statistical analysis of detected photon coincidences to estimate the number of independent fluorescent labels in the observation volume [1]. CoPS does exploit the photon antibunching effect: a single photon emitter can only generate one photon at a time. Originally developed for point measurements, CoPS has recently been extended to an imaging scheme [2]. Using a confocal fluorescence microscopy setup with pulsed excitation, four single-photon, detectors and parallellized time-correlated single photon counting electronics (MicroTime 200, PicoQuant), we prove the applicability of the method with artificial model systems (immobilized DNA origami) and present first steps towards biological samples. [1] Ta, H., Wolfrum, J., Herten, D.-P., An extended scheme for counting fluorescent molecules by photon-antibunching Laser Phys. 20:119 (2010). [2] Ta, H. et al., Mapping molecules in scanning far-field fluorescence nanoscopy. Nat. Commun. 6:7977 (2015).

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