Mapping molecules quantitatively in confocal fluorescence microscopy (Conference Presentation)

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

doi:10.1117/12.2507391

4 March 2019 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, Single Molecule Spectroscopy and Superresolution Imaging XII; 1088409 (2019) https://doi.org/10.1117/12.2507391
Event: SPIE BiOS, 2019, San Francisco, California, United States

Abstract

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).

Conference Presentation

Citation Download Citation

Marcelle Koenig, Caroline Berlage, Paja Reisch, Felix Koberling, Uwe Ortmann, Haisen Ta, and Rainer Erdmann "Mapping molecules quantitatively in confocal fluorescence microscopy (Conference Presentation)", Proc. SPIE 10884, Single Molecule Spectroscopy and Superresolution Imaging XII, 1088409 (4 March 2019); https://doi.org/10.1117/12.2507391

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KEYWORDS

Confocal microscopy

Luminescence

Microscopy

Molecules

Statistical analysis

Associative arrays

Molecular lasers

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