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Nanometer-scale colocalization microscopy of Streptococcus pneumoniae filaments
Author(s): Christoph Polzer; Stefan Ness; Mojtaba Mohseni; Joachim Rädler; Markus Hilleringmann; Thomas Hellerer
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Paper Abstract

We present numerical simulations and experiments employing two-photon excited fluorescence in a sum-frequency mixing scheme which could be used for colocalization experiments in biophysics. By means of numerical calculations using the Debye approximation, the point spread functions (PSF) of each focused laser beam as well as the resulting PSF of the two-color two-photon (2C2P) excitation are calculated and discussed. Experiments are performed on Streptococcus pneumoniae and on surface filaments (“pili”) of the bacteria. Two different fluorescent labels were used for staining the bacteria themselves as well as the surface filaments as structure of interest. Since one fluorophore is excited by one single laser and the other label only in the combination of both lasers, intrinsic colocalization of the signals on the nanometer scale is ensured. The two-color two-photon excitation is performed by an ultrashort fiber laser system with synchronized emission wavelengths at λ1 = 780 nm and λ2 = 1030 nm and pulse durations around 100 fs. The multiphoton microscopy approach provides high resolution and allows for three-dimensional imaging of bacteria in a volume of (5 x 5 x 5) μm3.

Paper Details

Date Published: 22 February 2019
PDF: 10 pages
Proc. SPIE 10882, Multiphoton Microscopy in the Biomedical Sciences XIX, 108822S (22 February 2019); doi: 10.1117/12.2509927
Show Author Affiliations
Christoph Polzer, Munich Univ. of Applied Sciences (Germany)
Ludwig-Maximilians-Univ. München (Germany)
Stefan Ness, Univ. of Applied Sciences Munich (Germany)
Mojtaba Mohseni, Munich Univ. of Applied Sciences (Germany)
Joachim Rädler, Ludwig-Maximilians-Univ. München (Germany)
Markus Hilleringmann, Univ. of Applied Sciences Munich (Germany)
Thomas Hellerer, Ludwig-Maximilians-Univ. München (Germany)

Published in SPIE Proceedings Vol. 10882:
Multiphoton Microscopy in the Biomedical Sciences XIX
Ammasi Periasamy; Peter T. C. So; Karsten König, Editor(s)

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