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Proceedings Paper

Adaptive holographic region of interest illumination with oblique angles for use in single molecule localization microscopy
Author(s): Alexander Jügler; Jan Becker; Ronny Förster; Rainer Heintzmann
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Paper Abstract

To improve the localization accuracy and to reduce the overall photo-damage in Single Molecule Localization Microscopy (SMLM), we introduce a holography based Region-of-Interest (ROI) illumination with oblique angles. The ROI illumination is realized in two different application modes which offer different advantages. Both modes (A,B) are implemented within the same setup by modifying the illumination light using a phase-only spatial light modulator (SLM) twice. This allows to adaptively modify the size and the (excitation) angle of the required ROI illumination, resulting in reduced out-of focus signal and less overall phototoxicity. Mode A generates a nearly speckle free, circular ROI which can be obtained instantly (no iterative algorithm needed). Mode B allows to realize an arbitrarily shaped ROI but comes at the cost of a higher presence of speckle structures as well as in an increased calculation time of the holograms. In both cases illumination angles up to 60° with high NA objectives are realizable, enabling the effective selection of specific parts of a biological probe to be illuminated.

Paper Details

Date Published: 22 July 2019
PDF: 3 pages
Proc. SPIE 11076, Advances in Microscopic Imaging II, 1107602 (22 July 2019); doi: 10.1117/12.2525253
Show Author Affiliations
Alexander Jügler, Leibniz-Institut für Photonische Technologien e.V. (Germany)
Jan Becker, Leibniz-Institut für Photonische Technologien e.V. (Germany)
Ronny Förster, Leibniz-Institut für Photonische Technologien e.V. (Germany)
Rainer Heintzmann, Leibniz-Institut für Photonische Technologien e.V. (Germany)
Friedrich-Schiller-Univ. Jena (Germany)

Published in SPIE Proceedings Vol. 11076:
Advances in Microscopic Imaging II
Emmanuel Beaurepaire; Francesco Saverio Pavone; Peter T. C. So, Editor(s)

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