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Proceedings Paper

Fabrication of two-color annular hybrid wave plate for three-dimensional super-resolution microscopy
Author(s): Hiroshi Kumagai; Yoshinori Iketaki; Kornel Jahn; Nador Bokor
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Paper Abstract

In super-resolution microscopy, we use fluorescence depletion, where an erase beam quenches a molecule in the S1 state generated by a pump beam, and then prevents fluorescence from the S1 state. When a tight doughnut shaped erase beam with is focused on the dyed sample together with a Gaussian pump beam, the remaining fluorescence spot in the focal plane becomes smaller than the diffraction-limited size. Applying destructive interference to the erase beam, erase beam has a minute three-dimensional dark spot surrounded by the light near the focal region. Since this spot introduces fluorescence depletion along the optical axis as in the focal plane, we can achieve three-dimensional super-resolution microscopy. However, to overcome the diffraction limit, an extremely precise optical alignment is required for projecting the focused pump beam into the dark spot of the erase beam. To resolve this technical issue, we fabricated a two-color annular hybrid wave plate (TAHWP) by combining two multi-order wave quartz plates. Although the pump and erase beams co-axially pass through the plate; the pump beam retains its original Gaussian shape, while the erase beam undergoes destructive interference. Inserting the TAHWP into a commercial scanning laser microscope, a three-dimensional spherical fluorescence spot with a volume of (~100 nm)3 can be created. Beside eliminating alignment problems and yielding a compact setup, the TAHWP makes our proposed method very suitable for commercial microscope systems. In this study, we report about detailed fabrication procedure and three-dimensional image properties given by the TAHWP.

Paper Details

Date Published: 9 March 2016
PDF: 6 pages
Proc. SPIE 9713, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXIII, 971311 (9 March 2016); doi: 10.1117/12.2211871
Show Author Affiliations
Hiroshi Kumagai, Kitasato Univ. (Japan)
Yoshinori Iketaki, Kitasato Univ. (Japan)
Olympus Corp. (Japan)
Kornel Jahn, Budapest Univ. of Technology and Economics (Hungary)
Nador Bokor, Budapest Univ. of Technology and Economics (Hungary)


Published in SPIE Proceedings Vol. 9713:
Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXIII
Thomas G. Brown; Carol J. Cogswell; Tony Wilson, Editor(s)

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