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Proceedings Paper

Development of fast two-dimensional standing wave microscopy using acousto-optic deflectors
Author(s): Olga Gliko; Duemani G. Reddy; William E. Brownell; Peter Saggau
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Paper Abstract

A novel scheme for two-dimensional (2D) standing wave fluorescence microscopy (SWFM) using acousto-optic deflectors (AODs) is proposed. Two laser beams were coupled into an inverted microscope and focused at the back focal plane of the objective lens. The position of each of two beams at the back focal plane was controlled by a pair of AODs. This resulted in two collimated beams that interfered in the focal plane, creating a lateral periodic excitation pattern with variable spacing and orientation. The phase of the standing wave pattern was controlled by phase delay between two RF sinusoidal signals driving the AODs. Nine SW patterns of three different orientations about the optical axis and three different phases were generated. The excitation of the specimen using these patterns will result in a SWFM image with enhanced 2D lateral resolution with a nearly isotropic effective point-spread function. Rotation of the SW pattern relative to specimen and varying the SW phase do not involve any mechanical movements and are only limited by the time required for the acoustic wave to fill the aperture of AOD. The resulting total acquisition time can be as short as 100 µs and is only further limited by speed and sensitivity of the employed CCD camera. Therefore, this 2D SWFM can provide a real time imaging of subresolution processes such as docking and fusion of synaptic vesicles. In addition, the combination of 2D SWFM with variable angle total internal reflection (TIR) can extend this scheme to fast microscopy with enhanced three-dimensional (3D) resolution.

Paper Details

Date Published: 12 February 2008
PDF: 8 pages
Proc. SPIE 6861, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XV, 68610B (12 February 2008); doi: 10.1117/12.761891
Show Author Affiliations
Olga Gliko, Baylor College of Medicine (United States)
Duemani G. Reddy, Rice Univ. (United States)
William E. Brownell, Baylor College of Medicine (United States)
Peter Saggau, Baylor College of Medicine (United States)
Rice Univ. (United States)


Published in SPIE Proceedings Vol. 6861:
Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XV
Jose-Angel Conchello; Carol J. Cogswell; Tony Wilson; Thomas G. Brown, Editor(s)

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