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Instrumentation, Measurement, and Metrology

Spatially modulated illumination microscopy using one objective lens

[+] Author Affiliations
Hans Mathée

Universität Heidelberg, Applied Optics and Information Processing, Kirchhoff-Institut für Physik, INF 227, 69120 Heidelberg, Germany

Christoph Wotzlaw

Universität Duisburg-Essen, Institut für Physiologie, Hufelandstrasse 55, 45122 Essen, Germany

Christoph Cremer

Universität Heidelberg, Applied Optics and Information Processing, Kirchhoff-Institut für Physik, INF 227, 69120 Heidelberg, Germany and Institute for Molecular Biophysics, The Jackson Laboratory, Bar Harbor, Maine

Udo Birk

Universität Heidelberg, Applied Optics and Information Processing, Kirchhoff-Institut für Physik, INF 227, 69120 Heidelberg, Germany

David Baddeley

Universität Heidelberg, Applied Optics and Information Processing, Kirchhoff-Institut für Physik, INF 227, 69120 Heidelberg, Germany

Opt. Eng. 46(8), 083603 (August 03, 2007). doi:10.1117/1.2768085
History: Received September 18, 2006; Revised February 08, 2007; Accepted February 22, 2007; Published August 03, 2007
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The spatially modulated illumination (SMI) microscope is a wide-field fluorescence microscope featuring axially structured illumination, through which access to information about subresolution object structures is obtained. We present a simplified setup where the interference pattern is generated by reflecting the laser beam with a mirror. We characterize our setup by presenting measurements on fluorescent microspheres with diameters ranging from 44 to 200nm. The results agree well with the sizes provided by the manufacturer. Furthermore, the spheres are analyzed with 458-, 514-, 488-, and 568-nm excitation wavelengths, giving good agreement of the sizes determined at the respective wavelengths. A measurement of the same objects using different excitation wavelengths leads to a size difference of a few nano-meters only. The potential of SMI microscopy for the fast analysis of many fluorescent objects is also addressed. In addition, the applicability to biological specimens is shown on fluorescently labeled, specific chromatin domains. The results obtained using the presented mirror geometry agree well with data obtained using a standard SMI microscope setup.

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© 2007 Society of Photo-Optical Instrumentation Engineers

Citation

Hans Mathée ; Christoph Wotzlaw ; Christoph Cremer ; Udo Birk and David Baddeley
"Spatially modulated illumination microscopy using one objective lens", Opt. Eng. 46(8), 083603 (August 03, 2007). ; http://dx.doi.org/10.1117/1.2768085


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