Abstract
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 200 nm. 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 nanometers 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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