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Using spatially modulated illumination (SMI) light microscopy it is possible to measure the sizes of fluorescent structures that have an extension far below the conventional optical resolution limit (“subresolution size”). Presently, the sizes are determined as the object extension along the optical axis of the SMI microscope. For this, however, “a priori” assumptions on the fluorochrome distribution (“shape”) within the examined fluorescent structure have to be made. Usually it is assumed that the fluorochrome follows a Gauss-distribution or a spherical distribution. In this report we overcome the necessity to make an assumption on the shape of the fluorochrome distribution. We introduce two new experimentally obtained parameters which allow the determination of a shape measure to describe the spatial distribution of the fluorescent dye. This becomes possible by independent measurements with different excitation wavelengths. As an example, we present shape parameter measurements on individual fluorescent microspheres with a nominal geometrical diameter (“size”) of 190 nm. In the case investigated, the experimental shape correlated well with a homogeneous fluorochrome distribution (“spherical shape”) but not with a variety of other “shapes”.

Although light microscopy and three-dimensional image analysis have made considerable progress during the last decade, it is still challenging to analyze the genome nano-architecture of specific gene domains in three-dimensional cell nuclei by fluorescence microscopy. Here, we present for the first time chromatin compaction measurements in human lymphocyte cell nuclei for three different, specific gene domains using a novel light microscopic approach called Spatially Modulated Illumination microscopy. Gene domains for p53, p58, and c-myc were labeled by fluorescence in situ hybridization and the sizes of the fluorescence in situ hybridization "spots" were measured. The mean diameters of the gene domains were determined to 103 nm (c-myc), 119 nm (p53), and 123 nm (p58) and did not correlate to the genomic, labeled sequence length. Assuming a spherical domain shape, these values would correspond to volumes of 5.7 x 10(-4) microm(3) (c-myc), 8.9 x 10(-4) microm(3) (p53), and 9.7 x 10(-4) microm(3) (p58). These volumes are approximately 2 orders of magnitude smaller than the diffraction limited illumination or observation volume, respectively, in a confocal laser scanning microscope using a high numerical aperture objective lens. By comparison of the labeled sequence length to the domain size, compaction ratios were estimated to 1:129 (p53), 1:235 (p58), and 1:396 (c-myc). The measurements demonstrate the advantage of the SMI technique for the analysis of gene domain nano-architecture in cell nuclei. The data indicate that chromatin compaction is subjected to a large variability which may be due to different states of genetic activity or reflect the cell cycle state.

An improved size measurement method using spatially modulated illumination (SMI) microscopy enhances subwavelength size determination of fluorescent objects. In this new approach the point spread function of the SMI microscope is reconstructed in each measurement. For this, reference objects with known dye distribution have to be put additionally to the unknown objects on the object slide or on the cover slip. We present data from measurements on fluorescent microspheres with diameters of 140 and 200 nm using an excitation wavelength of 488 nm.

Recently developed far field light optical methods are a powerful tool to analyze biological nanostructures and their dynamics, in particular including the interior of three-dimensionally conserved cells. In this article, the recently described method of spatially modulated illumination (SMI) microscopy has been further extended to the online determination of the extension of small, subwavelength sized, fluorescent objects (nanosizing). Using fluorescence excitation with 488 nm, the determination of fluorescent labeled object diameters down to 40 nm corresponding to about 1/12th of the wavelength used for one-photon excitation could be shown. The results of the SMI nanosizing procedure for a detailed, systematic variation of the object diameter are presented together with a fast algorithm for online size evaluation. In addition, we show a direct comparison of the diameter of “colocalization volumes” between SMI nanosizing and conventional confocal laser scanning microscopy.

Spatially modulated illumination fluorescence microscopy can in theory measure the sizes of objects with a diameter ranging between 10 and 200 nm and has allowed accurate size measurement of subresolution fluorescent beads ( approximately 40-100 nm). Biological structures in this size range have so far been measured by electron microscopy. Here, we have labeled sites containing the active, hyperphosphorylated form of RNA polymerase II in the nucleus of HeLa cells by using the antibody H5. The spatially modulated illumination-microscope was compared with confocal laser scanning and electron microscopes and found to be suitable for measuring the size of cellular nanostructures in a biological setting. The hyperphosphorylated form of polymerase II was found in structures with a diameter of approximately 70 nm, well below the 200-nm resolution limit of standard fluorescence microscopes.