Measuring the Size of Biological Nanostructures with Spatially Modulated Illumination Microscopy

Abstract

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 (∼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 ∼70 nm, well below the 200-nm resolution limit of standard fluorescence microscopes.