Measuring the size of biological nanostructures with Spatially Modulated Illumination Microscopy

Sonya Martin¹, Antonio Virgilio Failla², Udo Spöri³, Christoph Cremer³, and Ana Pombo⁴^*

¹ MRC - Clinical Sciences Centre, Faculty of Medicine, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UK, Contributed equally
² Applied Optics and Information Processing, Kirchoff Institute for Physics, University of Heidelberg, Im Neuenheimer Feld 227, 69120 Heidelberg, Germany; Physics of Condensed Matter, Institute of Physics, University of Potsdam, Am Neuen Palais 10, 14469 Potsdam, Germany, Contributed equally
³ Applied Optics and Information Processing, Kirchoff Institute for Physics, University of Heidelberg, Im Neuenheimer Feld 227, 69120 Heidelberg, Germany
⁴ MRC - Clinical Sciences Centre, Faculty of Medicine, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UK

^* Corresponding author. E-mail address: ana.pombo{at}csc.mrc.ac.uk.

Spatially modulated illumination fluorescence microscopy canin theory measure the sizes of objects with a diameter rangingbetween 10-200 nm, and has allowed accurate size measurementof subresolution fluorescent beads ( $~$ 40-100 nm). Biological structuresin this size range have so far been measured by electron microscopy.Here we have labeled sites containing the active, hyperphosphorylatedform of RNA polymerase II in the nucleus of HeLa cells usingthe antibody H5. The spatially modulated illumination microscopewas compared with confocal laser scanning and electron microscopesand found to be suitable for measuring the size of cellularnanostructures in a biological setting. The hyperphosphorylatedform of polymerase II was found in structures with $~$ 70 nm diameterwell below the 200 nm-resolution limit of standard fluorescencemicroscopes.