THREE-DIMENSIONAL SPECTRAL PRECISION DISTANCE (SPDM): PRINCIPLE AND METHODS

Christoph Cremer 1,2,3

1Institute of Molecular Biology (IMB), Ackermannweg 4, 55128 , ; 2Kirchhoff Institute for (KIP), and 3Institute for Pharmacy and Molecular Biotechnology (IPMB), University of Heidelberg, Im Neuenheimer Feld, D-69120 Heidelberg, Germany

e-mail:[email protected]; [email protected]

Novel developments in optical technology and photophysics made it possible to radically overcome the diffraction limit of conventional far-field fluorescence microscopy1. Here, the focus will be on a special variant of localization microscopy, called ‘Spectral Precision Distance Microscopy’ (SPDM). As other far-field localization microscopy methods using fluorescent emitters, like GSDIM, PALM/FPALM, STORM, dSTORM etc., it is based on the high precision localization of point sources possible even in the case that the distance between them is smaller than the conventional resolution limit of about 200 nm. In the original SPDM method described this was achieved by using photostable flurochromes with differences in their absorption/emission spectra, or their fluorescence life times2,3 . Applying the Rayleigh criterion of optical (two-point) resolution, in this way the positions of individual adjacent point sources were determined with a localization precision down to the few tens of nanometer range, both in the object plane and in 3D. Although in this way, an enhancement of structural resolution (as defined by the Nyquist theorem) is still difficult to achieve, localization microscopy using photostable emitters may contribute substantially to the spatial analysis of biological nanostructures, especially if combined with photoswitching based localization microscopy approaches and other superresolution techniques.

1) C. Cremer, B.R. Masters (2013) Resolution enhancement techniques in microscopy. Eur. Phys. J. H 38: 281–344. 2) A. Esa et al. (2000) 3D-spectral precision distance microscopy (SPDM) of chromatin nanostructures after triple-colour labeling: a study of the BCR region on chromosome 22 and the Philadelphia chromosome. J. Microscopy 199: 96 – 105. 3) M. Heilemann et al. (2002) High-Resolution colocalization of single dye molecules by fluorescence lifetime imaging microscopy. Anal. Chemistry 74: 3511- 3517. 4) J. Reymann et al. (2008) High precision structural analysis of subnuclear complexes in fixed and live cells via Spatially Modulated Illumination (SMI) microscopy. Chromosome Research 16: 367 –382.