Nanostructure Analysis Using Spatially Modulated Illumination Microscopy

Abstract

For an improved understanding of cellular processes, it is highly desirable to develop light optical methods for the analysis of biological nanostructures and their dynamics in the interior of three-dimensionally (3D) conserved cells. Here, important structural parameters to be considered are the topology, i.e. the mutual positions and distances, as well as the sizes of the constituting subunits. This has become possible by the development of a novel method of far-field light fluorescence microscopy, spatially modulated illumination (SMI) microscopy. Using this approach, axial distances between fluorescence-labeled targets can be measured with an accuracy close to 1 nm; their sizes can be determined down to a few tens of nanometers. This approach can be extended to the determination of 3D positions and mutual 3D distances and sizes of any number of small objects/subunits that can be discriminated due to their spectral signatures. Consequently, the new approach allows an ‘in situ nanostructure elucidation, until now regarded to be beyond the possibilities of far-field light microscopy. Application examples discussed are: colocalization/nanosizing and topological analysis of large protein-protein complexes, of nucleic acid-protein complexes (such as transcription factories), or of the highly complex DNA-protein nanostructures of which active/ inactive gene regions in the eukaryotic cell nucleus are constituted.

Resolution enhancement techniques in microscopy

“…However, in combination with high-precision axial positioning this technique of farfield light microscopy allowed the nondestructive high precision localization analysis of complex spatial arrangements [Albrecht et al 2001 inside relatively thick transparent specimens such as the cell nucleus and enabled size measurements at molecular dimensions of a few tens of nanometers [Failla et al 2002b[Failla et al , 2003Martin et al 2004; and 3D position measurements down to the 1 nm range [Albrecht et al 2001Failla et al 2001;Baddeley et al 2007]. It may be noted that the SMI precision localization measurements were explicitly seen by the authors as a step towards resolution enhancement by localization microscopy [e.g.…”

High-precision structural analysis of subnuclear complexes in fixed and live cells via spatially modulated illumination (SMI) microscopy

“…The fundamental principles of SMI microscopy have been discussed in detail in previous publications (Albrecht et al 2002, Failla et al 2002b, 2003. The SMI PSF is the product of the detection PSF corresponding to an ordinary wide-field PSF and the illumination PSF, which is given by the cos 2 shape.…”

“…In contrast to focused laser light techniques (Hell et al 1994, Klar et al 2000, Dyba & Hell 2002, Egner et al 2002 or structured illumination (Gustafsson et al 1995, Frohn et al 2000, the optical resolution itself is not enhanced. Nevertheless, in combination with high-precision axial positioning this technique of far-field light microscopy allows the nondestructive analysis of complex spatial arrangements (Albrecht et al 2002) inside relatively thick transparent specimens such as the cell nucleus and enables size measurements at molecular dimensions of a few tens of nanometers (Failla et al 2002b(Failla et al , 2003. SMI microscopy is now an established method for the analysis of topological arrangements of mammalian genome structure.…”

Superresolution size determination in fluorescence microscopy: A comparison between spatially modulated illumination and confocal laser scanning microscopy

“…28 Beyond the analysis of biological nanostructures, SMI microscopy might find interesting applications also in other fields of nanostructure research. This limit can be overcome with multispectral labeling.…”