Abstract text (incl. figure legends and references)

Introduction:

In biological context subcellular activity always shares both structural and functional components. In consequence, the examination of a biological function will gain benefit from a spatially resolved image of the location of activity. In turn, the interpretation of biological patterns is substantially supported by information regarding the function expected. This is commonly addressed by recording images of different spatial resolution and different functional information values. A combination of both different image types makes it possible to superpose function and location. We remove this process of superposition by spanning up a common functional and spatial context and interpret the electron microscopy records using the help of single molecule light microscopy-derived functional information and subsequent topology analysis.

Objective:

The approach sketched here is based on the assumption that functionality and spatial organization are coupled if a common size regime is covered. This assumption makes it possible to us to follow a biological function inside a native environment with a resolution nearby the one typical for electron microscopy. We subsequently interpret the higher resolved electron microscopic recording in terms of the functional information derived from the first part of the examination.

Material & Methods:

The selection of a biological function of interest is done via conventional immunostaining with fluorescently and gold labelled antibodies. The specimens are recorded by single molecule light microscopy (SMLM). We record the dye molecule blinking signals and the gold mediated fluorescence. The resulting signal matrix is not (only) converted into an image but also a template for topology based analyses. Subsequent to this first procedure to extract biological function, the specimen is stained by ruthenium oxide. The large difference in atomic mass between ruthenium and gold makes it possible to identify the locus of the function stained via fluorescent antibodies inside the spatial organization as given by the ruthenium stain.

Results:

We localize the biological function inside an environment contrasted by heavy metal staining. In difference to conventional immune-gold applications the electron microscopy image is interpreted by a set of information derived from the same specimen but not the same locality. The presence of gold makes it possible to coordinate this independent information in a common functional space inside a cell. The topology analyses do not improve the spatial resolution of the electron microscopy imaging but complement it with functional information.

Discussion:

With our approach we span a common context between single molecule light microscopy and electron microscopy. This context makes it possible to interpret the appearance of an electron microscopic record under terms of the biological function supported by the electron microscopic derived structure. For instance, the information regarding spatial organization derived from topology analysis can give a reference parameter to estimate whether the electron microscopy imaged pattern is a true basis for biological function or a superposition of original biological structure and preparation mediated artifacts.