Systematic, spatial imaging of large multimolecular assemblies and the emerging principles of supramolecular order in biological systems

Abstract

Understanding biological systems at the level of their relational (emergent) molecular properties in functional protein networks relies on imaging methods, able to spatially resolve a tissue or a cell as a giant, non-random, topologically defined collection of interacting supermolecules executing myriads of subcellular mechanisms. Here, the development and findings of parameter-unlimited functional super-resolution microscopy are described—a technology based on the fluorescence imaging cycler (IC) principle capable of co-mapping thousands of distinct biomolecular assemblies at high spatial resolution and differentiation (<40 nm distances). It is shown that the subcellular and transcellular features of such supermolecules can be described at the compositional and constitutional levels; that the spatial connection, relational stoichiometry, and topology of supermolecules generate hitherto unrecognized functional self-segmentation of biological tissues; that hierarchical features, common to thousands of simultaneously imaged supermolecules, can be identified; and how the resulting supramolecular order relates to spatial coding of cellular functionalities in biological systems. A large body of observations with IC molecular systems microscopy collected over 20 years have disclosed principles governed by a law of supramolecular segregation of cellular functionalities. This pervades phenomena, such as exceptional orderliness, functional selectivity, combinatorial and spatial periodicity, and hierarchical organization of large molecular systems, across all species investigated so far. This insight is based on the high degree of specificity, selectivity, and sensitivity of molecular recognition processes for fluorescence imaging beyond the spectral resolution limit, using probe libraries controlled by ICs. © 2013 The Authors. Journal of Molecular Recognition published by John Wiley & Sons, Ltd.

Using toponomics to characterize phenotypic diversity in alveolar macrophages from male mice treated with exogenous SP-A1

“…The TIS technology used here, as noted in the Introduction, has advantages over other more recently introduced multiplexing methods [41,51,55,56]. In these systems the antibodies are tagged with Cy dyes and the fluorescence is quenched by exposing the sections to H 2 O 2 at pH > 10.…”

“…We opted to use TIS for several reasons. Exposing the sections to H 2 O 2 at pH > 10 to quench the fluorescence [41,51,56,57], unlike photobleaching used in TIS, has been found to alter epitopes of some proteins and may decrease, eliminate, or enhance the fluorescence signal [41,57]. Unlike TIS the newer systems are yet to be automated and importantly lack the capabilities offered by the image processing software developed for use with TIS and referred to above.…”

“…Here we applied TIS (or MELC) [36,[41][42][43][44] to use Combinatorial Molecular Phenotypes or CMPs to characterize the heterogeneity of AM. A CMP is a designation indicating the presence or absence of all markers in a given pixel.…”

“…In all images there are 2 n possible CMPs where n = the number of markers used. TIS is a high throughput robotically-controlled microscopic system developed by Schubert [36,[41][42][43]. It enables immunophenotyping cells in their native environment by using robotically controlled reiterative cycles of immunostaining (tagging), imaging, and photobleaching of FITC-conjugated antibodies.…”

A Platform for Parameter Unlimited Molecular Geometry Imaging Obviously Enabling Life Saving Measures in ALS

“…These spatial blueprints, in turn, typically include so-called emergent systems [10], where emergence is understood as the process of complex pattern formation from more basic constituent parts [10]: large combinatorial molecular structures acquire, e.g. through the differential interplay of the numerous different protein classes, new properties in health and disease [10].…”