Quenched Stochastic Optical Reconstruction Microscopy (qSTORM) with Graphene Oxide

Abstract

Quenched Stochastic Optical Reconstruction Microscopy (qSTORM) was demonstrated with graphene oxide sheets, peptides and bacteria; a method of contrast enhancement with super-resolution fluorescence microscopy. Individual sheets of graphene oxide (GO) were imaged with a resolution of 16 nm using the quenching of fluorescence emission by GO via its large Resonant Energy Transfer (RET) efficiency. The method was then extended to image self-assembled peptide aggregates (resolution 19 nm) and live bacterial cells (resolution 55 nm, the capsular structure of E. coli from urinary tract infections) with extremely low backgrounds and high contrasts (between one and two orders of magnitude contrast factor improvements that depended on the thickness of the graphene oxide layer used). Graphene oxide films combined with STORM imaging thus provide an extremely convenient method to image samples with large backgrounds due to non-specifically bound fluorophores (either due to excess labelling or autofluorescent molecules), which is a common occurrence in studies of both biological cells and soft-condensed matter. The GO quenches the fluorescence across a thin layer at distances of less than 15 nm. Graphene oxide films coated with thin layers (≤15 nm) of polystyrene, polymethylmethacrylate and polylysine are shown to be effective in producing high contrast qSTORM images, providing a convenient modulation of sample/substrate interactions. The GO coatings can also provide an increased image resolution and a factor of 2.3 improvement was observed with the peptide fibres using a feature of interest metric,when there was a large non-specifically bound background.

Super-Resolution Fluorescence Microscopy Study of the Production of K1 Capsules byEscherichia coli: Evidence for the Differential Distribution of the Capsule at the Poles and the Equator of the Cell

“…This PLLGO coating improves the signal-to-noise ratio (i.e., the contrast) for fluorescence imaging by a factor of 100. 41…”

“…In addition, confocal and super-resolution (dSTORM) microscopies were used without the washing step with the immunostaining methods by incubating both primary (1:8000) and secondary (1:10 000) antibodies at a lower concentration with the bacteria due to the benefits of using PLLGO-coated slides, i.e., the PLLGO coating removed the background due to nonspecifically bound fluorophores and provided a 100 factor of improvement in contrast. 1 The polylysine coating has a negligible effect on the proliferation of the bacteria over a period of 3 h (the time for a complete capsule to form, Supporting Information (SI 5)).…”

A versatile route to edge-specific modifications to pristine graphene by electrophilic aromatic substitution

“…The fluorescence from the graphene is initially unexpected. All fluorophores less than 10 nm from graphene or GO are expected to quench their fluorescence due to resonant energy transfer (RET) [87][88][89], an effect that has been used for contrast enhancement in STORM [66]. This RET is suppressed at edges and other defects in graphene [90]; however, the control experiment in which the dye conjugation procedure was repeated on CVD G-SO 3…”

Super-Resolution Imaging with Graphene

“…Moreover, it can be used to cooperate with quenched stochastic optical reconstruction microscopy (qSTORM) to achieve the super-resolution imaging effect of self-assembled peptide fibrils and Escherichia coli. In 2018, Li et al experimentally demonstrated that GO coating with qSTORM could increase the imaging resolution and contrast [ 102 ] when imaging the peptides and bacteria via the strong quenching effect. Due to the same hexagonal lattice and similar electronic properties for both graphene and GO, the quenching effect is advisable with GO.…”

“…( b ) Reconstructed STORM image of peptide fibers. Reprinted with permission from [ 102 ]. Copyright 2018 Springer Nature.…”