Apertureless scanning near-field optical microscopy of sparsely labeled tobacco mosaic viruses and the intermediate filament desmin

Abstract

SummaryBoth fluorescence imaging and atomic force microscopy (AFM) are highly versatile and extensively used in applications ranging from nanotechnology to life sciences. In fluorescence microscopy luminescent dyes serve as position markers. Moreover, they can be used as active reporters of their local vicinity. The dipolar coupling of the tip with the incident light and the fluorophore give rise to a local field and fluorescence enhancement. AFM topographic imaging allows for resolutions down to the atomic scale. It can be operated in vacuum, under ambient conditions and in liquids. This makes it ideal for the investigation of a wide range of different samples. Furthermore an illuminated AFM cantilever tip apex exposes strongly confined non-propagating electromagnetic fields that can serve as a coupling agent for single dye molecules. Thus, combining both techniques by means of apertureless scanning near-field optical microscopy (aSNOM) enables concurrent high resolution topography and fluorescence imaging. Commonly, among the various (apertureless) SNOM approaches metallic or metallized probes are used. Here, we report on our custom-built aSNOM setup, which uses commercially available monolithic silicon AFM cantilevers. The field enhancement confined to the tip apex facilitates an optical resolution down to 20 nm. Furthermore, the use of standard mass-produced AFM cantilevers spares elaborate probe production or modification processes. We investigated tobacco mosaic viruses and the intermediate filament protein desmin. Both are mixed complexes of building blocks, which are fluorescently labeled to a low degree. The simultaneous recording of topography and fluorescence data allows for the exact localization of distinct building blocks within the superordinate structures.

Molecular insights into cardiomyopathies associated with desmin (DES) mutations

“…The assembly of recombinant desmin can be initiated in vitro by adding sodium chloride (Kreplak and Bar 2009). Transmission electron microscopy (TEM) or atomic force microscopy (AFM) can be applied for visualization of the assembly process (Brodehl et al 2012a;Harder et al 2013;Bar et al 2006). Of note, desmin contains one cysteine residue (p.C333), which can be used for site-specific labeling with chemical fluorescent dyes (Harder et al 2013).…”

“…Transmission electron microscopy (TEM) or atomic force microscopy (AFM) can be applied for visualization of the assembly process (Brodehl et al 2012a;Harder et al 2013;Bar et al 2006). Of note, desmin contains one cysteine residue (p.C333), which can be used for site-specific labeling with chemical fluorescent dyes (Harder et al 2013). Apertureless scanning near-field microscopy (aSNOM) was previously used in combination with Atto740-conjugated recombinant desmin to investigate the co-assembly of mutant and wild-type desmin (Brodehl et al 2016a;Harder et al 2013) (Fig.…”

“…Of note, desmin contains one cysteine residue (p.C333), which can be used for site-specific labeling with chemical fluorescent dyes (Harder et al 2013). Apertureless scanning near-field microscopy (aSNOM) was previously used in combination with Atto740-conjugated recombinant desmin to investigate the co-assembly of mutant and wild-type desmin (Brodehl et al 2016a;Harder et al 2013) (Fig. 5e).…”

“…d Cellular stretching of transfected cells expressing mutant desmin (Plodinec et al 2011). e Schematic overview of apertureless scanning near-field optical microscopy (aSNOM) (Harder et al 2013). f Longitudinal stretching of desmin filaments using the withdrawing meniscus of a buffer droplet.…”

Optical near-fields & nearfield optics

“…In spite of the high efficiency of plasmonic metal nanostructures, the near-field enhancement of dielectric structures is preferable for some applications. Walhorn et al [ 8 ] have developed a method for the simultaneous recording of topography and fluorescence that allows for the localization of distinct building blocks of supramolecular structures below the usual optical resolution. This is facilitated by using the optical near-field around a sharp dielectric AFM tip to excite the fluorescent molecules – a metallic tip would quench the fluorescence.…”