Nuclear pores as versatile reference standards for quantitative superresolution microscopy

Abstract

Quantitative fluorescence and superresolution microscopy are often limited by insufficient data quality or artifacts. In this context, it is essential to have biologically relevant control samples to benchmark and optimize the quality of microscopes, labels and imaging conditions.Here we exploit the stereotypic arrangement of proteins in the nuclear pore complex as in situ reference structures to characterize the performance of a variety of microscopy modalities. We created four genome edited cell lines in which we endogenously labeled the nucleoporin Nup96 with mEGFP, SNAP-tag or HaloTag or the photoconvertible fluorescent protein mMaple. We demonstrate their use a) as 3D resolution standards for calibration and quality control, b) to quantify absolute labeling efficiencies and c) as precise reference standards for molecular counting.These cell lines will enable the broad community to assess the quality of their microscopes and labels, and to perform quantitative, absolute measurements.

Molecular organization and mechanics of single vimentin filaments revealed by super-resolution imaging

“…The GFP-terminal ends were labeled with GFP-nanobodies-AF647, which are fluorescently-labeled single-chain antibody domains with high affinity for GFP that are widely used for SRM (33). With the absence of unlabeled endogenous vimentin, the labeling of vimentin was maximized allowing to perform quantitative dSTORM imaging in optimal conditions (23,34). We performed 2D-dSTORM imaging of fixed cells (Fig.…”

“…1D-E and 2D). To quantify the possible errors coming from SRM measurements, we measured the nuclear pores radius, which has become a standard for quantitative SRM (34), using a similar labeling technique (imaging of Nup96-SNAP labeled with SNAP-AF647), the same 3D-dSTORM microscopy setup and quantification method (Fig. S5).…”

Interrogating Synaptic Architecture: Approaches for Labeling Organelles and Cytoskeleton Components

“…When such fluorescent derivatives are added to cells expressing SNAP tag, the latter catalyzes self-labeling with the fluorophore by covalently attaching the fluorophore with the benzyl group to a cysteine residue in SNAP tag sequence. The reaction is highly specific and can be highly effective, resulting in nearly all present SNAP tags labeling, but recent studies report much lower efficiency (Thevathasan et al, 2019). The labeling reaction can be triggered in live cells when membrane permeable dyes such as tetramethyl-rhodamine-Star or 647-SiR are used.…”

Between Life and Death: Reducing Phototoxicity in Super-Resolution Microscopy

“…However, while many specialised fluorophores exist for fixed specimens (62), there are far fewer options available for live-cell imaging. An inappropriate choice of fluorophore for live-cell SRM will not only lead to low quality images downstream (63), but also inevitably impact acquisition settings and hence phototoxicity (10,64). As for most fluorescence microscopy techniques, the two classes of fluorophores used in SRM are fluorescent proteins (FPs) ( Fig.…”