Quantum mechanics

Context (archaeology)

Cytoplasm

Cell biology

Biology

Cell nucleus

Materials science

Microscope

Microscopy

Calibration

Computer science

Resolution (logic)

Paleontology

Physics

Nanotechnology

Optics

Nuclear pore

Fluorescence

Artificial intelligence

Fluorescence microscope

Nuclear transport

Nucleoporin

Biological system

Nature Methods

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, HaloTag or the photoconvertible fluorescent protein mMaple. We demonstrate their use (1) as three-dimensional resolution standards for calibration and quality control, (2) to quantify absolute labeling efficiencies and (3) as precise reference standards for molecular counting. These cell lines will enable the broader community to assess the quality of their microscopes and labels, and to perform quantitative, absolute measurements.

Nuclear pores as versatile reference standards for quantitative superresolution microscopy