Labeling Strategies Matter for Super-Resolution Microscopy: A Comparison between HaloTags and SNAP-tags

Erdmann, Roman S.; Baguley, Stephanie Wood; Richens, Jennifer H.; Wissner, Rebecca F.; Xi, Zhiqun; Allgeyer, Edward S.; Zhong, Sheng; Thompson, Alexander D.; Lowe, Nick; Butler, Richard J.; Bewersdorf, Joerg; Rothman, James E.; Johnston, Daniel St; Schepartz, Alanna; Toomre, Derek

doi:10.1016/j.chembiol.2019.01.003

Cited by 109 publications

(111 citation statements)

References 47 publications

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“…This can be due to incomplete folding of the enzymatic tags, inhibition of the tags by fixatives or intracellular components, or by incomplete activation and detection of the fluorophores, imperfect ligands, or bleaching during the initial off-switching step in SMLM, and warrants further investigation and optimization. Incomplete intracellular labeling for SNAP-tag and HaloTag was reported previously 53 , with the choice of dye strongly affecting the ELE. Also the incomplete maturation of photoconvertible proteins has been reported before 32 and should be a target for further optimization.…”

Section: Discussionmentioning

confidence: 94%

Nuclear pores as versatile reference standards for quantitative superresolution microscopy

Thevathasan¹,

Kahnwald²,

Cieslinski³

et al. 2019

Preprint

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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.

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Section: Discussionmentioning

confidence: 94%

Nuclear pores as versatile reference standards for quantitative superresolution microscopy

Thevathasan¹,

Kahnwald²,

Cieslinski³

et al. 2019

Preprint

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“…For protein tag conjugated fluorophores, a strong influence of the protein tag on the fluorophore substrate was recently reported and has to be taken into account when comparing the brightness of different fluorophores after conjugation to protein tags. 30 To identify promising labelling strategies suited for PBSA of cellular targets, we then compared the measured photostabilities as well as the nominal brightness of each fluorophore with ATTO 647N, which is a well suited fluorophore for in vitro measurements due to its high molecular brightness and excellent photostability.…”

Section: Resultsmentioning

confidence: 99%

Photobleaching step analysis for robust determination of protein complex stoichiometries

Hummert

Yserentant

Fink

et al. 2020

Preprint

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The composition of cellular structures on the nanoscale is a key determinant of macroscopic functions in cell biology and beyond. Different fluorescence single-molecule techniques have proven ideally suited for measuring protein copy numbers of cellular structures in intact biological samples. Of these, photobleaching step analysis poses minimal demands on the microscope and its counting range has significantly improved with more sophisticated algorithms for step detection, albeit at an increasing computational cost. Here, we present a comprehensive framework for photobleaching step analysis, optimizing both data acquisition and analysis. To make full use of the potential of photobleaching step analysis, we evaluate various labelling strategies with respect to their molecular brightness and photostability. The developed analysis algorithm focuses on automation and computational efficiency. Moreover, we benchmark the framework with experimental data acquired on DNA origami labeled with defined fluorophore numbers to demonstrate counting of up to 35 fluorophores. Finally, we show the power of the combination of optimized trace acquisition and automated data analysis for robust protein counting by counting labelled nucleoporin 107 in nuclear pore complexes of intact U2OS cells. The successful in situ application promotes this framework as a new resource enabling cell biologists to robustly determine the stoichiometries of molecular assemblies at the single-molecule level in an automated fashion.

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“…This can be achieved experimentally, e.g. by using tags or unnatural amino acids as labels 17,18 . The aim of our study is to develop a template-based analysis approach to determine the distances between individual protomers, by making use of the correct assignment of localizations to individual protomers.…”

Section: Resultsmentioning

confidence: 99%

“…These constraints disqualify fluorescently labeled antibodies. Appropriate possibilities include small tags 17 and unnatural amino acids 18 . In principle, also switchable fluorescent proteins can be used for the analysis of oligomeric structures which are large compared to the size of the fluorescent protein.…”

Section: Discussionmentioning

confidence: 99%

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A workflow for sizing oligomeric biomolecules based on cryo single molecule localization microscopy

Schneider

Telschow

Mercier

et al. 2020

Preprint

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Single molecule localization microscopy (SMLM) has enormous potential for resolving subcellular structures below the diffraction limit of light microscopy: Localization precision in the low digit nanometer regime has been shown to be achievable. In order to record localization microscopy data, however, sample fixation is inevitable to prevent molecular motion during the rather long recording times of minutes up to hours. Eventually, it turns out that preservation of the sample's ultrastructure during fixation becomes the limiting factor. We propose here a workflow for data analysis, which is based on SMLM performed at cryogenic temperatures. Since molecular dipoles of the fluorophores are fixed at low temperatures, such an approach offers the possibility to use the orientation of the dipole as an additional information for image analysis. In particular, assignment of localizations to individual dye molecules becomes possible with high reliability. We quantitatively characterized the new approach based on the analysis of simulated oligomeric structures. Side lengths can be determined with a relative error of less than 1% for tetramers with a nominal side length of 5 nm, even if the assumed localization precision for single molecules is more than 2 nm.

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Labeling Strategies Matter for Super-Resolution Microscopy: A Comparison between HaloTags and SNAP-tags

Cited by 109 publications

References 47 publications

Nuclear pores as versatile reference standards for quantitative superresolution microscopy

Nuclear pores as versatile reference standards for quantitative superresolution microscopy

Photobleaching step analysis for robust determination of protein complex stoichiometries

A workflow for sizing oligomeric biomolecules based on cryo single molecule localization microscopy

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