A Simple Method for GFP- and RFP-based Dual Color Single-Molecule Localization Microscopy

Platonova, Evgenia; Winterflood, Christian M.; Ewers, Helge

doi:10.1021/acschembio.5b00046

Cited by 37 publications

(34 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We also note that nanobody-staining performed equally well after fixation with most common reagents: paraformaldehyde (this study, [32], glutaraldehyde [32], methanol [33], …”

Section: Sample Preparation and Smlm Imagingsupporting

confidence: 52%

“…This method provides high resolution for both colors, minimizes cross-talk, and is free from chromatic aberrations and thus overall allows for very accurate colocalization analysis in SMLM [5,23]. We here aimed to demonstrate the quality of labeling via nanobodies by accessing two distinct members of a small compact multiprotein structure.…”

Section: Resolution In Dual Color Nanobody-mediated Single Molecule Lmentioning

confidence: 99%

See 1 more Smart Citation

Single-molecule microscopy of molecules tagged with GFP or RFP derivatives in mammalian cells using nanobody binders

Platonova

Winterflood

Junemann

et al. 2015

Methods

Self Cite

View full text Add to dashboard Cite

With the recent development of single-molecule localization-based superresolution microscopy, the imaging of cellular structures at a resolution below the diffraction-limit of light has become a widespread technique. While single fluorescent molecules can be resolved in the nanometer range, the delivery of these molecules to the authentic structure in the cell via traditional antibody-mediated techniques can add substantial error due to the size of the antibodies. Accurate and quantitative labeling of cellular molecules has thus become one of the bottlenecks in the race for highest resolution of target structures. Here we illustrate in detail how to use small, high affinity nanobody binders against GFP and RFP family proteins for highly generic labeling of fusion constructs with bright organic dyes. We provide detailed protocols and examples for their application in superresolution imaging and single particle tracking and demonstrate advantages over conventional labeling approaches.

show abstract

“…We also note that nanobody-staining performed equally well after fixation with most common reagents: paraformaldehyde (this study, [32], glutaraldehyde [32], methanol [33], …”

Section: Sample Preparation and Smlm Imagingsupporting

confidence: 52%

Section: Resolution In Dual Color Nanobody-mediated Single Molecule Lmentioning

confidence: 99%

Single-molecule microscopy of molecules tagged with GFP or RFP derivatives in mammalian cells using nanobody binders

Platonova

Winterflood

Junemann

et al. 2015

Methods

Self Cite

View full text Add to dashboard Cite

show abstract

“…Here, it is imperative that this microscopy improves speed of image acquisition, which has hampered plant cell biology applications. It is encouraging that localization microscopy is compatible with conventional fluorophores [81][82][83], and it should be possible to use existing lines with labelled CesAs and CalS. Here, it will be interesting to investigate whether also the CalS moves during callose deposition similar to what has been observed for the CesAs.…”

Section: Discussionmentioning

confidence: 84%

Cellulose and callose synthesis and organization in focus, what's new?

Schneider

Hanak

Persson

et al. 2016

Current Opinion in Plant Biology

104

View full text Add to dashboard Cite

Article available under the terms of the CC-BY-NC-ND licence (https://creativecommons.org/licenses/by-nc-nd/4.0/) eprints@whiterose.ac.uk https://eprints.whiterose.ac.uk/ Reuse Unless indicated otherwise, fulltext items are protected by copyright with all rights reserved. The copyright exception in section 29 of the Copyright, Designs and Patents Act 1988 allows the making of a single copy solely for the purpose of non-commercial research or private study within the limits of fair dealing. The publisher or other rights-holder may allow further reproduction and re-use of this version -refer to the White Rose Research Online record for this item. Where records identify the publisher as the copyright holder, users can verify any specific terms of use on the publisher's website. TakedownIf you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing eprints@whiterose.ac.uk including the URL of the record and the reason for the withdrawal request. Abstract Plant growth and development are supported by plastic but strong cell walls. These walls consist largely of polysaccharides that vary in content and structure. Most of the polysaccharides are produced in the Golgi apparatus and are then secreted to the apoplast and built into the growing walls. However, the two glucan polymers cellulose and callose are synthesized at the plasma membrane by cellulose or callose synthase complexes, respectively. Cellulose is the most common cell wall polymer in land plants and provides strength to the walls to support directed cell expansion. In contrast, callose is integral to specialized cell walls, such as the cell plate that separates dividing cells and growing pollen tube walls, and maintains important functions during abiotic and biotic stress responses. The last years have seen a dramatic increase in our understanding of how cellulose and callose are manufactured, and new factors that regulate the synthases have been identified. Much of this knowledge has been amassed via various microscopy-based techniques, including various confocal techniques and super-resolution imaging. Here, we summarize and synthesize recent findings in the fields of cellulose and callose synthesis in plant biology. Title

show abstract

“…Cells were plated on glass-bottom dishes and imaged in imaging medium (Fluorobrite DMEM, 10% FBS, GlutaMAX, 10-30 mM HEPES; Thermo Fisher Scientific) 16-48 h after transfection.Particle functionalization and live cell experiments. For live cell experimentstargeting GFP or YFP, LaG-16 anti-GFP nanobodies32 were recombinantly produced in house and labeled with α-Biotin-ω-(succinimidyl propionate) 24(ethylene glycol) (Iris Biotech) as previously described33,34 . The succinimidyl ester was added to the nanobody at 2-fold molar excess for the labeling reaction.…”

mentioning

confidence: 99%

Directed manipulation of membrane proteins by fluorescent magnetic nanoparticles

Santos‐Otte

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

The plasma membrane is the interface through which cells interact with their environment. Membrane proteins are embedded in the lipid bilayer of the plasma membrane and their function in this context is often linked to their specific location and dynamics within the membrane. However, few methods are available for nanoscale manipulation of membrane protein location at the single molecule level. Here, we report the use of fluorescent magnetic nanoparticles (FMNPs) to track membrane molecules and to manipulate their movement. FMNPs allow single-particle tracking (SPT) at 10 nm spatial and 5 ms temporal resolution, and using a magnetic needle, we pull membrane components laterally through the membrane with femtonewton-range forces. In this way, we successfully dragged lipid-anchored and transmembrane proteins over the surface of living cells. Doing so, we detected submembrane barriers and in combination with super-resolution microscopy could localize these barriers to the actin cytoskeleton. We present here a versatile approach to probe membrane processes in live cells via the magnetic control of membrane protein motion.

show abstract

A Simple Method for GFP- and RFP-based Dual Color Single-Molecule Localization Microscopy

Cited by 37 publications

References 26 publications

Single-molecule microscopy of molecules tagged with GFP or RFP derivatives in mammalian cells using nanobody binders

Single-molecule microscopy of molecules tagged with GFP or RFP derivatives in mammalian cells using nanobody binders

Cellulose and callose synthesis and organization in focus, what's new?

Directed manipulation of membrane proteins by fluorescent magnetic nanoparticles

Contact Info

Product

Resources

About