scholarly journals Choosing the right label for single-molecule tracking in live bacteria: Side-by-side comparison of photoactivatable fluorescent protein and Halo tag dyes

2018 ◽  
Author(s):  
Nehir Banaz ◽  
Jarno Mäkelä ◽  
Stephan Uphoff
Keyword(s):  
Single Molecule ◽  
High Speed ◽  
Cell Function ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Single Molecule Tracking ◽  
Advantages And Disadvantages ◽  
Fluorescent Ligands ◽  
The Right

AbstractVisualizing and quantifying molecular motion and interactions inside living cells provides crucial insight into the mechanisms underlying cell function. This has been achieved by super-resolution localization microscopy and single-molecule tracking in conjunction with photoactivatable fluorescent proteins. An alternative labelling approach relies on genetically-encoded protein tags with cell-permeable fluorescent ligands which are brighter and less prone to photobleaching than fluorescent proteins but require a laborious labelling process. Either labelling method is associated with significant advantages and disadvantages that should be taken into consideration depending on the microscopy experiment planned. Here, we describe an optimised procedure for labelling Halo-tagged proteins in live Escherichia coli cells. We provide a side-by-side comparison of Halo tag with different fluorescent ligands against the popular photoactivatable fluorescent protein PAmCherry. Using test proteins with different intracellular dynamics, we evaluated fluorescence intensity, background, photostability, and single-molecule localization and tracking results. Capitalising on the brightness and extended spectral range of fluorescent Halo ligands, we also demonstrate high-speed and dual-colour single-molecule tracking.

Development of a green reversibly photoswitchable variant of Eos fluorescent protein with fixation resistance

2021 ◽  
pp. mbc.E21-01-0044
Author(s):  
Mitsuo Osuga ◽  
Tamako Nishimura ◽  
Shiro Suetsugu
Keyword(s):  
Single Molecule ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Excitation Light ◽  
Antibody Staining ◽  
Aldehyde Fixation ◽  
Green Fluorescent ◽  
Super Resolution Microscopy

Super-resolution microscopy determines the localization of fluorescent proteins with high precision, beyond the diffraction limit of light. Super-resolution microscopic techniques include photoactivated localization microscopy (PALM), which can localize a single protein by the stochastic activation of its fluorescence. In the determination of single-molecule localization by PALM, the number of molecules that can be analyzed per image is limited. Thus, many images are required to reconstruct the localization of numerous molecules in the cell. However, most fluorescent proteins lose their fluorescence upon fixation. Here, we combined the amino acid substitutions of two Eos protein derivatives, Skylan-S and mEos4b, which are a green reversibly photoswitchable fluorescent protein (RSFP) and a fixation-resistant green-to-red photo-convertible fluorescent protein, respectively, resulting in the fixation-resistant Skylan-S (frSkylan-S), a green RSFP. The frSkylan-S protein is inactivated by excitation light and re-activated by irradiation with violet light, and retained more fluorescence after aldehyde fixation than Skylan-S. The qualities of the frSkylan-S fusion proteins were sufficiently high in PALM observations, as examined using α-tubulin and clathrin light chain. Furthermore, frSkylan-S can be combined with antibody staining for multicolor imaging. Therefore, frSkylan-S is a green fluorescent protein suitable for PALM imaging under aldehyde-fixation conditions.

scholarly journals Mapping the 3D genome through high speed single-molecule tracking of functional transcription factors in single living cells

2019 ◽  
Author(s):  
Adam J. M. Wollman ◽  
Erik G. Hedlund ◽  
Sviatlana Shashkova ◽  
Mark C. Leake
Keyword(s):  
Single Molecule ◽  
High Speed ◽  
Fluorescent Protein ◽  
Super Resolution ◽  
Living Cells ◽  
3D Genome ◽  
Straightforward Method ◽  
Single Genome ◽  
Position Data ◽  
Single Living Cells

AbstractHow genomic DNA is organized within the cell nucleus is a long-standing question. We describe a single-molecule bioimaging method with super-resolution localization precision and very rapid millisecond temporal resolution, coupled to fully quantitative image analysis tools, to help to determine genome organization and dynamics using budding yeast Saccharomyces cerevisiae as a model eukaryotic organism. We utilize astigmatism imaging, a robust technique that enables extraction of 3D position data, on genomically encoded fluorescent protein reporters that bind to DNA. Our relatively straightforward method enables snapshot reconstructions of 3D architectures of single genome conformations directly in single functional living cells.

Structural Evidence of Photoisomerization Pathways in Fluorescent Proteins

2019 ◽  
Author(s):  
Jeffrey Chang ◽  
Matthew Romei ◽  
Steven Boxer
Keyword(s):  
Rational Design ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Unit Cell Size ◽  
Chlorine Substituent ◽  
Gfp Chromophore ◽  
The One ◽  
Green Fluorescent ◽  
Super Resolution Microscopy

<p>Double-bond photoisomerization in molecules such as the green fluorescent protein (GFP) chromophore can occur either via a volume-demanding one-bond-flip pathway or via a volume-conserving hula-twist pathway. Understanding the factors that determine the pathway of photoisomerization would inform the rational design of photoswitchable GFPs as improved tools for super-resolution microscopy. In this communication, we reveal the photoisomerization pathway of a photoswitchable GFP, rsEGFP2, by solving crystal structures of <i>cis</i> and <i>trans</i> rsEGFP2 containing a monochlorinated chromophore. The position of the chlorine substituent in the <i>trans</i> state breaks the symmetry of the phenolate ring of the chromophore and allows us to distinguish the two pathways. Surprisingly, we find that the pathway depends on the arrangement of protein monomers within the crystal lattice: in a looser packing, the one-bond-flip occurs, whereas in a tighter packing (7% smaller unit cell size), the hula-twist occurs.</p><p> </p><p> </p><p> </p><p> </p><p> </p><p> </p> <p> </p>

scholarly journals Single-Molecule Studies on a FRET Biosensor: Lessons from a Comparison of Fluorescent Protein Equipped versus Dye-Labeled Species

Molecules ◽  
2018 ◽  
Vol 23 (12) ◽  
pp. 3105 ◽  
Author(s):  
Henning Höfig ◽  
Michele Cerminara ◽  
Ilona Ritter ◽  
Antonie Schöne ◽  
Martina Pohl ◽  
...  
Keyword(s):  
Conformational Change ◽  
Single Molecule ◽  
Fluorescent Dyes ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Strong Increase ◽  
Single Molecule Level ◽  
Labeling Scheme

Bacterial periplasmic binding proteins (PBPs) undergo a pronounced ligand-induced conformational change which can be employed to monitor ligand concentrations. The most common strategy to take advantage of this conformational change for a biosensor design is to use a Förster resonance energy transfer (FRET) signal. This can be achieved by attaching either two fluorescent proteins (FPs) or two organic fluorescent dyes of different colors to the PBPs in order to obtain an optical readout signal which is closely related to the ligand concentration. In this study we compare a FP-equipped and a dye-labeled version of the glucose/galactose binding protein MglB at the single-molecule level. The comparison demonstrates that changes in the FRET signal upon glucose binding are more pronounced for the FP-equipped sensor construct as compared to the dye-labeled analog. Moreover, the FP-equipped sensor showed a strong increase of the FRET signal under crowding conditions whereas the dye-labeled sensor was not influenced by crowding. The choice of a labeling scheme should therefore be made depending on the application of a FRET-based sensor.

scholarly journals tdLanYFP, a yellow, bright, photostable and pH insensitive fluorescent protein for live cell imaging and FRET-based sensing strategies

2021 ◽  
Author(s):  
Y. Bousmah ◽  
H. Valenta ◽  
G. Bertolin ◽  
U. Singh ◽  
V. Nicolas ◽  
...  
Keyword(s):  
Single Molecule ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy ◽  
Live Cell ◽  
Live Cells

AbstractYellow fluorescent proteins (YFP) are widely used as optical reporters in Förster Resonance Energy Transfer (FRET) based biosensors. Although great improvements have been done, the sensitivity of the biosensors is still limited by the low photostability and the poor fluorescence performances of YFPs at acidic pHs. In fact, today, there is no yellow variant derived from the EYFP with a pK1/2 below ∼5.5. Here, we characterize a new yellow fluorescent protein, tdLanYFP, derived from the tetrameric protein from the cephalochordate B. lanceolatum, LanYFP. With a quantum yield of 0.92 and an extinction coefficient of 133 000 mol−1.L.cm−1, it is, to our knowledge, the brightest dimeric fluorescent protein available, and brighter than most of the monomeric YFPs. Contrasting with EYFP and its derivatives, tdLanYFP has a very high photostability in vitro and preserves this property in live cells. As a consequence, tdLanYFP allows the imaging of cellular structures with sub-diffraction resolution with STED nanoscopy. We also demonstrate that the combination of high brightness and strong photostability is compatible with the use of spectro-microscopies in single molecule regimes. Its very low pK1/2 of 3.9 makes tdLanYFP an excellent tag even at acidic pHs. Finally, we show that tdLanYFP can be a FRET partner either as donor or acceptor in different biosensing modalities. Altogether, these assets make tdLanYFPa very attractive yellow fluorescent protein for long-term or single-molecule live-cell imaging that is also suitable for FRET experiment including at acidic pH.

scholarly journals High-Speed Single-Molecule Tracking of CXCL13 in the B-Follicle

2018 ◽  
Vol 9 ◽  
Author(s):  
Helen Miller ◽  
Jason Cosgrove ◽  
Adam J. M. Wollman ◽  
Emily Taylor ◽  
Zhaokun Zhou ◽  
...  
Keyword(s):  
Single Molecule ◽  
High Speed ◽  
Single Molecule Tracking
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