scholarly journals Nanobody Detection of Standard Fluorescent Proteins Enables Multi-Target DNA-PAINT with High Resolution and Minimal Displacement Errors

2018 ◽  
Author(s):  
Shama Sograte-Idrissi ◽  
Nazar Oleksiievets ◽  
Sebastian Isbaner ◽  
Mariana Eggert-Martinez ◽  
Jörg Enderlein ◽  
...  
Keyword(s):  
Fluorescent Proteins ◽  
Super Resolution ◽  
Resolving Power ◽  
Acquisition Time ◽  
Efficient Manner ◽  
Target Dna ◽  
Multiplexed Imaging ◽  
Dna Strands ◽  
Linkage Error ◽  
20 Nm

AbstractDNA-PAINT is a rapidly developing fluorescence super-resolution technique which allows for reaching spatial resolutions below 10 nm. It also enables the imaging of multiple targets in the same sample. However, using DNA-PAINT to observe cellular structures at such resolution remains challenging. Antibodies, which are commonly used for this purpose, lead to a displacement between the target protein and the reporting fluorophore of 20-25 nm, thus limiting the resolving power. Here, we used nanobodies to minimize this linkage error to ~4 nm. We demonstrate multiplexed imaging by using 3 nanobodies, each able to bind to a different family of fluorescent proteins. We couple the nanobodies with single DNA strands via a straight forward and stoichiometric chemical conjugation. Additionally, we built a versatile computer-controlled microfluidic setup to enable multiplexed DNA-PAINT in an efficient manner. As a proof of principle, we labeled and imaged proteins on mitochondria, the Golgi apparatus, and chromatin. We obtained super-resolved images of the 3 targets with 20 nm resolution, and within only 35 minutes acquisition time.

scholarly journals Nanobody Detection of Standard Fluorescent Proteins Enables Multi-Target DNA-PAINT with High Resolution and Minimal Displacement Errors

Cells ◽  
2019 ◽  
Vol 8 (1) ◽  
pp. 48 ◽  
Author(s):  
Shama Sograte-Idrissi ◽  
Nazar Oleksiievets ◽  
Sebastian Isbaner ◽  
Mariana Eggert-Martinez ◽  
Jörg Enderlein ◽  
...  
Keyword(s):  
Fluorescent Proteins ◽  
Super Resolution ◽  
Resolving Power ◽  
Acquisition Time ◽  
Efficient Manner ◽  
Target Dna ◽  
Dna Strands ◽  
Linkage Error ◽  
Nanoscale Topography ◽  
20 Nm

DNA point accumulation for imaging in nanoscale topography (PAINT) is a rapidly developing fluorescence super-resolution technique, which allows for reaching spatial resolutions below 10 nm. It also enables the imaging of multiple targets in the same sample. However, using DNA-PAINT to observe cellular structures at such resolution remains challenging. Antibodies, which are commonly used for this purpose, lead to a displacement between the target protein and the reporting fluorophore of 20–25 nm, thus limiting the resolving power. Here, we used nanobodies to minimize this linkage error to ~4 nm. We demonstrate multiplexed imaging by using three nanobodies, each able to bind to a different family of fluorescent proteins. We couple the nanobodies with single DNA strands via a straight forward and stoichiometric chemical conjugation. Additionally, we built a versatile computer-controlled microfluidic setup to enable multiplexed DNA-PAINT in an efficient manner. As a proof of principle, we labeled and imaged proteins on mitochondria, the Golgi apparatus, and chromatin. We obtained super-resolved images of the three targets with 20 nm resolution, and within only 35 minutes acquisition time.

scholarly journals A photoswitchable fluorescent protein for hours-time-lapse and sub-second-resolved super-resolution imaging

Microscopy ◽  
2021 ◽  
Author(s):  
Tetsuichi Wazawa ◽  
Ryohei Noma ◽  
Shusaku Uto ◽  
Kazunori Sugiura ◽  
Takashi Washio ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Ph Dependence ◽  
Super Resolution ◽  
Time Lapse ◽  
Light Irradiation ◽  
Acquisition Time ◽  
Polarization Angle ◽  
Cellular Dynamics

Abstract Reversibly photoswitchable fluorescent proteins (RSFPs) are a class of fluorescent proteins whose fluorescence can be turned on and off by light irradiation. RSFPs have become essential tools for super-resolution (SR) imaging. Because most SR imaging techniques require high-power-density illumination, mitigating phototoxicity in cells due to intense light irradiation has been a challenge. Although we previously developed an RSFP named Kohinoor to achieve SR imaging with low phototoxicity, the photoproperties were insufficient to move a step further to explore the cellular dynamics by SR imaging. Here, we show an improved version of RSFP, Kohinoor2.0, which is suitable for SR imaging of cellular processes. Kohinoor2.0 shows a 2.6-fold higher fluorescence intensity, 2.5-fold faster chromophore maturation and 1.5-fold faster off-switching than Kohinoor. The analysis of the pH dependence of the visible absorption band revealed that Kohinoor2.0 and Kohinoor were in equilibria among multiple fluorescently bright and dark states, with the mutations introduced into Kohinoor2.0 bringing about a higher stabilization of the fluorescently bright states compared to Kohinoor. Using Kohinoor2.0 with our SR imaging technique, super-resolution polarization demodulation/on-state polarization angle narrowing, we conducted 4-h time-lapse SR imaging of an actin filament network in mammalian cells with a total acquisition time of 480 s without a noticeable indication of phototoxicity. Furthermore, we demonstrated the SR imaging of mitochondria dynamics at a time resolution of 0.5 s, in which the fusion and fission processes were clearly visualized. Thus, Kohinoor2.0 is shown to be an invaluable RSFP for the SR imaging of cellular dynamics.

In-Situ Observation of Clay Minerals Hydrated and Intercalated With Liquid Chemicals Using Film-Sealed Environmental Cell

1980 ◽  
Vol 38 ◽  
pp. 208-209 ◽  
Author(s):  
K. Fukushima ◽  
N. Kohyama ◽  
A. Fukami
Keyword(s):  
Carbon Film ◽  
Resolving Power ◽  
In Situ Observation ◽  
Interlayer Water ◽  
Saturated Water ◽  
Structure Changes ◽  
Environmental Cell ◽  
Gas Layer ◽  
20 Nm

A film-sealed high resolution environmental cell(E.C) for observing hydrated materials had been developed by us(l). Main specification of the E.C. is as follows: 1) Accelerated voltage; 100 kV. 2) Gas in the E.C.; saturated water vapour with carrier gas of 50 Torr. 3) Thickness of gas layer; 50 μm. 4) Sealing film; evaporated carbon film(20 nm thick) with plastic microgrid. 5) Resolving power; 1 nm. 6) Transmittance of electron beam; 60% at 100 kV. The E.C. had been successfully applied to the study of hydrated halloysite(2) (3). Kaolin minerals have no interlayer water and are basically non-expandable but form intercalation compounds with some specific chemicals such as hydrazine, formamide and etc. Because of these compounds being mostly changed in vacuum, we tried to reveal the structure changes between in wet air and in vacuum of kaolin minerals intercalated with hydrazine and of hydrated state of montmori1lonite using the E.C. developed by us.

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 BEAR reveals that increased fidelity variants can successfully reduce the mismatch tolerance of adenine but not cytosine base editors

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
András Tálas ◽  
Dorottya A. Simon ◽  
Péter I. Kulcsár ◽  
Éva Varga ◽  
Sarah L. Krausz ◽  
...  
Keyword(s):  
High Throughput ◽  
Genome Engineering ◽  
Sequence Context ◽  
Base Editing ◽  
Target Dna ◽  
Dna Strands ◽  
Deaminase Domain ◽  
Target Effects ◽  
Target Sets

AbstractAdenine and cytosine base editors (ABE, CBE) allow for precision genome engineering. Here, Base Editor Activity Reporter (BEAR), a plasmid-based fluorescent tool is introduced, which can be applied to report on ABE and CBE editing in a virtually unrestricted sequence context or to label base edited cells for enrichment. Using BEAR-enrichment, we increase the yield of base editing performed by nuclease inactive base editors to the level of the nickase versions while maintaining significantly lower indel background. Furthermore, by exploiting the semi-high-throughput potential of BEAR, we examine whether increased fidelity SpCas9 variants can be used to decrease SpCas9-dependent off-target effects of ABE and CBE. Comparing them on the same target sets reveals that CBE remains active on sequences, where increased fidelity mutations and/or mismatches decrease the activity of ABE. Our results suggest that the deaminase domain of ABE is less effective to act on rather transiently separated target DNA strands, than that of CBE explaining its lower mismatch tolerance.

scholarly journals Limits for resolving tandem mass tag reporter ions with identical integer mass using phase constrained spectrum deconvolution

2018 ◽  
Author(s):  
Christian D. Kelstrup ◽  
Konstantin Aizikov ◽  
Tanveer S. Batth ◽  
Arne Kreutzman ◽  
Dmitry Grinfeld ◽  
...  
Keyword(s):  
Mass Spectrometer ◽  
Peptide Sequencing ◽  
Mass Resolution ◽  
Resolving Power ◽  
Acquisition Time ◽  
Tandem Mass ◽  
Optimal Method ◽  
Orbitrap Mass Spectrometer ◽  
Mass Resolving Power ◽  
Spectrum Deconvolution

ABSTRACTA popular method for peptide quantification relies on isobaric labeling such as tandem mass tags (TMT) which enables multiplexed proteome analyses. Quantification is achieved by reporter ions generated by fragmentation in a tandem mass spectrometer. However, with higher degrees of multiplexing, the smaller mass differences between the reporter ions increase the mass resolving power requirements. This contrasts with faster peptide sequencing capabilities enabled by lowered mass resolution on Orbitrap instruments. It is therefore important to determine the mass resolution limits for highly multiplexed quantification when maximizing proteome depth. Here we defined the lower boundaries for resolving TMT reporter ions with 0.0063 Da mass differences using an ultra-high-field Orbitrap mass spectrometer. We found the optimal method depends on the relative ratio between closely spaced reporter ions and that 64 ms transient acquisition time provided sufficient resolving power for separating TMT reporter ions with absolute ratio changes up to 16-fold. Furthermore, a 32 ms transient processed with phase-constrained spectrum deconvolution provides >50% more identifications with >99% quantified, but with a slight loss in quantification precision and accuracy. These findings should guide decisions on what Orbitrap resolution settings to use in future proteomics experiments relying on TMT reporter ion quantification with identical integer masses.

Fluorescent Proteins and Super-Resolution Microscopy

2017 ◽  
Vol 37 (3) ◽  
pp. 0318008
Author(s):  
彭鼎铭 Peng Dingming ◽  
付志飞 Fu Zhifei ◽  
徐平勇 Xu Pingyong
Keyword(s):  
Fluorescent Proteins ◽  
Super Resolution ◽  
Super Resolution Microscopy

scholarly journals Nanoscale imaging of bacterial infections by sphingolipid expansion microscopy

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Ralph Götz ◽  
Tobias C. Kunz ◽  
Julian Fink ◽  
Franziska Solger ◽  
Jan Schlegel ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Bacterial Infections ◽  
Super Resolution ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Nanoscale Imaging ◽  
Intracellular Organelles ◽  
Resolution Imaging ◽  
Simkania Negevensis ◽  
20 Nm

AbstractExpansion microscopy (ExM) enables super-resolution imaging of proteins and nucleic acids on conventional microscopes. However, imaging of details of the organization of lipid bilayers by light microscopy remains challenging. We introduce an unnatural short-chain azide- and amino-modified sphingolipid ceramide, which upon incorporation into membranes can be labeled by click chemistry and linked into hydrogels, followed by 4× to 10× expansion. Confocal and structured illumination microscopy (SIM) enable imaging of sphingolipids and their interactions with proteins in the plasma membrane and membrane of intracellular organelles with a spatial resolution of 10–20 nm. As our functionalized sphingolipids accumulate efficiently in pathogens, we use sphingolipid ExM to investigate bacterial infections of human HeLa229 cells by Neisseria gonorrhoeae, Chlamydia trachomatis and Simkania negevensis with a resolution so far only provided by electron microscopy. In particular, sphingolipid ExM allows us to visualize the inner and outer membrane of intracellular bacteria and determine their distance to 27.6 ± 7.7 nm.

scholarly journals Sequence-Specific Detection of DNA Strands Using a Solid-State Nanopore Assisted by Microbeads

Micromachines ◽  
2020 ◽  
Vol 11 (12) ◽  
pp. 1097
Author(s):  
Yin Zhang ◽  
Zengdao Gu ◽  
Jiabin Zhao ◽  
Liying Shao ◽  
Yajing Kan
Keyword(s):  
Low Cost ◽  
Local Concentration ◽  
Dna Molecules ◽  
Dna Translocation ◽  
Specific Sequence ◽  
Free Dna ◽  
Target Dna ◽  
Dna Strands ◽  
Ionic Pulse ◽  
Biotinylated Probes

Simple, rapid, and low-cost detection of DNA with specific sequence is crucial for molecular diagnosis and therapy applications. In this research, the target DNA molecules are bonded to the streptavidin-coated microbeads, after hybridizing with biotinylated probes. A nanopore with a diameter significantly smaller than the microbeads is used to detect DNA molecules through the ionic pulse signals. Because the DNA molecules attached on the microbead should dissociate from the beads before completely passing through the pore, the signal duration time for the target DNA is two orders of magnitude longer than free DNA. Moreover, the high local concentration of target DNA molecules on the surface of microbeads leads to multiple DNA molecules translocating through the pore simultaneously, which generates pulse signals with amplitude much larger than single free DNA translocation events. Therefore, the DNA molecules with specific sequence can be easily identified by a nanopore sensor assisted by microbeads according to the ionic pulse signals.

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