scholarly journals Three dimensional cross-modal image inference: label-free methods for subcellular structure prediction

2017 ◽  
Author(s):  
Chek Ounkomol ◽  
Daniel A. Fernandes ◽  
Sharmishtaa Seshamani ◽  
Mary M. Maleckar ◽  
Forrest Collman ◽  
...  
Keyword(s):  
Structure Prediction ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Dyes ◽  
Three Dimensional ◽  
Live Cells ◽  
Label Free ◽  
State Changes ◽  
Microscopy Images

AbstractFluorescence microscopy has enabled imaging of key subcellular structures in living cells; however, the use of fluorescent dyes and proteins is often expensive, time-consuming, and damaging to cells. Here, we present a tool for the prediction of fluorescently labeled structures in live cells solely from 3D brightfield microscopy images. We show the utility of this approach in predicting several structures of interest from the same static 3D brightfield image, and show that the same tool can prospectively be used to predict the spatiotemporal position of these structures from a bright-field time series. This approach could also be useful in a variety of application areas, such as cross-modal image registration, quantification of live cell imaging, and determination of cell state changes.

scholarly journals CRISPR-mediated Multiplexed Live Cell Imaging of Nonrepetitive Genomic Loci

2020 ◽  
Author(s):  
Patricia A. Clow ◽  
Nathaniel Jillette ◽  
Jacqueline J. Zhu ◽  
Albert W. Cheng
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Repetitive Sequences ◽  
Binary Sequence ◽  
Three Dimensional ◽  
Live Cell ◽  
Live Cells ◽  
Imaging Method ◽  
Genomic Locus ◽  
Time Dynamics

AbstractThree-dimensional (3D) structures of the genome are dynamic, heterogeneous and functionally important. Live cell imaging has become the leading method for chromatin dynamics tracking. However, existing CRISPR- and TALE-based genomic labeling techniques have been hampered by laborious protocols and low signal-to-noise ratios (SNRs), and are thus mostly applicable to repetitive sequences. Here, we report a versatile CRISPR/Casilio-based imaging method, with an enhanced SNR, that allows for one nonrepetitive genomic locus to be labeled using a single sgRNA. We constructed Casilio dual-color probes to visualize the dynamic interactions of cohesin-bound elements in single live cells. By forming a binary sequence of multiple Casilio probes (PISCES) across a continuous stretch of DNA, we track the dynamic 3D folding of a 74kb genomic region over time. This method offers unprecedented resolution and scalability for delineating the dynamic 4D nucleome.One Sentence SummaryCasilio enables multiplexed live cell imaging of nonrepetitive DNA loci for illuminating the real-time dynamics of genome structures.

scholarly journals Label-free and live cell imaging by interferometric scattering microscopy

2018 ◽  
Vol 9 (10) ◽  
pp. 2690-2697 ◽  
Author(s):  
Jin-Sung Park ◽  
Il-Buem Lee ◽  
Hyeon-Min Moon ◽  
Jong-Hyeon Joo ◽  
Kyoung-Hoon Kim ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Complex Structure ◽  
Live Cell ◽  
Fluorescent Label ◽  
Live Cells ◽  
Label Free ◽  
Cytoplasmic Organelles ◽  
Microscopic Techniques

Despite recent remarkable advances in microscopic techniques, it still remains very challenging to directly observe the complex structure of cytoplasmic organelles in live cells without a fluorescent label.

Conformation of Ribosomes from Escherichia Coli

1974 ◽  
Vol 32 ◽  
pp. 210-211
Author(s):  
M. Boublik ◽  
W. Hellmann ◽  
F. Jenkins
Keyword(s):  
Binding Sites ◽  
Ribosomal Proteins ◽  
Fluorescent Dyes ◽  
Protein Biosynthesis ◽  
Three Dimensional ◽  
Spatial Arrangement ◽  
Dimensional Structure ◽  
Complete Understanding ◽  
And Function

The present knowledge of the three-dimensional structure of ribosomes is far too limited to enable a complete understanding of the various roles which ribosomes play in protein biosynthesis. The spatial arrangement of proteins and ribonuclec acids in ribosomes can be analysed in many ways. Determination of binding sites for individual proteins on ribonuclec acid and locations of the mutual positions of proteins on the ribosome using labeling with fluorescent dyes, cross-linking reagents, neutron-diffraction or antibodies against ribosomal proteins seem to be most successful approaches. Structure and function of ribosomes can be correlated be depleting the complete ribosomes of some proteins to the functionally inactive core and by subsequent partial reconstitution in order to regain active ribosomal particles.

A single probe to sense Al(iii) colorimetrically and Cd(ii) by turn-on fluorescence in physiological conditions and live cells, corroborated by X-ray crystallographic and theoretical studies

2015 ◽  
Vol 44 (9) ◽  
pp. 4123-4132 ◽  
Author(s):  
Chirantan Kar ◽  
Soham Samanta ◽  
Sudeep Goswami ◽  
Aiyagari Ramesh ◽  
Gopal Das
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Selective Recognition ◽  
Live Cells ◽  
Paper Strip ◽  
Theoretical Studies ◽  
X Ray ◽  
Physiological Conditions ◽  
Turn On ◽  
Single Probe

Selective recognition of Al3+and Cd2+by UV-Vis and fluorescence based techniques using a cinnamaldehyde functionalized conjugated ligand, and its applications in paper strip and live cell imaging.

Dye selection for live cell imaging of intact siRNA

2012 ◽  
Vol 393 (1-2) ◽  
pp. 23-35 ◽  
Author(s):  
Markus Hirsch ◽  
Dennis Strand ◽  
Mark Helm
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Dyes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Lapse ◽  
Live Cell ◽  
High Yield ◽  
Ph Variation ◽  
Rnai Efficiency

Abstract Investigations into the fate of small interfering RNA (siRNA) after transfection may unravel new ways to improve RNA interference (RNAi) efficiency. Because intracellular degradation of RNA may prevent reliable observation of fluorescence-labeled siRNA, new tools for fluorescence microscopy are warranted to cover the considerable duration of the RNAi effect. Here, the characterization and application of new fluorescence resonance energy transfer (FRET) dye pairs for sensing the integrity of duplex siRNA is reported, which allows an assessment of the degradation status of an siRNA cell population by live cell imaging. A panel of high-yield fluorescent dyes has been investigated for their suitability as FRET pairs for the investigation of RNA inside the cell. Nine dyes in 13 FRET pairs were evaluated based on the performance in assays of photostability, cross-excitation, bleed-through, as well as on quantified changes of fluorescence as a consequence of, e.g., RNA strand hybridization and pH variation. The Atto488/Atto590 FRET pair has been applied to live cell imaging, and has revealed first aspects of unusual trafficking of intact siRNA. A time-lapse study showed highly dynamic movement of siRNA in large perinuclear structures. These and the resulting optimized FRET labeled siRNA are expected to have significant impact on future observations of labeled RNAs in living cells.

scholarly journals Live-Cell Imaging of Caspase Activation for High-Content Screening

2009 ◽  
Vol 14 (8) ◽  
pp. 956-969 ◽  
Author(s):  
Christophe Antczak ◽  
Toshimitsu Takagi ◽  
Christina N. Ramirez ◽  
Constantin Radu ◽  
Hakim Djaballah
Keyword(s):  
Cell Death ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Exposure Period ◽  
Live Cells ◽  
Assay Method ◽  
Caspase Activation ◽  
Fluorogenic Substrate ◽  
Automated Imaging ◽  
First Time

Caspases are central to the execution of programmed cell death, and their activation constitutes the biochemical hallmark of apoptosis. In this article, the authors report the successful adaptation of a high-content assay method using the DEVDNucView488™ fluorogenic substrate, and for the first time, they show caspase activation in live cells induced by either drugs or siRNA. The fluorogenic substrate was found to be nontoxic over an exposure period of several days, during which the authors demonstrate automated imaging and quantification of caspase activation of the same cell population as a function of time. Overexpression of the antiapoptotic protein Bcl-XL, alone or in combination with the inhibitor Z-VAD-FMK, attenuated caspase activation in HeLa cells exposed to doxorubicin, etoposide, or cell death siRNA. This method was further validated against 2 well-characterized NSCLC cell lines reported to be sensitive (H3255) or refractory (H2030) to erlotinib, where the authors show a differential time-dependent activation was observed for H3255 and no significant changes in H2030, consistent with their respective chemosensitivity profile. In summary, the results demonstrate the feasibility of using this newly adapted and validated high-content assay to screen chemical or RNAi libraries for the identification of previously uncovered enhancers and suppressors of the apoptotic machinery in live cells. ( Journal of Biomolecular Screening 2009:956-969)

Cost-Effective High-Speed, Three-Dimensional Live-Cell Imaging of HIV-1 Transfer at the T Cell Virological Synapse

2021 ◽  
Author(s):  
Alice Sandmeyer ◽  
Lili Wang ◽  
Wolfgang Hübner ◽  
Marcel Müller ◽  
Benjamin Chen ◽  
...  
Keyword(s):  
T Cell ◽  
Live Cell Imaging ◽  
High Speed ◽  
Cell Imaging ◽  
Three Dimensional ◽  
Cost Effective ◽  
Live Cell ◽  
Virological Synapse ◽  
Hiv 1

scholarly journals Label-Free Live-Cell Imaging with Confocal Raman Microscopy

2012 ◽  
Vol 102 (2) ◽  
pp. 360-368 ◽  
Author(s):  
Katharina Klein ◽  
Alexander M. Gigler ◽  
Thomas Aschenbrenner ◽  
Roberto Monetti ◽  
Wolfram Bunk ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Raman Microscopy ◽  
Live Cell ◽  
Confocal Raman Microscopy ◽  
Label Free ◽  
Confocal Raman

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 ER sheet persistence is coupled to myosin 1c–regulated dynamic actin filament arrays

2014 ◽  
Vol 25 (7) ◽  
pp. 1111-1126 ◽  
Author(s):  
Merja Joensuu ◽  
Ilya Belevich ◽  
Olli Rämö ◽  
Ilya Nevzorov ◽  
Helena Vihinen ◽  
...  
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Network Architecture ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Three Dimensional ◽  
Actin Depolymerization ◽  
Specific Subset ◽  
3D Network ◽  
3D Electron Microscopy

The endoplasmic reticulum (ER) comprises a dynamic three-dimensional (3D) network with diverse structural and functional domains. Proper ER operation requires an intricate balance within and between dynamics, morphology, and functions, but how these processes are coupled in cells has been unclear. Using live-cell imaging and 3D electron microscopy, we identify a specific subset of actin filaments localizing to polygons defined by ER sheets and tubules and describe a role for these actin arrays in ER sheet persistence and, thereby, in maintenance of the characteristic network architecture by showing that actin depolymerization leads to increased sheet fluctuation and transformations and results in small and less abundant sheet remnants and a defective ER network distribution. Furthermore, we identify myosin 1c localizing to the ER-associated actin filament arrays and reveal a novel role for myosin 1c in regulating these actin structures, as myosin 1c manipulations lead to loss of the actin filaments and to similar ER phenotype as observed after actin depolymerization. We propose that ER-associated actin filaments have a role in ER sheet persistence regulation and thus support the maintenance of sheets as a stationary subdomain of the dynamic ER network.

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