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Live Cell Visualization of Multiple Protein–Protein Interactions with BiFC Rainbow

Live Cell Visualization of Multiple Protein–Protein Interactions with BiFC Rainbow | News Imagerie cellulaire - Cellular imaging | Scoop.it
Sheng Wang, Miao Ding, Boxin Xue, Yingping Hou and Yujie Sun

As one of the most powerful tools to visualize PPIs in living cells, bimolecular fluorescence complementation (BiFC) has gained great advancement during recent years, including deep tissue imaging with far-red or near-infrared fluorescent proteins or super-resolution imaging with photochromic fluorescent proteins. However, little progress has been made toward simultaneous detection and visualization of multiple PPIs in the same cell, mainly due to the spectral crosstalk. In this report, we developed novel BiFC assays based on large-Stokes-shift fluorescent proteins (LSS-FPs) to detect and visualize multiple PPIs in living cells. With the large excitation/emission spectral separation, LSS-FPs can be imaged together with normal Stokes shift fluorescent proteins to realize multicolor BiFC imaging using a simple illumination scheme. We also further demonstrated BiFC rainbow combining newly developed BiFC assays with previously established mCerulean/mVenus-based BiFC assays to achieve detection and visualization of four PPI pairs in the same cell. Additionally, we prove that with the complete spectral separation of mT-Sapphire and CyOFP1, LSS-FP-based BiFC assays can be readily combined with intensity-based FRET measurement to detect ternary protein complex formation with minimal spectral crosstalk. Thus, our newly developed LSS-FP-based BiFC assays not only expand the fluorescent protein toolbox available for BiFC but also facilitate the detection and visualization of multiple protein complex interactions in living cells.


ACS Chem. Biol., Article ASAP

DOI: 10.1021/acschembio.7b00931

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SAVE THE DATE   --  14 novembre 2019 - Journée RIC Paris-Saclay

SAVE THE DATE   --  14 novembre 2019 - Journée RIC Paris-Saclay | News Imagerie cellulaire - Cellular imaging | Scoop.it

Jeudi 14 novembre prochain aura lieu

la 5ème journée d’imagerie du RIC Paris-Saclay.

 

Trois thèmes : Imagerie ionique - Imagerie de la transcription - Imagerie de la mitochondrie

 

Lieu : Faculté de médecine Paris-Sud 

 

Programme : communiqué très prochainement.

 

Inscription : gratuite mais obligatoire

  

 

SAVE THE DATE : 14 NOVEMBRE 2019 -  (9h00 / 16h30)

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EB1 contributes to microtubule bundling and organization, along with root growth, in Arabidopsis thaliana

EB1 contributes to microtubule bundling and organization, along with root growth, in Arabidopsis thaliana | News Imagerie cellulaire - Cellular imaging | Scoop.it
From the Université Paris-Saclay
 
Arthur T. Molines1, Jessica Marion1, Salem Chabout2, Laetitia Besse3, Jim P. Dompierre3, Grégory Mouille2, Frédéric M. Coquelle1
 

1  Department of Cell Biology, Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, 91198, Gif-sur-Yvette Cedex, France

2  Institut Jean-Pierre Bourgin (IJPB), INRA - AgroParisTech, 78026 Versailles Cedex, France

3  Light Microscopy Facility, Imagerie-Gif, Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, 91198, Gif-sur-Yvette Cedex, France

 

 

 

Microtubules are involved in plant development and adaptation to their environment, but the sustaining molecular mechanisms remain elusive. Microtubule-end-binding 1 (EB1) proteins participate in directional root growth in Arabidopsis thaliana . However, a connection to the underlying microtubule array has not been established yet. We show here that EB1 proteins contribute to the organization of cortical microtubules in growing epidermal plant cells, without significant modulation of microtubule dynamics. Using super-resolution stimulated emission depletion (STED) microscopy and an original quantification approach, we also demonstrate a significant reduction of apparent microtubule bundling in cytoplasmic-EB1-deficient plants, suggesting a function for EB1 in the interaction between adjacent microtubules. Furthermore, we observed root growth defects in EB1-deficient plants, which are not related to cell division impairment. Altogether, our results support a role for EB1 proteins in root development, in part by maintaining the organization of cortical microtubules.

 

Biol Open. 2018 Aug 2;7(8)

DOI : https://doi.org/10.1242/bio.030510

PubMed : 29945874

Open access : http://bio.biologists.org/content/biolopen/7/8/bio030510.full.pdf

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Optoacoustic microscopy at multiple discrete frequencies

Optoacoustic microscopy at multiple discrete frequencies | News Imagerie cellulaire - Cellular imaging | Scoop.it

Stephan Kellnberger, Dominik Soliman, George J. Tserevelakis, Markus Seeger, Hong Yang, Angelos Karlas, Ludwig Prade, Murad Omar & Vasilis Ntziachristos

 

Optoacoustic (photoacoustic) sensing employs illumination of transient energy and is typically implemented in the time domain using nanosecond photon pulses. However, the generation of high-energy short photon pulses requires complex laser technology that imposes a low pulse repetition frequency (PRF) and limits the number of wavelengths that are concurrently available for spectral imaging. To avoid the limitations of working in the time domain, we have developed frequency-domain optoacoustic microscopy (FDOM), in which light intensity is modulated at multiple discrete frequencies. We integrated FDOM into a hybrid system with multiphoton microscopy, and we examine the relationship between image formation and modulation frequency, showcase high-fidelity images with increasing numbers of modulation frequencies from phantoms and in vivo, and identify a redundancy in optoacoustic measurements performed at multiple frequencies. We demonstrate that due to high repetition rates, FDOM achieves signal-to-noise ratios similar to those obtained by time-domain methods, using commonly available laser diodes. Moreover, we experimentally confirm various advantages of the frequency-domain implementation at discrete modulation frequencies, including concurrent illumination at two wavelengths that are carried out at different modulation frequencies as well as flow measurements in microfluidic chips and in vivo based on the optoacoustic Doppler effect. Furthermore, we discuss how FDOM redefines possibilities for optoacoustic imaging by capitalizing on the advantages of working in the frequency domain.

 

Light: Science & Applications volume 7, Article number: 109 (2018)

https://doi.org/10.1038/s41377-018-0101-2

Open Access : https://www.nature.com/articles/s41377-018-0101-2.pdf

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Deep learning enables cross-modality super-resolution in fluorescence microscopy

Deep learning enables cross-modality super-resolution in fluorescence microscopy | News Imagerie cellulaire - Cellular imaging | Scoop.it

Hongda Wang, Yair Rivenson, Yiyin Jin, Zhensong Wei, Ronald Gao, Harun Günaydın, Laurent A. Bentolila, Comert Kural & Aydogan Ozcan

 

We present deep-learning-enabled super-resolution across different fluorescence microscopy modalities. This data-driven approach does not require numerical modeling of the imaging process or the estimation of a point-spread-function, and is based on training a generative adversarial network (GAN) to transform diffraction-limited input images into super-resolved ones. Using this framework, we improve the resolution of wide-field images acquired with low-numerical-aperture objectives, matching the resolution that is acquired using high-numerical-aperture objectives. We also demonstrate cross-modality super-resolution, transforming confocal microscopy images to match the resolution acquired with a stimulated emission depletion (STED) microscope. We further demonstrate that total internal reflection fluorescence (TIRF) microscopy images of subcellular structures within cells and tissues can be transformed to match the results obtained with a TIRF-based structured illumination microscope. The deep network rapidly outputs these super-resolved images, without any iterations or parameter search, and could serve to democratize super-resolution imaging.

 

Nature Methods (2018) Published: 17 December 2018

https://doi.org/10.1038/s41592-018-0239-0

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CNRS on Instagram: “#RETRO2017 Replis de la #muqueuse intestinale d’une souris observés en #microscopie confocale multi-couleurs. ”

CNRS on Instagram: “#RETRO2017 Replis de la #muqueuse intestinale d’une souris observés en #microscopie confocale multi-couleurs. ” | News Imagerie cellulaire - Cellular imaging | Scoop.it
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Scanning Fluorescence Correlation Spectroscopy for Quantification of the Dynamics and Interactions in Tube Organelles of Living Cells

Scanning Fluorescence Correlation Spectroscopy for Quantification of the Dynamics and Interactions in Tube Organelles of Living Cells | News Imagerie cellulaire - Cellular imaging | Scoop.it

 

 

Joseph D. Unsay, Fabronia Murad, Eduard Hermann, Jonas Ries,
Ana J. García‐Sáez
 

Single‐molecule spectroscopic quantification of protein‐protein interactions directly in the organelles of living cells is highly desirable but remains challenging. Bulk methods, such as Förster resonance energy transfer (FRET), currently only give a relative quantification of the strength of protein‐protein interactions. Here, we introduce tube scanning fluorescence cross‐correlation spectroscopy (tubeSFCCS) for the absolute quantification of diffusion and complex formation of fluorescently labeled molecules in the mitochondrial compartments. We determined the extent of association between the apoptosis regulators Bcl‐xL and tBid at the mitochondrial outer membrane of living cells and discovered that practically all mitochondria‐bound Bcl‐xL and tBid are associated with each other, in contrast to undetectable association in the cytosol. Furthermore, we show further applicability of our method to other mitochondrial proteins, as well as to proteins in the endoplasmic reticulum (ER) membrane.

 

ChemPhysChem 2018, 19, 3273–327

https://doi.org/10.1002/cphc.201800705

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Imaging of Receptor Dimers in Zebrafish and Living Cells via Aptamer Recognition and Proximity-Induced Hybridization Chain Reaction

Imaging of Receptor Dimers in Zebrafish and Living Cells via Aptamer Recognition and Proximity-Induced Hybridization Chain Reaction | News Imagerie cellulaire - Cellular imaging | Scoop.it

Liping Wang, Wei Li, Jin Sun, Su-Yun Zhang, Sheng Yang, Jingying Li, Juan Li and Huang-Hao Yang

 

On cell-membrane surfaces, receptor-protein dimers play fundamental roles in many signaling pathways that are crucial for normal biological processes and cancer development. Efficient and sensitive analysis of receptor dimers in the native environment is highly desirable. Herein, we present a strategy for amplified imaging of receptor dimers in zebrafish and living cells that relies on aptamer recognition and proximity-induced hybridization chain reaction. Taking advantage of specific aptamer recognition and enzyme-free signal amplification, this strategy is successfully applied to the visualization of c-Met-receptor dimers in an HGF-independent or -dependent manner. Therefore, the developed imaging strategy paves the way for further investigation of the dimerization or oligomerization states of cell-surface receptors and their corresponding activation processes in zebrafish and living cells.

 

Anal. Chem., Article ASAP Publication Date (Web): Nov. 16, 2018
Doi : 10.1021/acs.analchem.8b04015
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Fluorescent Benzothiazinone Analogues Efficiently and Selectively Label Dpre1 in Mycobacteria and Actinobacteria

Fluorescent Benzothiazinone Analogues Efficiently and Selectively Label Dpre1 in Mycobacteria and Actinobacteria | News Imagerie cellulaire - Cellular imaging | Scoop.it

Raphael Sommer,  Joao Neres, Jérémie Piton, Neeraj Dhar, Astrid van der Sar, Raju Mukherjee, Thierry Laroche, Paul J. Dyson, John D. McKinney, Wilbert Bitter, Vadim Makarov and Stewart T. Cole

 

Benzothiazinones (BTZ) are highly potent bactericidal inhibitors of mycobacteria and the lead compound, BTZ043, and the optimized drug candidate, PBTZ169, have potential for the treatment of tuberculosis. Here, we exploited the tractability of the BTZ scaffold by attaching a range of fluorophores to the 2-substituent of the BTZ ring via short linkers. We show by means of fluorescence imaging that the most advanced derivative, JN108, is capable of efficiently labeling its target, the essential flavoenzyme DprE1, both in cell-free extracts and after purification as well as in growing cells of different actinobacterial species. DprE1 displays a polar localization in Mycobacterium tuberculosis, M. marinum, M. smegmatis, and Nocardia farcinica but not in Corynebacterium glutamicum. Finally, mutation of the cysteine residue in DprE1 in these species, to which BTZ covalently binds, abolishes completely the interaction with JN108, thereby highlighting the specificity of this fluorescent probe.

 

J. Am. Chem. Soc., Just Accepted Manuscript Nov. 28, 2018
Doi : 10.1021/jacs.8b10783
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Whole-cell, 3D and multi-color STED imaging with exchangeable fluorophores

Whole-cell, 3D and multi-color STED imaging with exchangeable fluorophores | News Imagerie cellulaire - Cellular imaging | Scoop.it

Christoph Spahn, Jonathan B. Grimm, Luke D. Lavis, Marko Lampe, and Mike Heilemann

 

We demonstrate STED microscopy of whole bacterial and eukaryotic cells using fluorogenic labels that reversibly bind to their target structure. A constant exchange of labels guarantees the removal of photobleached fluorophores and their replacement by intact fluorophores, thereby circumventing bleaching-related limitations of STED super-resolution imaging. We achieve a constant labeling density and demonstrate a fluorescence signal for long and theoretically unlimited acquisition times. Using this concept, we demonstrate whole-cell, 3D, multi-color and live cell STED microscopy.

 

Nano Lett., Just Accepted Manuscript Dec. 10, 2018
DOI: 10.1021/acs.nanolett.8b04385
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Bioconjugation strategy for cell surface labelling with gold nanostructures designed for highly localized pH measurement

Bioconjugation strategy for cell surface labelling with gold nanostructures designed for highly localized pH measurement | News Imagerie cellulaire - Cellular imaging | Scoop.it

Leonardo Puppulin, Shigekuni Hosogi, Hongxin Sun, Kazuhiko Matsuo, Toshio Inui, Yasuaki Kumamoto, Toshinobu Suzaki, Hideo Tanaka & Yoshinori Marunaka

 

Regulation of intracellular pH is critically important for many cellular functions. The quantification of proton extrusion in different types of cells and physiological conditions is pivotal to fully elucidate the mechanisms of pH homeostasis. Here we show the use of gold nanoparticles (AuNP) to create a high spatial resolution sensor for measuring extracellular pH in proximity of the cell membrane. We test the sensor on HepG2 liver cancer cells and MKN28 gastric cancer cells before and after inhibition of Na+/H+ exchanger. The gold surface conjugation strategy is conceived with a twofold purpose: i) to anchor the AuNP to the membrane proteins and ii) to quantify the local pH from AuNP using surface enhanced Raman spectroscopy (SERS). The nanometer size of the cell membrane anchored sensor and the use of SERS enable us to visualize highly localized variation of pH induced by H+ extrusion, which is particularly upregulated in cancer cells.

 

Nature Communications volume 9, Article number: 5278 (2018)

https://doi.org/10.1038/s41467-018-07726-5

Open Access : https://www.nature.com/articles/s41467-018-07726-5.pdf

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simple, one-step polychromatic staining method for epoxy-embedded semithin tissue sections

simple, one-step polychromatic staining method for epoxy-embedded semithin tissue sections | News Imagerie cellulaire - Cellular imaging | Scoop.it

Shunichi Morikawa, Azusa Sato and Taichi Ezaki

 

 

Although conventional toluidine blue staining is a common technique used for rapid observation of semithin sections prior to transmission electron microscopy, it is monochromatic and insufficient for accurate identification of different tissue components by light microscopy. Additionally, polychromatic staining methods generally require step-by-step processes involving different dyes, and it is often difficult to balance the color tone of each step. In this study, we developed a simple polychromatic staining method for epoxy-embedded tissue sections. We stained preheated sections with an aqueous ethanol solution of azure B and basic fuchsin, with the addition of sodium tetraborate to enhance the staining efficacy. We optimized various staining conditions to enable sufficient coloration easily and consistently in a single, rapid staining step, using a single staining-mixture solution. Our method enabled clear differentiation of various tissue structures according to color tone and stain intensity, thereby facilitating the detection of fine structural differences, including various organelle and inclusion bodies. This technique represents a simple polychrome-staining method to allow more informative and convincing histological investigation in various fields of research and education.

 

Microscopy, 2018, Volume 67, Issue 6, 331– 344

https://doi.org/10.1093/jmicro/dfy037

Open Access : https://academic-oup-com.gate2.inist.fr/jmicro/article-pdf/67/6/331/27008925/dfy037.pdf

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A gradient-based, GPU-accelerated, high-precision contour-segmentation algorithm with application to cell membrane fluctuation spectroscopy

A gradient-based, GPU-accelerated, high-precision contour-segmentation algorithm with application to cell membrane fluctuation spectroscopy | News Imagerie cellulaire - Cellular imaging | Scoop.it

Michael Mell, Francisco Monroy

 

We present a novel intensity-gradient based algorithm specifically designed for nanometer-segmentation of cell membrane contours obtained with high-resolution optical microscopy combined with high-velocity digital imaging. The algorithm relies on the image oversampling performance and computational power of graphical processing units (GPUs). Both, synthetic and experimental data are used to quantify the sub-pixel precision of the algorithm, whose analytic performance results comparatively higher than in previous methods. Results from the synthetic data indicate that the spatial precision of the presented algorithm is only limited by the signal-to-noise ratio (SNR) of the contour image. We emphasize on the application of the new algorithm to membrane fluctuations (flickering) in eukaryotic cells, bacteria and giant vesicle models. The method shows promising applicability in several fields of cellular biology and medical imaging for nanometer-precise boundary-determination and mechanical fingerprinting of cellular membranes in optical microscopy images. Our implementation of this high-precision flicker spectroscopy contour tracking algorithm (HiPFSTA) is provided as open-source at www.github.com/michaelmell/hipfsta.

 

PLoS ONE 13 (12): e0207376.

https://doi.org/10.1371/journal.pone.0207376

Open Access : https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0207376&type=printable

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Quantification of protein mobility and associated reshuffling of cytoplasm during chemical fixation

Quantification of protein mobility and associated reshuffling of cytoplasm during chemical fixation | News Imagerie cellulaire - Cellular imaging | Scoop.it

Jan Huebinger, Jessica Spindler, Kristin J. Holl & Björn Koos 

 

To understand cellular functionalities, it is essential to unravel spatio-temporal patterns of molecular distributions and interactions within living cells. The technological progress in fluorescence microscopy now allows in principle to measure these patterns with sufficient spatial resolution. However, high resolution imaging comes with long acquisition times and high phototoxicity. Therefore, physiological live cell imaging is often unfeasible and chemical fixation is employed. Yet, fixation methods have not been rigorously investigated, in terms of pattern preservation, at the resolution at which cells can now be imaged. A key parameter for this is the time required until fixation is complete. During this time, cells are under unphysiological conditions and patterns decay. We demonstrate here that formaldehyde fixation takes more than one hour for cytosolic proteins in cultured cells. Other small aldehydes, glyoxal and acrolein, did not perform better. Associated with this, we found a distinct displacement of proteins and lipids, including their loss from cells. Fixations using glutaraldehyde were faster than four minutes and retained most cytoplasmic proteins. Surprisingly, autofluorescence produced by glutaraldehyde was almost completely absent with supplementary addition of formaldehyde without compromising fixation speed. These findings indicate, which cellular processes can actually be reliably imaged after a certain chemical fixation.

 

Scientific Reports volume 8, Article number: 17756 (2018)

https://doi.org/10.1038/s41598-018-36112-w

Open Access : https://www.nature.com/articles/s41598-018-36112-w.pdf

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Quantification of protein mobility and associated reshuffling of cytoplasm during chemical fixation

Quantification of protein mobility and associated reshuffling of cytoplasm during chemical fixation | News Imagerie cellulaire - Cellular imaging | Scoop.it

Jan Huebinger, Jessica Spindler, Kristin J. Holl and  Björn Koos

 

Department of Systemic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Str.11, 44227, Dortmund, Germany.

 

To understand cellular functionalities, it is essential to unravel spatio-temporal patterns of molecular distributions and interactions within living cells. The technological progress in fluorescence microscopy now allows in principle to measure these patterns with sufficient spatial resolution. However, high resolution imaging comes with long acquisition times and high phototoxicity. Therefore, physiological live cell imaging is often unfeasible and chemical fixation is employed. Yet, fixation methods have not been rigorously investigated, in terms of pattern preservation, at the resolution at which cells can now be imaged. A key parameter for this is the time required until fixation is complete. During this time, cells are under unphysiological conditions and patterns decay. We demonstrate here that formaldehyde fixation takes more than one hour for cytosolic proteins in cultured cells. Other small aldehydes, glyoxal and acrolein, did not perform better. Associated with this, we found a distinct displacement of proteins and lipids, including their loss from cells. Fixations using glutaraldehyde were faster than four minutes and retained most cytoplasmic proteins. Surprisingly, autofluorescence produced by glutaraldehyde was almost completely absent with supplementary addition of formaldehyde without compromising fixation speed. These findings indicate, which cellular processes can actually be reliably imaged after a certain chemical fixation.

 

Sci Rep. 2018; 8: 17756

doi : 10.1038/s41598-018-36112-w

Open access article : https://www.nature.com/articles/s41598-018-36112-w.pdf

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Multifocal multiphoton volumetric imaging approach for high-speed time-resolved Förster resonance energy transfer imaging in vivo

Multifocal multiphoton volumetric imaging approach for high-speed time-resolved Förster resonance energy transfer imaging in vivo | News Imagerie cellulaire - Cellular imaging | Scoop.it

Simon P. Poland, Grace K. Chan, James A. Levitt, Nikola Krstajić, Ahmet T. Erdogan, Robert K. Henderson, Maddy Parsons, and Simon M. Ameer-Beg

 

In this Letter, we will discuss the development of a multifocal multiphoton fluorescent lifetime imaging system where four individual fluorescent intensity and lifetime planes are acquired simultaneously, allowing us to obtain volumetric data without the need for sequential scanning at different axial depths. Using a phase-only spatial light modulator (SLM) with an appropriate algorithm to generate a holographic pattern, we project a beamlet array within a sample volume of a size, which can be preprogrammed by the user. We demonstrate the capabilities of the system to image live-cell interactions. While only four planes are shown, this technique can be rescaled to a large number of focal planes, enabling full 3D acquisition and reconstruction.

 

Optics Letters Vol. 43, Issue 24, pp. 6057-6060 (2018)

https://doi.org/10.1364/OL.43.006057

Open Access : https://www.osapublishing.org/ol/viewmedia.cfm?uri=ol-43-24-6057&seq=0 

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Super-resolution enhancement by quantum image scanning microscopy

Super-resolution enhancement by quantum image scanning microscopy | News Imagerie cellulaire - Cellular imaging | Scoop.it

Ron Tenne, Uri Rossman, Batel Rephael, Yonatan Israel, Alexander Krupinski-Ptaszek, Radek Lapkiewicz, Yaron Silberberg & Dan Oron

 

The principles of quantum optics have yielded a plethora of ideas to surpass the classical limitations of sensitivity and resolution in optical microscopy. While some ideas have been applied in proof-of-principle experiments, imaging a biological sample has remained challenging, mainly due to the inherently weak signal measured and the fragility of quantum states of light. In principle, however, these quantum protocols can add new information without sacrificing the classical information and can therefore enhance the capabilities of existing super-resolution techniques. Image scanning microscopy, a recent addition to the family of super-resolution methods, generates a robust resolution enhancement without reducing the signal level. Here, we introduce quantum image scanning microscopy: combining image scanning microscopy with the measurement of quantum photon correlation allows increasing the resolution of image scanning microscopy up to twofold, four times beyond the diffraction limit. We introduce the Q-ISM principle and obtain super-resolved optical images of a biological sample stained with fluorescent quantum dots using photon antibunching, a quantum effect, as a resolution-enhancing contrast mechanism.

 

Nature Methods (2018) Published: 17 December 2018

https://doi.org/10.1038/s41566-018-0324-z

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Imaging cellular ultrastructures using expansion microscopy (U-ExM)

Imaging cellular ultrastructures using expansion microscopy (U-ExM) | News Imagerie cellulaire - Cellular imaging | Scoop.it

Davide Gambarotto, Fabian U. Zwettler, Maeva Le Guennec, Marketa Schmidt-Cernohorska, Denis Fortun, Susanne Borgers, Jörn Heine, Jan-Gero Schloetel, Matthias Reuss, Michael Unser, Edward S. Boyden, Markus Sauer, Virginie Hamel & Paul Guichard

 

Determining the structure and composition of macromolecular assemblies is a major challenge in biology. Here we describe ultrastructure expansion microscopy (U-ExM), an extension of expansion microscopy that allows the visualization of preserved ultrastructures by optical microscopy. This method allows for near-native expansion of diverse structures in vitro and in cells; when combined with super-resolution microscopy, it unveiled details of ultrastructural organization, such as centriolar chirality, that could otherwise be observed only by electron microscopy.

 

Nature Methods (2018) Published: 17 December 2018

https://doi.org/10.1038/s41592-018-0238-1

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Single-Cell Imaging of Metastatic Potential of Cancer Cells

Single-Cell Imaging of Metastatic Potential of Cancer Cells | News Imagerie cellulaire - Cellular imaging | Scoop.it
Krishna Midde, Nina Sun, Cristina Rohena, Linda Joosen, Harsharan Dhillon, Pradipta Ghosh
 

Molecular imaging of metastatic “potential” is an unvanquished challenge. To engineer biosensors that can detect and measure the metastatic “potential” of single living cancer cells, we carried out a comprehensive analysis of the pan-cancer phosphoproteome to search for actin remodelers required for cell migration, which are enriched in cancers but excluded in normal cells. Only one phosphoprotein emerged, tyr-phosphorylated CCDC88A (GIV/Girdin), a bona fide metastasis-related protein across a variety of solid tumors. We designed multi-modular biosensors that are partly derived from GIV, and because GIV integrates prometastatic signaling by multiple oncogenic receptors, we named them “‘integrators of metastatic potential (IMP).” IMPs captured the heterogeneity of metastatic potential within primary lung and breast tumors at steady state, detected those few cells that have acquired the highest metastatic potential, and tracked their enrichment during metastasis. These findings provide proof of concept that IMPs can measure the diversity and plasticity of metastatic potential of tumor cells in a sensitive and unbiased way.

 

iScience Volume 10, 21 December 2018, Pages 53-65
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Dynamic Imaging of Small Molecule Induced Protein–Protein Interactions in Living Cells with a Fluorophore Phase Transition Based Approach

Dynamic Imaging of Small Molecule Induced Protein–Protein Interactions in Living Cells with a Fluorophore Phase Transition Based Approach | News Imagerie cellulaire - Cellular imaging | Scoop.it

Chan-I Chung, Qiang Zhang and Xiaokun Shu

 

Protein–protein interactions (PPIs) mediate signal transduction in cells. Small molecules that regulate PPIs are important tools for biology and biomedicine. Dynamic imaging of small molecule induced PPIs characterizes and verifies these molecules in living cells. It is thus important to develop cellular assays for dynamic visualization of small molecule induced protein–protein association and dissociation in living cells. Here we have applied a fluorophore phase transition based principle and designed a PPI assay named SPPIER (separation of phases-based protein interaction reporter). SPPIER utilizes the green fluorescent protein (GFP) and is thus genetically encoded. Upon small molecule induced PPI, SPPIER rapidly forms highly fluorescent GFP droplets in living cells. SPPIER detects immunomodulatory drug (IMiD) induced PPI between cereblon and the transcription factor Ikaros. It also detects IMiD analogue (e.g., CC-885) induced PPI between cereblon and GSPT1. Furthermore, SPPIER can visualize bifunctional molecules (e.g. PROTAC)-induced PPI between an E3 ubiquitin ligase and a target protein. Lastly, SPPIER can be modified to image small molecule induced protein–protein dissociation, such as nutlin-induced dissociation between HDM2 and p53. The intense brightness and rapid kinetics of SPPIER enable robust and dynamic visualization of PPIs in living cells.

 

Anal. Chem., Article ASAP Publication Date (Web): Nov. 15, 2018
Doi : 10.1021/acs.analchem.8b03476
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Multispectral Atomic Force Microscopy-Infrared Nano-Imaging of Malaria Infected Red Blood Cells

Multispectral Atomic Force Microscopy-Infrared Nano-Imaging of Malaria Infected Red Blood Cells | News Imagerie cellulaire - Cellular imaging | Scoop.it

David Perez-Guaita, Kamila Kochan, Mitchell Batty, Christian Doerig, Jose Garcia-Bustos, Shirly Espinoza, Don McNaughton, Phil Heraud and Bayden R. Wood

 

Atomic force microscopy-infrared (AFM-IR) spectroscopy is a powerful new technique that can be applied to study molecular composition of cells and tissues at the nanoscale. AFM-IR maps are acquired using a single wavenumber value: they show either the absorbance plotted against a single wavenumber value or a ratio of two absorbance values. Here, we implement multivariate image analysis to generate multivariate AFM-IR maps and use this approach to resolve subcellular structural information in red blood cells infected with Plasmodium falciparum at different stages of development. This was achieved by converting the discrete spectral points into a multispectral line spectrum prior to multivariate image reconstruction. The approach was used to generate compositional maps of subcellular structures in the parasites, including the food vacuole, lipid inclusions, and the nucleus, on the basis of the intensity of hemozoin, hemoglobin, lipid, and DNA IR marker bands, respectively. Confocal Raman spectroscopy was used to validate the presence of hemozoin in the regions identified by the AFM-IR technique. The high spatial resolution of AFM-IR combined with hyperspectral modeling enables the direct detection of subcellular components, without the need for cell sectioning or immunological/biochemical staining. Multispectral-AFM-IR thus has the capacity to probe the phenotype of the malaria parasite during its intraerythrocytic development. This enables novel approaches to studying the mode of action of antimalarial drugs and the phenotypes of drug-resistant parasites, thus contributing to the development of diagnostic and control measures

 

Anal. Chem., 2018, 90 (5), pp 3140–3148

Doi : 10.1021/acs.analchem.7b04318

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Combined Use of Unnatural Amino Acids Enables Dual Color Super-Resolution Imaging of Proteins via Click Chemistry

Combined Use of Unnatural Amino Acids Enables Dual Color Super-Resolution Imaging of Proteins via Click Chemistry | News Imagerie cellulaire - Cellular imaging | Scoop.it

Kim -A. Saal, Frank Richter, Peter Rehling, and Silvio O. Rizzoli

 

Recent advances in optical nanoscopy have brought the imaging resolution to the size of the individual macromolecules, thereby setting stringent requirements for the fluorescent labels. Such requirements are optimally fulfilled by the incorporation of unnatural amino acids (UAAs) in the proteins of interest (POI), followed by fluorophore conjugation via click chemistry. However, this approach has been limited to single POIs in mammalian cells. Here we solve this problem by incorporating different UAAs in different POIs, which are expressed in independent cell sets. The cells are then fused, thereby combining the different proteins and organelles, and are easily imaged by dual-color super-resolution microscopy. This procedure, which we termed Fuse2Click, is simple, requires only the well-established Amber codon, and allows the use of all previously optimized UAAs and tRNA/RS pairs. This should render it a tool of choice for multi-color click-based imaging.

 

ACS Nano, Just Accepted Manuscript Dec. 10, 2018
Doi : 10.1021/acsnano.8b06047
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Membrane water for probing neuronal membrane potentials and ionic fluxes at the single cell level

Membrane water for probing neuronal membrane potentials and ionic fluxes at the single cell level | News Imagerie cellulaire - Cellular imaging | Scoop.it

M. E. P. Didier, O. B. Tarun, P. Jourdain, P. Magistretti & S. Roke

 

Neurons communicate through electrochemical signaling within a complex network. These signals are composed of changes in membrane potentials and are traditionally measured with the aid of (toxic) fluorescent labels or invasive electrical probes. Here, we demonstrate an improvement in label-free second harmonic neuroimaging sensitivity by ~3 orders of magnitude using a wide-field medium repetition rate illumination. We perform a side-by-side patch-clamp and second harmonic imaging comparison to demonstrate the theoretically predicted linear correlation between whole neuron membrane potential changes and the square root of the second harmonic intensity. We assign the ion induced changes to the second harmonic intensity to changes in the orientation of membrane interfacial water, which is used to image spatiotemporal changes in the membrane potential and K+ ion flux. We observe a non-uniform spatial distribution and temporal activity of ion channels in mouse brain neurons.

 

Nature Communications volume 9, Article number: 5287 (2018)

https://doi.org/10.1038/s41467-018-07713-w

Open Access : https://www.nature.com/articles/s41467-018-07713-w.pdf

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combined treatment with erythrocyte lysis solution and Sudan Black B reduces tissue autofluorescence in double-labeling immunofluorescence

combined treatment with erythrocyte lysis solution and Sudan Black B reduces tissue autofluorescence in double-labeling immunofluorescence | News Imagerie cellulaire - Cellular imaging | Scoop.it

 

The autofluorescence of animal tissues complicates the results obtained using fluorescence microscopy. Many techniques have been used to reduce autofluorescence; however, all these techniques have the disadvantage of reducing the intensity of immunofluorescence staining. We observed the features of autofluorescence in formalin-fixed paraffin-embedded (FFPE) vascularized liver and kidney sections and assessed the effects of an intravascular treatment with erythrocyte lysis solution (ELS) before a routine perfusion with normal saline (NS) and Sudan Black B (SBB) treatment after antigen retrieval on reducing autofluorescence reduction and the visualization of antigens to establish an optimal method for reducing autofluorescence. Erythrocytes exhibited bright autofluorescence in FFPE liver and kidney sections, which altered the results of actin and destrin immunofluorescence staining. The SBB treatment significantly reduced background autofluorescence and exerted a moderate effect on reducing the autofluorescence of erythrocytes, and the intravascular ELS treatment eliminated erythrocyte autofluorescence in FFPE liver and kidney sections. A combined treatment with ELS and SBB further reduced autofluorescence but did not decrease actin and destrin immunofluorescence staining in double-labeled FFPE liver and kidney sections. In conclusion, the application of an intravascular ELS treatment before the NS perfusion combined with an SBB treatment after antigen retrieval is a simple and efficient strategy for reducing autofluorescence in FFPE vascularized tissues and can be broadly used in fluorescence microscopy analyses.

 

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A review of techniques for visualising soft tissue microstructure deformation and quantifying strain Ex Vivo

A review of techniques for visualising soft tissue microstructure deformation and quantifying strain Ex Vivo | News Imagerie cellulaire - Cellular imaging | Scoop.it

C.M. DISNEY, P.D. LEE, J.A. HOYLAND, M.J. SHERRATT & B.K. BAY

 

Many biological tissues have a complex hierarchical structure allowing them to function under demanding physiological loading conditions. Structural changes caused by ageing or disease can lead to loss of mechanical function. Therefore, it is necessary to characterise tissue structure to understand normal tissue function and the progression of disease. Ideally intact native tissues should be imaged in 3D and under physiological loading conditions. The current published in situ imaging methodologies demonstrate a compromise between imaging limitations and maintaining the samples native mechanical function. This review gives an overview of in situ imaging techniques used to visualise microstructural deformation of soft tissue, including three case studies of different tissues (tendon, intervertebral disc and artery). Some of the imaging techniques restricted analysis to observational mechanics or discrete strain measurement from invasive markers. Full‐field local surface strain measurement has been achieved using digital image correlation. Volumetric strain fields have successfully been quantified from in situ X‐ray microtomography (micro‐CT) studies of bone using digital volume correlation but not in soft tissue due to low X‐ray transmission contrast. With the latest developments in micro‐CT showing in‐line phase contrast capability to resolve native soft tissue microstructure, there is potential for future soft tissue mechanics research where 3D local strain can be quantified. These methods will provide information on the local 3D micromechanical environment experienced by cells in healthy, aged and diseased tissues. It is hoped that future applications of in situ imaging techniques will impact positively on the design and testing of potential tissue replacements or regenerative therapies.

 

Journal of Microscopy, Vol. 272, Issue 3 2018, pp. 165–179

https://doi.org/10.1111/jmi.12701

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FRET biosensor allows spatio-temporal observation of shear stress-induced polar RhoGDIα activation

FRET biosensor allows spatio-temporal observation of shear stress-induced polar RhoGDIα activation | News Imagerie cellulaire - Cellular imaging | Scoop.it

Shuai Shao, Xiaoling Liao, Fei Xie, Sha Deng, Xue Liu, Tapani Ristaniemi & Bo Liu

 

Rho GDP-dissociation inhibitor α (RhoGDIα) is a known negative regulator of the Rho family that shuts off GDP/GTP cycling and cytoplasm/membrane translocation to regulate cell migration. However, to our knowledge, no reports are available that focus on how the RhoGDIα-Rho GTPases complex is activated by laminar flow through exploring the activation of RhoGDIα itself. Here, we constructed a new biosensor using fluorescence resonance energy transfer (FRET) technology to measure the spatio-temporal activation of RhoGDIα in its binding with Rho GTPases in living HeLa cells. Using this biosensor, we find that the dissociation of the RhoGDIα-Rho GTPases complex is increased by shear stress, and its dissociation rate varies with subcellular location. Moreover, this process is mediated by membrane fluidity, cytoskeleton and Src activity, which indicates that the regulation of RhoGDIα activation under shear stress application represents a relatively separate pathway from the shear stress-induced Rho pathway.

 

Communications Biology volume 1, Article number: 224 (2018)

https://doi.org/10.1038/s42003-018-0232-2

Open Access : https://www.nature.com/articles/s42003-018-0232-2.pdf

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