News Imagerie cellulaire - Cellular imaging
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Super-multiplexed fluorescence microscopy via photostability contrast

Super-multiplexed fluorescence microscopy via photostability contrast | News Imagerie cellulaire - Cellular imaging | Scoop.it

Antony Orth, Richik N. Ghosh, Emma Wilson, Timothy Doughney, Hannah Brown, Philipp Reineck, Jeremy Thompson and Brant C. Gibson


Many areas of biological research rely heavily on fluorescence microscopy to observe and quantify the inner workings of the cell. Traditionally, multiple types of cellular structures or biomolecules are visualized simultaneously with spectrally distinct fluorescent labels. A high degree of multiplexing is desirable as it affords the experiment greater information content, speeding up research timelines. Multiplexing can be increased by imaging a larger number of spectral channels, however, the wide emission spectra of most fluorophores limits multiplexing to four or five labels in standard fluorescence microscopes. Further multiplexing requires another dimension of contrast. Here, we show that photostability differences can be used to distinguish between fluorescent labels. By combining photobleaching characteristics with a novel unmixing algorithm, we resolve up to three fluorescent labels in a single spectral channel and unmix fluorescent labels with nearly identical emission spectra. We apply our technique to organic dyes, autofluorescent biomolecules and fluorescent proteins, and show that the latter are particularly well suited to our method as their bleaching is often reversible. Our approach has the potential to triple the multiplexing capabilities of any digital widefield or confocal fluorescence microscope with no additional hardware, making it readily accessible to a wide range of researchers.


bioRxiv preprint first posted online Feb. 2, 2018;

doi: https://doi.org/10.1101/258889


https://www.biorxiv.org/content/biorxiv/early/2018/02/02/258889.full.pdf

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Automated muscle histopathology analysis using CellProfiler

Automated muscle histopathology analysis using CellProfiler | News Imagerie cellulaire - Cellular imaging | Scoop.it

Yeh Siang Lau, Li Xu, Yandi Gao and Renzhi Han

 

Histological assessment of skeletal muscle sections is important for the research of muscle physiology and diseases. Quantifiable measures of skeletal muscle often include mean fiber diameter, fiber size distribution, and centrally nucleated muscle fibers. These parameters offer insights into the dynamic adaptation of skeletal muscle cells during repeated cycles of degeneration and regeneration associated with many muscle diseases and injuries. Computational programs designed to obtain these parameters would greatly facilitate such efforts and offer significant advantage over manual image analysis, which is very labor-intensive and often subjective. Here, we describe a customized pipeline termed MuscleAnalyzer for muscle histology analysis based upon CellProfiler, a free, open-source software for measuring and analyzing cell images.

 

Skeletal Muscle 2018 8 : 32

https://doi.org/10.1186/s13395-018-0178-6

Open Access : https://skeletalmusclejournal.biomedcentral.com/track/pdf/10.1186/s13395-018-0178-6

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A novel computational method for automatic segmentation, quantification and comparative analysis of immunohistochemically labeled tissue sections

A novel computational method for automatic segmentation, quantification and comparative analysis of immunohistochemically labeled tissue sections | News Imagerie cellulaire - Cellular imaging | Scoop.it

Elena Casiraghi, Veronica Huber, Marco Frasca, Mara Cossa, Matteo Tozzi, Licia Rivoltini, Biagio Eugenio Leone, Antonello Villa and Barbara Vergani

 

In the clinical practice, the objective quantification of histological results is essential not only to define objective and well-established protocols for diagnosis, treatment, and assessment, but also to ameliorate disease comprehension.
The software MIAQuant_Learn presented in this work segments, quantifies and analyzes markers in histochemical and immunohistochemical images obtained by different biological procedures and imaging tools. MIAQuant_Learn employs supervised learning techniques to customize the marker segmentation process with respect to any marker color appearance. Our software expresses the location of the segmented markers with respect to regions of interest by mean-distance histograms, which are numerically compared by measuring their intersection. When contiguous tissue sections stained by different markers are available, MIAQuant_Learn aligns them and overlaps the segmented markers in a unique image enabling a visual comparative analysis of the spatial distribution of each marker (markers’ relative location). Additionally, it computes novel measures of markers’ co-existence in tissue volumes depending on their density.
Applications of MIAQuant_Learn in clinical research studies have proven its effectiveness as a fast and efficient tool for the automatic extraction, quantification and analysis of histological sections. It is robust with respect to several deficits caused by image acquisition systems and produces objective and reproducible results. Thanks to its flexibility, MIAQuant_Learn represents an important tool to be exploited in basic research where needs are constantly changing.

 

BMC Bioinformatics 2018 19 : 2302

https://doi.org/10.1186/s12859-018-2302-3

Open Access : https://bmcbioinformatics.biomedcentral.com/track/pdf/10.1186/s12859-018-2302-3

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Application of black phosphorus nanodots to live cell imaging

Application of black phosphorus nanodots to live cell imaging | News Imagerie cellulaire - Cellular imaging | Scoop.it

Yong Cheol Shin, Su-Jin Song, Yu Bin Lee, Moon Sung Kang, Hyun Uk Lee, Jin-Woo Oh and Dong-Wook Han

 

Black phosphorus (BP) has emerged as a novel class of nanomaterials owing to its unique optical and electronic properties. BP, a two-dimensional (2D) nanomaterial, is a structure where phosphorenes are stacked together in layers by van der Waals interactions. However, although BP nanodots have many advantages, their biosafety and biological effect have not yet been elucidated as compared to the other nanomaterials. Therefore, it is particularly important to assess the cytotoxicity of BP nanodots for exploring their potentials as novel biomaterials.

Biomaterials Research 2018 22 : 31

https://doi.org/10.1186/s40824-018-0142-x

Open Access : https://biomaterialsres.biomedcentral.com/track/pdf/10.1186/s40824-018-0142-x

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Conformational dynamics of the ABC transporter McjD seen by single‐molecule FRET

Conformational dynamics of the ABC transporter McjD seen by single‐molecule FRET | News Imagerie cellulaire - Cellular imaging | Scoop.it

Florence Husada, Kiran Bountra, Konstantinos Tassis, Marijn de Boer, Maria Romano, Sylvie Rebuffat, Konstantinos Beis & Thorben Cordes

 

ABC transporters utilize ATP for export processes to provide cellular resistance against toxins, antibiotics, and harmful metabolites in eukaryotes and prokaryotes. Based on static structure snapshots, it is believed that they use an alternating access mechanism, which couples conformational changes to ATP binding (outward‐open conformation) and hydrolysis (inward‐open) for unidirectional transport driven by ATP. Here, we analyzed the conformational states and dynamics of the antibacterial peptide exporter McjD from Escherichia coli using single‐molecule Förster resonance energy transfer (smFRET). For the first time, we established smFRET for an ABC exporter in a native‐like lipid environment and directly monitor conformational dynamics in both the transmembrane‐ (TMD) and nucleotide‐binding domains (NBD). With this, we unravel the ligand dependences that drive conformational changes in both domains. Furthermore, we observe intrinsic conformational dynamics in the absence of ATP and ligand in the NBDs. ATP binding and hydrolysis on the other hand can be observed via NBD conformational dynamics. We believe that the progress made here in combination with future studies will facilitate full understanding of ABC transport cycles.

 

The EMBO Journal, e100056, Published online: September 20, 2018

DOI : 10.15252/embj.2018100056

Open Access : http://emboj.embopress.org/content/early/2018/09/20/embj.2018100056

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ClearT immersion optical clearing method for intact 3D spheroids imaging through confocal laser scanning microscopy

ClearT immersion optical clearing method for intact 3D spheroids imaging through confocal laser scanning microscopy | News Imagerie cellulaire - Cellular imaging | Scoop.it
Elisabete C. Costa, André F. Moreira, Duarte de Melo-Diogo, Ilídio J. Correia
 

Spheroids are 3D in vitro platforms that fill the gap between the 2D cell cultures and animal models on the therapeutics development pipeline. Yet, the methods and equipment used in the in vitro assays are optimized for the analysis of cells cultured as monolayers. For instance, confocal laser scanning microscopy (CLSM) does not allow the observation of thick intact spheroids due to light penetration issues. To overcome this limitation, spheroids treatment with clearing agents started to be explored. Herein, we demonstrate for the first time the application of ClearT clearing method for the imaging of propidium iodide (PI) stained spheroids by CLSM. The results demonstrate that the ClearT is a reversible clearing method that does not influence the structure of the spheroid and significantly improved the PI signal penetration depth in about 43%. Additionally, ClearT also enhanced the cells imaging within the spheroid by increasing the cross-penetration depth in 46.6% at 100 µm of depth. Overall, the results show that ClearT method may allow the improvement of the CLSM accuracy on the evaluation of the cellular death within spheroids prompted by therapeutics.

 

Optics & Laser Technology Vol. 106, October 2018, pp94-99

https://doi.org/10.1016/j.optlastec.2018.04.002

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NATIVE: Nanobody-assisted tissue immunostaining for volumetric EM.

NATIVE: Nanobody-assisted tissue immunostaining for volumetric EM. | News Imagerie cellulaire - Cellular imaging | Scoop.it

Tao Fang, Xiaotang Lu, Daniel Berger, Hidde Ploegh & Jeff Lichtman

 

Morphological and molecular characteristics determine the function of biological tissues. Attempts to combine immunofluorescence and electron microscopy invariably compromise the quality of the ultrastructure of tissue sections. We developed NATIVE, a correlated light and electron microscopy approach that preserves ultrastructure while showing the locations of multiple molecular moieties even deep within tissues. This technique allowed the large-scale 3D reconstruction of a volume of mouse hippocampal CA3 tissue at nanometer resolution.

 

Protocol Exchange (2018) Published online 17 October 2018

doi:10.1038/protex.2018.108

Open Access : https://www.nature.com/protocolexchange/protocols/7039

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Real-Time Determination of the Cell-Cycle Position of Individual Cells within Live Tumors Using FUCCI Cell-Cycle Imaging.

Real-Time Determination of the Cell-Cycle Position of Individual Cells within Live Tumors Using FUCCI Cell-Cycle Imaging. | News Imagerie cellulaire - Cellular imaging | Scoop.it
Shuya Yano and Robert M. Hoffman

 

Most cytotoxic agents have limited efficacy for solid cancers. Cell-cycle phase analysis at the single-cell level in solid tumors has shown that the majority of cancer cells in tumors is not cycling and is therefore resistant to cytotoxic chemotherapy. Intravital cell-cycle imaging within tumors demonstrated the cell-cycle position and distribution of cancer cells within a tumor, and cell-cycle dynamics during chemotherapy. Understanding cell-cycle dynamics within tumors should provide important insights into novel treatment strategies.

 

Cells 2018, 7(10), 168

http://dx.doi.org/10.3390/cells7100168

Open Access : https://www.mdpi.com/2073-4409/7/10/168/pdf

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Assessing photodamage in live-cell STED microscopy

Assessing photodamage in live-cell STED microscopy | News Imagerie cellulaire - Cellular imaging | Scoop.it

Nicole Kilian, Alexander Goryaynov, Mark D. Lessard, Giles Hooker,

Derek Toomre, James E. Rothman & Joerg Bewersdorf

 

The recent breakthroughs in the development of optical nanoscopy have provided unprecedented views of the inner workings of cells. Stimulated emission depletion (STED) microscopy, in particular, allows real-time observation of living cells at resolutions of 50 nm or less1,2. However, the high irradiation intensities used in STED nanoscopy have raised concerns about the validity of live-cell observations obtained with this and similar approaches3,4. We report here that, under the right conditions, living cells can be imaged by STED nanoscopy without substantial photodamage.

 

Nature Methods volume 15, pages755–756 (2018)

https://doi.org/10.1038/s41592-018-0145-5

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Synchrotron-Based X-Ray Fluorescence Microscopy as a Technique for Imaging of Elements in Plants

Synchrotron-Based X-Ray Fluorescence Microscopy as a Technique for Imaging of Elements in Plants | News Imagerie cellulaire - Cellular imaging | Scoop.it

Peter M. Kopittke, Tracy Punshon, David J. Paterson, Ryan V. Tappero, Peng Wang, F. Pax C. Blamey, Antony van der Ent, Enzo Lombi

 

Understanding the distribution of elements within plant tissues is important across a range of fields in plant science. In this review, we examine synchrotron-based x-ray fluorescence microscopy (XFM) as an elemental imaging technique in plant sciences, considering both its historical and current uses as well as discussing emerging approaches. XFM offers several unique capabilities of interest to plant scientists, including in vivo analyses at room temperature and pressure, good detection limits (approximately 1–100 mg kg−1), and excellent resolution (down to 50 nm). This has permitted its use in a range of studies, including for functional characterization in molecular biology, examining the distribution of nutrients in food products, understanding the movement of foliar fertilizers, investigating the behavior of engineered nanoparticles, elucidating the toxic effects of metal(loid)s in agronomic plant species, and studying the unique properties of hyperaccumulating plants. We anticipate that continuing technological advances at XFM beamlines also will provide new opportunities moving into the future, such as for high-throughput screening in molecular biology, the use of exotic metal tags for protein localization, and enabling time-resolved, in vivo analyses of living plants. By examining current and potential future applications, we hope to encourage further XFM studies in plant sciences by highlighting the versatility of this approach.

 

Plant Physiol. Vol. 178, 2018, pp507-523

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Nondestructive fluorescence lifetime imaging and time-resolved fluorescence spectroscopy detect cartilage matrix depletion and correlate with mechanical properties

Nondestructive fluorescence lifetime imaging and time-resolved fluorescence spectroscopy detect cartilage matrix depletion and correlate with mechanical properties | News Imagerie cellulaire - Cellular imaging | Scoop.it

A.K. Haudenschild, B.E. Sherlock, X. Zhou, J.C. Hu, J.K. Leach, L. Marcu and K.A. Athanasiou

 

Tissue engineers utilize a battery of expensive, time-consuming and destructive techniques to assess the composition and function of engineered tissues. A nondestructive solution to monitor tissue maturation would reduce costs and accelerate product development. As a first step toward this goal, two nondestructive, label-free optical techniques, namely multispectral fluorescent lifetime imaging (FLIm) and time-resolved fluorescence spectroscopy (TRFS), were investigated for their potential in evaluating the biochemical and mechanical properties of articular cartilage.

 

European Cells and Materials, Vol. 36 2018 : 30-43

DOI: 10.22203/eCM.v036a03

Open Access : http://www.ecmjournal.org/papers/vol036/pdf/v036a03.pdf

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Imaging beyond the super-resolution limits using ultrastructure expansion microscopy (UltraExM)

Imaging beyond the super-resolution limits using ultrastructure expansion microscopy (UltraExM) | News Imagerie cellulaire - Cellular imaging | Scoop.it

Davide Gambarotto, Fabian Zwettler, Marketa Cernohorska, Denis Fortun, Susanne Borgers, Jorn Heine, Jan-Gero Schloetel, Matthias Reuss, Michael Unser, Edward Boyden, Markus Sauer, Virginie Hamel, Paul Guichard

 

For decades, electron microscopy (EM) was the only method able to reveal the ultrastructure of cellular organelles and molecular complexes because of the diffraction limit of optical microscopy. In recent past, the emergence of super-resolution fluorescence microscopy enabled the visualization of cellular structures with so far unmatched spatial resolution approaching virtually molecular dimensions. Despite these technological advances, currently super-resolution microscopy does not permit the same resolution level as provided by electron microscopy, impeding the attribution of a protein to an ultrastructural element. Here, we report a novel method of near-native expansion microscopy (UltraExM), enabling the visualization of preserved ultrastructures of macromolecular assemblies with subdiffraction-resolution by standard optical microscopy. UltraExM revealed for the first time the ultrastructural localization of tubulin glutamylation in centrioles. Combined with super-resolution microscopy, UltraExM unveiled the centriolar chirality, an ultrastructural signature, which was only visualizable by electron microscopy.

 

bioRxiv preprint first posted online Apr. 25, 2018 not peer-reviewed

http://dx.doi.org/10.1101/308270

Open Access : https://www.biorxiv.org/content/biorxiv/early/2018/04/25/308270.full.pdf

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Invited Article: Comparison of hyperspectral coherent Raman scattering microscopies for biomedical applications.

Invited Article: Comparison of hyperspectral coherent Raman scattering microscopies for biomedical applications. | News Imagerie cellulaire - Cellular imaging | Scoop.it

T. Bocklitz, T. Meyer, M. Schmitt, I. Rimke, F. Hoffmann, F. von Eggeling, G. Ernst, O. Guntinas-Lichius and J. Popp

 

Raman scattering based imaging represents a very powerful optical tool for biomedical diagnostics. Different Raman signatures obtained by distinct tissue structures and disease induced changes provoke sophisticated analysis of the hyperspectral Raman datasets. While the analysis of linear Raman spectroscopic tissue data is quite established, the evaluation of hyperspectral nonlinear Raman data has not yet been evaluated in great detail. The two most common nonlinear Raman methods are CARS (coherent anti-Stokes Raman scattering) and SRS (stimulated Raman scattering) spectroscopy. Specifically the linear concentration dependence of SRS as compared to the quadratic dependence of CARS has fostered the application of SRS tissue imaging. Here, we applied spectral processing to hyperspectral SRS and CARS data for tissue characterization. We could demonstrate for the first time that similar cluster distributions can be obtained for multispectral CARS and SRS data but that clustering is based on different spectral features due to interference effects in CARS and the different concentration dependence of CARS and SRS. It is shown that a direct combination of CARS and SRS data does not improve the clustering results.

 

APL Photonics, Volume 3, Issue 9 >10.1063/1.5030159

https://doi.org/10.1063/1.5030159

Open Access : http://aip.scitation.org/doi/pdf/10.1063/1.5030159?class=pdf

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Tutorial: Coherent Raman light matter interaction processes

Tutorial: Coherent Raman light matter interaction processes | News Imagerie cellulaire - Cellular imaging | Scoop.it

Hervé  Rigneault and Pascal Berto

 

Coherent Raman scattering processes such as coherent anti-Stokes Raman scattering and stimulated Raman scattering are described in a tutorial way keeping simple physical pictures and simple derivations. The simplicity of the presentation keeps however most of the key features of these coherent and resonant processes and their intimate relation with spontaneous Raman scattering. This tutorial provides a digest of introduction to the fundamental physics at work, and it does not focus on the numerous technological implementations; rather, it provides the concepts and the physical tools to understand the extensive literature in this field. The presentation is made simple enough for under-graduate students, graduate students, and newcomers with various scientific backgrounds.

 

APL Photonics, Volume 3, Issue 9 > 10.1063/1.5030335

https://doi.org/10.1063/1.5030335

Open Access : https://aip.scitation.org/doi/pdf/10.1063/1.5030335?class=pdf

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Measuring DNA content in live cells by fluorescence microscopy

Measuring DNA content in live cells by fluorescence microscopy | News Imagerie cellulaire - Cellular imaging | Scoop.it

Cecil J. Gomes, Michael W. Harman, Sara M. Centuori, Charles W. Wolgemuth and Jesse D. Martinez

 

Live-cell fluorescence microscopy (LCFM) is a powerful tool used to investigate cellular dynamics in real time. However, the capacity to simultaneously measure DNA content in cells being tracked over time remains challenged by dye-associated toxicities. The ability to measure DNA content in single cells by means of LCFM would allow cellular stage and ploidy to be coupled with a variety of imaging directed analyses. Here we describe a widely applicable nontoxic approach for measuring DNA content in live cells by fluorescence microscopy. This method relies on introducing a live-cell membrane-permeant DNA fluorophore, such as Hoechst 33342, into the culture medium of cells at the end of any live-cell imaging experiment and measuring each cell’s integrated nuclear fluorescence to quantify DNA content. Importantly, our method overcomes the toxicity and induction of DNA damage typically caused by live-cell dyes through strategic timing of adding the dye to the cultures; allowing unperturbed cells to be imaged for any interval of time before quantifying their DNA content. We assess the performance of our method empirically and discuss adaptations that can be implemented using this technique.

 

Cell Division 2018 13 : 6

https://doi.org/10.1186/s13008-018-0039-z

Open Access : https://celldiv.biomedcentral.com/track/pdf/10.1186/s13008-018-0039-z

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A deep learning-based algorithm for 2-D cell segmentation in microscopy images

A deep learning-based algorithm for 2-D cell segmentation in microscopy images | News Imagerie cellulaire - Cellular imaging | Scoop.it

Yousef Al-Kofahi, Alla Zaltsman, Robert Graves, Will Marshall and

Mirabela Rusu

 

Automatic and reliable characterization of cells in cell cultures is key to several applications such as cancer research and drug discovery. Given the recent advances in light microscopy and the need for accurate and high-throughput analysis of cells, automated algorithms have been developed for segmenting and analyzing the cells in microscopy images. Nevertheless, accurate, generic and robust whole-cell segmentation is still a persisting need to precisely quantify its morphological properties, phenotypes and sub-cellular dynamics.
We present a single-channel whole cell segmentation algorithm. We use markers that stain the whole cell, but with less staining in the nucleus, and without using a separate nuclear stain. We show the utility of our approach in microscopy images of cell cultures in a wide variety of conditions. Our algorithm uses a deep learning approach to learn and predict locations of the cells and their nuclei, and combines that with thresholding and watershed-based segmentation. We trained and validated our approach using different sets of images, containing cells stained with various markers and imaged at different magnifications. Our approach achieved a 86% similarity to ground truth segmentation when identifying and separating cells.
The proposed algorithm is able to automatically segment cells from single channel images using a variety of markers and magnifications.

 

BMC Bioinformatics 2018 19 : 365

https://doi.org/10.1186/s12859-018-2375-z

Open Access : https://bmcbioinformatics.biomedcentral.com/track/pdf/10.1186/s12859-018-2375-z

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Cryo-laser scanning confocal microscopy of diffusible plant compounds

Cryo-laser scanning confocal microscopy of diffusible plant compounds | News Imagerie cellulaire - Cellular imaging | Scoop.it

Kevin Vidot, Cédric Gaillard, Camille Rivard, René Siret and Marc Lahaye

 

The in vivo observation of diffusible components, such as ions and small phenolic compounds, remains a challenge in turgid plant organs. The analytical techniques used to localize such components in water-rich tissue with a large field of view are lacking. It remains an issue to limit compound diffusion during sample preparation and observation processes.
An experimental setup involving the infusion staining of plant tissue and the cryo-fixation and cryo-sectioning of tissue samples followed by fluorescence cryo-observation by laser scanning confocal microscopy (LSCM) was developed. This setup was successfully applied to investigate the structure of the apple fruit cortex and table grape berry and was shown to be relevant for localizing calcium, potassium and flavonoid compounds.
The cryo-approach was well adapted and opens new opportunities for imaging other diffusible components in hydrated tissues.

 

Plant Methods (2018) 14:89

https://doi.org/10.1186/s13007-018-0356-x

Open Access : https://plantmethods.biomedcentral.com/track/pdf/10.1186/s13007-018-0356-x

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Real-time visualization of oxidative stress-mediated neurodegeneration of individual spinal motor neurons in vivo

Real-time visualization of oxidative stress-mediated neurodegeneration of individual spinal motor neurons in vivo | News Imagerie cellulaire - Cellular imaging | Scoop.it

Isabel  Formella, Adam J. Svahn, Rowan A.W. Radford, Emily K. Don, Nicholas J. Cole, Alison Hogan, Albert Lee, Roger S. Chung,  

Marco Morsch

 

Generation of reactive oxygen species (ROS) has been shown to be important for many physiological processes, ranging from cell differentiation to apoptosis. With the development of the genetically encoded photosensitiser KillerRed (KR) it is now possible to efficiently produce ROS dose-dependently in a specific cell type upon green light illumination. Zebrafish are the ideal vertebrate animal model for these optogenetic methods because of their transparency and efficient transgenesis. Here we describe a zebrafish model that expresses membrane-targeted KR selectively in motor neurons. We show that KR-activated neurons in the spinal cord undergo stress and cell death after induction of ROS. Using single-cell resolution and time-lapse confocal imaging, we selectively induced neurodegeneration in KR-expressing neurons leading to characteristic signs of apoptosis and cell death. We furthermore illustrate a targeted microglia response to the induction site as part of a physiological response within the zebrafish spinal cord. Our data demonstrate the successful implementation of KR mediated ROS toxicity in motor neurons in vivo and has important implications for studying the effects of ROS in a variety of conditions within the central nervous system, including aging and age-related neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis.

 

Redox Biology Vol. 19, October 2018, pp 226-234

https://doi.org/10.1016/j.redox.2018.08.011

Open Access : https://www.sciencedirect.com/science/article/pii/S2213231718305974/pdfft?md5=f52d8c09a6f3b3d055aac53547f4321e&pid=1-s2.0-S2213231718305974-main.pdf

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Extended-resolution imaging of the interaction of lipid droplets and mitochondria

Extended-resolution imaging of the interaction of lipid droplets and mitochondria | News Imagerie cellulaire - Cellular imaging | Scoop.it
Maria Pribasnig, Benedikt Kien, Lisa Pusch, Guenter Haemmerle, Robert Zimmermann, Heimo Wolinski
 

Physical contacts between organelles play a pivotal role in intracellular trafficking of metabolites. Monitoring organelle interactions in living cells using fluorescence microscopy is a powerful approach to functionally assess these cellular processes. However, detailed target acquisition is typically limited due to light diffraction. Furthermore, subcellular compartments such as lipid droplets and mitochondria are highly dynamic and show significant subcellular movement. Thus, high-speed acquisition of these organelles with extended-resolution is appreciated. Here, we present an imaging informatics pipeline enabling spatial and time-resolved analysis of the dynamics and interactions of fluorescently labeled lipid droplets and mitochondria in a fibroblast cell line. The imaging concept is based on multispectral confocal laser scanning microscopy and includes high-speed resonant scanning for fast spatial acquisition of organelles. Extended-resolution is achieved by the recording of images at minimized pinhole size and by post-processing of generated data using a computational image restoration method. Computation of inter-organelle contacts is performed on basis of segmented spatial image data. We show limitations of the image restoration and segmentation part of the imaging informatics pipeline. Since both image processing methods are implemented in other related methodologies, our findings will help to identify artifacts and the false-interpretation of obtained morphometric data. As a proof-of-principle, we studied how lipid load and overexpression of PLIN5, considered to be involved in the tethering of LDs and mitochondria, affects organelle association.

 
BBA - Molecular and Cell Biology of Lipids 1863 (2018) 1285–129

https://doi.org/10.1016/j.bbalip.2018.07.008

Open Access : https://www.sciencedirect.com/science/article/pii/S1388198118301768/pdfft?md5=e3dfc2041b33c4622b4ef76e58796a14&pid=1-s2.0-S1388198118301768-main.pdf

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Revolutionary microscopy technique nets most lucrative prize in science

Revolutionary microscopy technique nets most lucrative prize in science | News Imagerie cellulaire - Cellular imaging | Scoop.it

A picture might be worth a thousand words — but inventing a way to take nanoscale pictures is worth US$3-million. The inventor of a ‘super-resolution’ microscopy technique that biologists are using to reveal the hidden molecular structures of cells is one of six big winners of this year’s Breakthrough Prizes — the most lucrative awards in science and mathematics. The winners were announced on 17 October.
The microscopy method’s lead inventor, Xiaowei Zhuang, is a biophysicist at Harvard University in Cambridge, Massachusetts, and an investigator at the Howard Hughes Medical Institute (HHMI) in Chevy Chase, Maryland. She was awarded one of four prizes in the life sciences for developing stochastic optical-reconstruction microscopy — known as STORM1 — just over a decade ago. The technique was one of the first to break a fundamental resolution limit of conventional light miscroscopy and is now used widely in the biology community.

 

Nature News, 17 October 2018

doi: 10.1038/d41586-018-07079-5

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Label free 3D analysis of organelles in living cells by refractive index shows pre-mitotic organelle spinning in mammalian stem cells

Label free 3D analysis of organelles in living cells by refractive index shows pre-mitotic organelle spinning in mammalian stem cells | News Imagerie cellulaire - Cellular imaging | Scoop.it

Patrick A Sandoz, Christopher Tremblay, Sebastien Equis, Sorin Pop, Lisa Pollaro, Yann Cotte, Gisou F van der Goot, Mathieu Frechin

 

Holo-tomographic microscopy (HTM) is a label-free non-phototoxic microscopy method reporting the fine changes of a cell's refractive indexes (RI) in 3D. By combining HTM with epifluorescence, we demonstrate that cellular organelles such as Lipid droplets and mitochondria show a specific RI signature that distinguishes them with high resolution and contrast. We further show that HTM allows to follow in unprecedented ways the dynamics of mitochondria, lipid droplets as well as that of endocytic structures in live cells over long period of time, which led us to observe to our knowledge for the first time a global organelle spinning occurring before mitosis.

 

bioRxiv preprint first posted online Sep. 4, 2018, 407239

https://doi.org/10.1101/407239

https://www.biorxiv.org/content/early/2018/09/04/407239

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Phototoxicity revisited

Editorial

 

As microscopy methods for studying biology in living samples advance and demand for them grows, assessment of light damage caused by imaging becomes increasingly important.

 

Nature Methods volume 15, page751 (2018)

https://doi.org/10.1038/s41592-018-0170-4

Open Access : https://www.nature.com/articles/s41592-018-0170-4.pdf

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The 2018 correlative microscopy techniques roadmap.

The 2018 correlative microscopy techniques roadmap. | News Imagerie cellulaire - Cellular imaging | Scoop.it

Toshio Ando, Satya Prathyusha Bhamidimarri, Niklas Brending, H Colin-York, Lucy Collinson, Niels De Jonge, P J de Pablo, Elke Debroye, Christian Eggeling, Christian Franck, Marco Fritzsche, Hans Gerritsen, Ben N G Giepmans, Kay Grunewald, Johan Hofkens, Jacob P Hoogenboom, Kris P F Janssen, Rainer Kaufman, Judith Klumpermann, Nyoman Kurniawan, Jana Kusch, Nalan Liv, Viha Parekh, Diana B Peckys, Florian Rehfeldt, David C Reutens, Maarten B J Roeffaers, Tim Salditt, Iwan A T Schaap, Ulrich S Schwarz, Paul Verkade, Michael W Vogel, Richard Wagner, Mathias Winterhalter, Haifeng Yuan and Giovanni Zifarelli

 

Developments in microscopy have been instrumental to progress in the life sciences, and many new techniques have been introduced and led to new discoveries throughout the last century. A wide and diverse range of methodologies is now available, including electron microscopy, atomic force microscopy, magnetic resonance imaging, small-angle x-ray scattering and multiple super-resolution fluorescence techniques, and each of these methods provides valuable read-outs to meet the demands set by the samples under study. Yet, the investigation of cell development requires a multi-parametric approach to address both the structure and spatio-temporal organization of organelles, and also the transduction of chemical signals and forces involved in cell–cell interactions. Although the microscopy technologies for observing each of these characteristics are well developed, none of them can offer read-out of all characteristics simultaneously, which limits the information content of a measurement. For example, while electron microscopy is able to disclose the structural layout of cells and the macromolecular arrangement of proteins, it cannot directly follow dynamics in living cells. The latter can be achieved with fluorescence microscopy which, however, requires labelling and lacks spatial resolution. A remedy is to combine and correlate different readouts from the same specimen, which opens new avenues to understand structure–function relations in biomedical research. At the same time, such correlative approaches pose new challenges concerning sample preparation, instrument stability, region of interest retrieval, and data analysis. Because the field of correlative microscopy is relatively young, the capabilities of the various approaches have yet to be fully explored, and uncertainties remain when considering the best choice of strategy and workflow for the correlative experiment. With this in mind, the Journal of Physics D: Applied Physics presents a special roadmap on the correlative microscopy techniques, giving a comprehensive overview from various leading scientists in this field, via a collection of multiple short viewpoints.

 

Journal of Physics D: Applied Physics, Volume 51, Number 44, 443001

https://doi.org/10.1088/1361-6463/aad055

Open Access : http://iopscience.iop.org/article/10.1088/1361-6463/aad055/pdf

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Wide-field multiphoton imaging through scattering media without correction

Wide-field multiphoton imaging through scattering media without correction | News Imagerie cellulaire - Cellular imaging | Scoop.it

Adrià Escobet-Montalbán, Roman Spesyvtsev, Mingzhou Chen, Wardiya Afshar Saber, Melissa Andrews, C. Simon Herrington, Michael Mazilu and Kishan Dholakia

 

Optical approaches to fluorescent, spectroscopic, and morphological imaging have made exceptional advances in the last decade. Super-resolution imaging and wide-field multiphoton imaging are now underpinning major advances across the biomedical sciences. While the advances have been startling, the key unmet challenge to date in all forms of optical imaging is to penetrate deeper. A number of schemes implement aberration correction or the use of complex photonics to address this need. In contrast, we approach this challenge by implementing a scheme that requires no a priori information about the medium nor its properties. Exploiting temporal focusing and single-pixel detection in our innovative scheme, we obtain wide-field two-photon images through various turbid media including a scattering phantom and tissue reaching a depth of up to seven scattering mean free path lengths. Our results show that it competes favorably with standard point-scanning two-photon imaging, with up to a fivefold improvement in signal-to-background ratio while showing significantly lower photobleaching.

 

Science Advances, 12 Oct 2018 : Vol. 4, no. 10, eaau1338
DOI: 10.1126/sciadv.aau1338

Open Access : http://advances.sciencemag.org/content/advances/4/10/eaau1338.full.pdf

 

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Invited Article: Label-free nerve imaging with a coherent anti-Stokes Raman scattering rigid endoscope using two optical fibers for laser delivery.

Invited Article: Label-free nerve imaging with a coherent anti-Stokes Raman scattering rigid endoscope using two optical fibers for laser delivery. | News Imagerie cellulaire - Cellular imaging | Scoop.it

Keigo Hirose, Shuichiro Fukushima, Taichi Furukawa, Hirohiko Niioka and Mamoru Hashimoto

 

A coherent anti-Stokes Raman scattering (CARS) rigid endoscope using two optical fibers to deliver excitation beams individually is developed. The use of two optical fibers allows the correction of longitudinal chromatic aberration and enhances the CARS signal by a factor of 2.59. The endoscope is used to image rat sciatic nerves with an imaging time of 10 s. Imaging of the rabbit prostatic fascia without sample slicing is also demonstrated, which reveals the potential for the application of the CARS endoscope to robot-assisted surgery.
 

APL Photonics, Volume 3, Issue 9 >10.1063/1.5031817

https://doi.org/10.1063/1.5031817

http://aip.scitation.org/doi/pdf/10.1063/1.5031817?class=pdf

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Visualizing protein synthesis in mice with in vivo labeling of deuterated amino acids using vibrational imaging

Visualizing protein synthesis in mice with in vivo labeling of deuterated amino acids using vibrational imaging | News Imagerie cellulaire - Cellular imaging | Scoop.it

Lingyan Shi, Yihui Shen and Wei Min

 

Proteins are one of the major components of biological systems, and understanding their metabolism is critical to study various biochemical processes in living systems. Despite extensive efforts to study protein metabolism such as autoradiography, mass spectrometry, and fluorescence microscopy, visualizing the spatial distribution of overall protein metabolism in mammals at subcellular resolution is still challenging. A recent study from our group reported imaging newly synthesized proteins in cultured mammalian cells, tissues, or even in mice using stimulated Raman scattering (SRS) microscopy coupled with metabolic labeling of deuterated amino acids (dAA). However, our previous method of dAA administration via drinking water, albeit convenient, is insufficient for in vivo studies. This is due to poor labeling efficiency and limited access to many important organs such as the brain, pancreas, or tumor. In this study, we have significantly improved and optimized the in vivo administration method by intra-carotid arterial injection of dAA in mice and obtained imaging contrast of protein metabolic activity in many more organs and tissues, such as cerebral and cerebellar cortex and hippocampal regions in the mouse brain. We also imaged newly formed proteins in the choroid plexus and pancreas at different time points, illustrating the metabolic dynamics of proteins in these important secretory organs. In addition, we visualized the metabolic heterogeneity of protein synthesis in colon tumor xenografts, which can be used to distinguish tumor and normal tissues. In summary, this combination of a new dAA administration technique and SRS imaging platform demonstrates an effective tool for the in vivo study of complex protein metabolism in mammals, in both physiological and pathological states.
 

APL Photonic, Volume 3, Issue 9 >10.1063/1.5028134

https://doi.org/10.1063/1.5028134

Open Access : http://aip.scitation.org/doi/pdf/10.1063/1.5028134?class=pdf

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