15 results on '"Nickels A. Jensen"'
Search Results
2. Rational control of off‐state heterogeneity in a photoswitchable fluorescent protein provides switching contrast enhancement
- Author
-
Angela Mantovanelli, A. Gorel, Dominique Bourgeois, M. Hilpert, Gabriela Nass Kovacs, Mark S. Hunter, Sébastien Boutet, Ninon Zala, Kiyoshi Ueda, Jason E. Koglin, Andrew Aquila, Michel Sliwa, Ilme Schlichting, M. Stricker, Stefan Jakobs, Kensuke Tono, Martin Weik, Virgile Adam, Anne-Sophie Banneville, Lucas Martinez Uriarte, T. Domratcheva, Michel Thépaut, Mengning Liang, Oleksandr Glushonkov, Martin Byrdin, Giorgio Schirò, Marie Luise Grünbein, Robert L. Shoeman, Kyprianos Hadjidemetriou, Thomas R. M. Barends, Sergio Carbajo, Nina Eleni Christou, Thomas J. Lane, Victor Bezchastnov, Daehyun You, Tadeo Moreno Chicano, Lutz Foucar, Marco Cammarata, Nickels A. Jensen, C.M. Roome, Jacques-Philippe Colletier, Marco Kloos, Franck Fieschi, Eugenio de la Mora, Mariam El Khatib, Nicolas Coquelle, Shigeki Owada, Matthew Seaberg, R. Bruce Doak, Karol Nass, Joyce Woodhouse, Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Department of NanoBiophotonics [Göttingen], Max Planck Institute for Biophysical Chemistry (MPI-BPC), Max-Planck-Gesellschaft-Max-Planck-Gesellschaft, Max Planck Institute for Medical Research [Heidelberg], Max-Planck-Gesellschaft, Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory (SLAC), Stanford University-Stanford University, Max-Planck-Institut für Medizinische Forschung, Institut de Physique de Rennes (IPR), Université de Rennes (UR)-Centre National de la Recherche Scientifique (CNRS), RIKEN SPring-8 Center [Hyogo] (RIKEN RSC), RIKEN - Institute of Physical and Chemical Research [Japon] (RIKEN), Japan Synchrotron Radiation Research Institute [Hyogo] (JASRI), Tohoku University [Sendai], Laboratoire Avancé de Spectroscopie pour les Intéractions la Réactivité et l'Environnement - UMR 8516 (LASIRE), Institut de Chimie du CNRS (INC)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Department of Chemistry, Lomonosov Moscow State University, Lomonosov Moscow State University (MSU), The XFEL experiments were carried out at BL2-EH3 of SACLA with the approval of the Japan Synchrotron Radiation Research Institute (JASRI, Proposal No. 2018 A8026, 27–29 July 2018) and at the CXI beamline at the LCLS (Proposal No. LM47 (23–27 June 2016) and LR38 (22–26 February 2018). We warmly thank the SACLA and LCLS staff for assistance. Use of the LCLS, SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract no. DE-AC02-76SF00515. Part of the sample injector used at LCLS for this research was funded by the National Institutes of Health, P41 GM103393, formerly P41RR001209. We acknowledge support from the Max Planck Society. The study was supported by travel grants from the CNRS (GoToXFEL) to MW, an ANR grant to MW, MC, MSl (BioXFEL), a PhD fellowship from Lille University to LMU and an MENESR – Univ. Grenoble Alpes fellowship to KH. This work was partially carried out at the platforms of the Grenoble Instruct-ERIC center (IBS and ISBG, UMS 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB)., ANR-17-CE11-0047,Cryo-PALM,Microscopie super-résolution par localisation de molécules uniques à température cryogénique.(2017), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Centre National de la Recherche Scientifique (CNRS), Laboratoire Physico-Chimie Curie [Institut Curie] (PCC), Institut Curie [Paris]-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), and Publica
- Subjects
serial femtosecond crystallography ,[SDV]Life Sciences [q-bio] ,RESOLFT ,Green Fluorescent Proteins ,nanoscopy ,Atomic ,quantum chemistry ,Particle and Plasma Physics ,Theoretical and Computational Chemistry ,Microscopy ,Side chain ,switching contrast ,Escherichia coli ,Nuclear ,Physical and Theoretical Chemistry ,[PHYS]Physics [physics] ,Chemical Physics ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,Chemistry ,photoswitchable fluorescent proteins ,Resolution (electron density) ,Molecular ,Chromophore ,Fluorescence ,Atomic and Molecular Physics, and Optics ,Luminescent Proteins ,Femtosecond ,Biophysics ,Generic health relevance ,Biological imaging ,Physical Chemistry (incl. Structural) - Abstract
Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance and novel applications. Here we establish a link between photoswitching contrast, a key parameter that largely dictates the achievable resolution in nanoscopy applications, and chromophore conformation in the non-fluorescent state of rsEGFP2, a widely employed label in REversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy. Upon illumination, the cis chromophore of rsEGFP2 isomerizes to two distinct off-state conformations, trans1 and trans2, located on either side of the V151 side chain. Reducing or enlarging the side chain at this position (V151A and V151L variants) leads to single off-state conformations that exhibit higher and lower switching contrast, respectively, compared to the rsEGFP2 parent. The combination of structural information obtained by serial femtosecond crystallography with high-level quantum chemical calculations and with spectroscopic and photophysical data determined in vitro suggests that the changes in switching contrast arise from blue- and red-shifts of the absorption bands associated to trans1 and trans2, respectively. Thus, due to elimination of trans2, the V151A variants of rsEGFP2 and its superfolding variant rsFolder2 display a more than two-fold higher switching contrast than their respective parent proteins, both in vitro and in E. coli cells. The application of the rsFolder2-V151A variant is demonstrated in RESOLFT nanoscopy. Our study rationalizes the connection between structural and photophysical chromophore properties and suggests a means to rationally improve fluorescent proteins for nanoscopy applications.
- Published
- 2022
3. The Positive Switching Fluorescent Protein Padron2 Enables Live-Cell Reversible Saturable Optical Linear Fluorescence Transitions (RESOLFT) Nanoscopy without Sequential Illumination Steps
- Author
-
Timo Konen, Isabelle Jansen, Mariano L. Bossi, Daniel Stumpf, Stefan W. Hell, Tim Grotjohann, Stefan Jakobs, Nickels A. Jensen, and Michael Weber
- Subjects
Materials science ,Green Fluorescent Proteins ,RESOLFT ,General Physics and Astronomy ,02 engineering and technology ,010402 general chemistry ,01 natural sciences ,Article ,super-resolution microscopy ,fluorescent protein ,Fluorescent protein ,General Materials Science ,Sequential switching ,Lighting ,Light response ,switching ,business.industry ,Super-resolution microscopy ,Padron ,General Engineering ,021001 nanoscience & nanotechnology ,Fluorescence ,0104 chemical sciences ,Luminescent Proteins ,Wavelength ,Microscopy, Fluorescence ,Optoelectronics ,Contrast ratio ,live cell ,0210 nano-technology ,business - Abstract
Reversibly switchable fluorescent proteins (RSFPs) can be repeatedly transferred between a fluorescent on- and a nonfluorescent off-state by illumination with light of different wavelengths. Negative switching RSFPs are switched from the on- to the off-state with the same wavelength that also excites fluorescence. Positive switching RSFPs have a reversed light response, where the fluorescence excitation wavelength induces the transition from the off- to the on-state. Reversible saturable optical linear (fluorescence) transitions (RESOLFT) nanoscopy utilizes these switching states to achieve diffraction-unlimited resolution but so far has primarily relied on negative switching RSFPs by using time sequential switching schemes. On the basis of the green fluorescent RSFP Padron, we engineered the positive switching RSFP Padron2. Compared to its predecessor, it can undergo 50-fold more switching cycles while displaying a contrast ratio between the on- and the off-states of more than 100:1. Because of its robust switching behavior, Padron2 supports a RESOLFT imaging scheme that entirely refrains from sequential switching as it only requires beam scanning of two spatially overlaid light distributions. Using Padron2, we demonstrate live-cell RESOLFT nanoscopy without sequential illumination steps.
- Published
- 2021
4. The Positive Switching RSFP Padron2 Enables Live-Cell RESOLFT Nanoscopy Without Sequential Irradiation Steps
- Author
-
Stefan W. Hell, Nickels A. Jensen, Isabelle Jansen, Timo Konen, Tim Grotjohann, and Stefan Jakobs
- Subjects
0303 health sciences ,Light response ,Excitation wavelength ,Materials science ,business.industry ,RESOLFT ,02 engineering and technology ,021001 nanoscience & nanotechnology ,Fluorescence ,03 medical and health sciences ,Wavelength ,Optoelectronics ,Contrast ratio ,Irradiation ,0210 nano-technology ,business ,Sequential switching ,030304 developmental biology - Abstract
Reversibly switchable fluorescent proteins (RSFPs) can be repeatedly transferred between a fluorescent on- and a non-fluorescent off-state in response to irradiation with light of different wavelengths. Negative switching RSFPs are switched from the on- to the off-state with the same wavelength which also excites fluorescence. Positive switching RSFPs have a reversed light response where the fluorescence excitation wavelength induces the transition from the off- to the on-state. Reversible saturable optical linear (fluorescence) transitions (RESOLFT) nanoscopy utilizes these switching states to achieve diffraction-unlimited resolution, but so far has primarily relied on negative switching RSFPs by using time sequential switching schemes.Based on the green fluorescent RSFP Padron, we engineered the positive switching RSFP Padron2. Compared to its predecessor, it can undergo 50-fold more switching cycles while displaying a contrast ratio between the on- and the off-state of more than 100:1. Because of its robust switching behavior, Padron2 supports a RESOLFT imaging scheme that entirely refrains from sequential switching as it only requires beam scanning of two spatially overlaid light distributions. Using Padron2, we demonstrate live-cell RESOLFT nanoscopy without sequential irradiation steps.
- Published
- 2020
5. Live‐cell RESOLFT nanoscopy of transgenic Arabidopsis thaliana
- Author
-
Sebastian Schnorrenberg, Tim Grotjohann, Thomas Teichmann, Nickels A. Jensen, Lars Frahm, Hassan Ghareeb, Stefan W. Hell, Stefan Jakobs, and Volker Lipka
- Subjects
0106 biological sciences ,RESOLFT ,plant ,Plant Science ,01 natural sciences ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,03 medical and health sciences ,Microtubule ,Live cell imaging ,Arabidopsis ,Fluorescence microscope ,fluorescence lifetime gating ,Ecology, Evolution, Behavior and Systematics ,Original Research ,030304 developmental biology ,0303 health sciences ,rsEGFP ,Ecology ,biology ,Super-resolution microscopy ,Chemistry ,Botany ,food and beverages ,super‐resolution microscopy ,biology.organism_classification ,Fluorescence ,live cell imaging ,Autofluorescence ,QK1-989 ,Biophysics ,010606 plant biology & botany - Abstract
Subdiffraction super‐resolution fluorescence microscopy, or nanoscopy, has seen remarkable developments in the last two decades. Yet, for the visualization of plant cells, nanoscopy is still rarely used. In this study, we established RESOLFT nanoscopy on living green plant tissue. Live‐cell RESOLFT nanoscopy requires and utilizes comparatively low light doses and intensities to overcome the diffraction barrier. We generated a transgenic Arabidopsis thaliana plant line expressing the reversibly switchable fluorescent protein rsEGFP2 fused to the mammalian microtubule‐associated protein 4 (MAP4) in order to ubiquitously label the microtubule cytoskeleton. We demonstrate the use of RESOLFT nanoscopy for extended time‐lapse imaging of cortical microtubules in Arabidopsis leaf discs. By combining our approach with fluorescence lifetime gating, we were able to acquire live‐cell RESOLFT images even close to chloroplasts, which exhibit very strong autofluorescence. The data demonstrate the feasibility of subdiffraction resolution imaging in transgenic plant material with minimal requirements for sample preparation.
- Published
- 2020
6. Reversibly Switchable Fluorescent Proteins for RESOLFT Nanoscopy
- Author
-
Nickels A. Jensen, Isabelle Jansen, Stefan Jakobs, and Maria Kamper
- Subjects
0303 health sciences ,03 medical and health sciences ,Photochromism ,Materials science ,Microscopy ,RESOLFT ,Biophysics ,02 engineering and technology ,021001 nanoscience & nanotechnology ,0210 nano-technology ,Fluorescence ,030304 developmental biology ,Green fluorescent protein - Abstract
Diffraction-limited lens-based optical microscopy fails to discern fluorescent features closer than $$\sim $$ ∼ 200 nm. All super-resolution microscopy (nanoscopy) approaches that fundamentally overcome the diffraction barrier rely on fluorophores that can adopt different states, typically a fluorescent ‘on-’state and a dark, non-fluorescent ‘off-’state. In reversible saturable optical linear fluorescence transitions (RESOLFT) nanoscopy, light is applied to induce transitions between two states and to switch fluorophores on and off at defined spatial coordinates. RESOLFT nanoscopy relies on metastable reversibly switchable fluorophores. Thereby, it is particularly suited for live-cell imaging, because it requires relatively low light levels to overcome the diffraction barrier. Most implementations of RESOLFT nanoscopy utilize reversibly photoswitchable fluorescent proteins (RSFPs), which are derivatives of proteins from the green fluorescent protein (GFP) family. In recent years, analysis of the molecular mechanisms of the switching processes have paved the way to a rational design of new RSFPs with superior characteristics for super-resolution microscopy. In this chapter, we focus on the newly developed RSFPs, the light-driven switching mechanisms and the use of RSFPs for RESOLFT nanoscopy.
- Published
- 2020
7. Near-infrared STED nanoscopy with an engineered bacterial phytochrome
- Author
-
Stefan Jakobs, Maria Kamper, Stefan W. Hell, Haisen Ta, and Nickels A. Jensen
- Subjects
0301 basic medicine ,STED ,bacterial phytochrome ,Materials science ,Infrared ,Cell Survival ,Infrared Rays ,Science ,Recombinant Fusion Proteins ,General Physics and Astronomy ,02 engineering and technology ,Protein Engineering ,General Biochemistry, Genetics and Molecular Biology ,Article ,Green fluorescent protein ,03 medical and health sciences ,Bacterial Proteins ,Microscopy ,Fluorescence microscope ,Humans ,Nanotechnology ,Absorption (electromagnetic radiation) ,lcsh:Science ,Multidisciplinary ,Phytochrome ,Near-infrared spectroscopy ,STED microscopy ,technology, industry, and agriculture ,General Chemistry ,021001 nanoscience & nanotechnology ,3. Good health ,Luminescent Proteins ,030104 developmental biology ,Microscopy, Fluorescence ,Biophysics ,lcsh:Q ,0210 nano-technology ,HeLa Cells - Abstract
The near infrared (NIR) optical window between the cutoff for hemoglobin absorption at 650 nm and the onset of increased water absorption at 900 nm is an attractive, yet largely unexplored, spectral regime for diffraction-unlimited super-resolution fluorescence microscopy (nanoscopy). We developed the NIR fluorescent protein SNIFP, a bright and photostable bacteriophytochrome, and demonstrate its use as a fusion tag in live-cell microscopy and STED nanoscopy. We further demonstrate dual color red-confocal/NIR-STED imaging by co-expressing SNIFP with a conventional red fluorescent protein., Super-resolution microscopy using wavelengths in the near infrared (NIR) optical window is particularly appealing for live cell and tissue imaging, yet largely unexplored. Here the authors present NIR-STED nanoscopy of living mammalian cells using the new bacteriophytochrome-based fluorescent protein SNIFP.
- Published
- 2018
8. Reversibel schaltbare fluoreszierende Proteine für die Superauflösung
- Author
-
Martin Andresen, Stefan Jakobs, and Nickels A. Jensen
- Subjects
0301 basic medicine ,03 medical and health sciences ,030104 developmental biology ,Chemistry ,Pharmacology toxicology ,RESOLFT ,Microscopy ,Biophysics ,Molecular Biology ,Fluorescence ,Biotechnology ,Cell biology - Abstract
Super-resolution microscopy, or nanoscopy, enables the visualization of cellular structures inaccessible to conventional light microscopy. RESOLFT nanoscopy is especially suitable for the imaging of living cells. It requires fluorescent proteins (RSFPs) that can be repeatedly switched between a fluorescent and a non-fluorescent state by light. We have analyzed the molecular switching mechanisms and generated a family of RSFPs specifically tailored for live cell RESOLFT nanoscopy.
- Published
- 2016
9. Quantification of cell infection caused by Listeria monocytogenes invasion
- Author
-
Muhammad Arif, Trinad Chakraborty, Tim Wilhelm Nattkemper, Ulrike Technow, Nicole Fisch, Karsten Niehaus, Nickels A. Jensen, and Nasir M. Rajpoot
- Subjects
medicine.drug_class ,Bioimage informatics ,Cytological Techniques ,Green Fluorescent Proteins ,Antibiotics ,Bioengineering ,medicine.disease_cause ,Applied Microbiology and Biotechnology ,Cell Line ,Microbiology ,Mice ,Immune system ,Listeria monocytogenes ,Pattern recognition ,Image Processing, Computer-Assisted ,medicine ,Animals ,Listeriosis ,Pathogen ,Cell Nucleus ,High content screen ,biology ,Macrophages ,Intracellular parasite ,General Medicine ,biology.organism_classification ,Bacterial Load ,Microscopy, Fluorescence ,Cell culture ,Listeria ,Cell segmentation ,Bacterial infection ,Algorithms ,Software ,Bacteria ,Biotechnology - Abstract
Listeria monocytogenes causes a life-threatening food-borne disease known as Listeriosis. Elderly,immunocompromised, and pregnant women are primarily the victims of this facultative intracellular Gram-positive pathogen. Since the bacteria survive intracellularly within the human host cells they are protected against the immune system and poorly accessed by many antibiotics. In order to screen pharmaceutical substances for their ability to interfere with the infection, persistence and release of L. monocytogenes a high content as say is required. We established a high content screen (HCS) using the RAW 264.7 mouse macrophage cell line seeded into 96-well glass bottom microplates. Cells were infected with GFP-expressing L. monocytogenes and stained thereafter with Hoechst 33342.Automated image acquisition was carried out by the Scan(R) screening station. We have developed an algorithm that automatically grades cells in microscopy images of fluorescent-tagged Listeria for the severity of infection. The grading accuracy of this newly developed algorithm is 97.1% as compared to a 74.3%grading accuracy we obtained using the commercial Olympus Scan(R) software.
- Published
- 2011
10. Live-cell multiplane three-dimensional super-resolution optical fluctuation imaging
- Author
-
F. Gisou van der Goot, Marcel Leutenegger, Theo Lasser, Azat Sharipov, Elena A. Dubikovskaya, Stefan Geissbuehler, Stefan Jakobs, Anja Huss, Aurélien Godinat, Patrick A. Sandoz, Noelia L. Bocchio, Nickels A. Jensen, and Joerg Enderlein
- Subjects
Diffraction ,Microscope ,Green Fluorescent Proteins ,General Physics and Astronomy ,02 engineering and technology ,cumulant imaging ,Article ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,law.invention ,Myoblasts ,Mice ,03 medical and health sciences ,Imaging, Three-Dimensional ,Optics ,Sampling (signal processing) ,law ,Microscopy ,Animals ,Humans ,Vimentin ,Fluorescent protein ,fluorescence nanoscopy ,Fluorescent Dyes ,030304 developmental biology ,Physics ,0303 health sciences ,Multidisciplinary ,business.industry ,General Chemistry ,Carbocyanines ,021001 nanoscience & nanotechnology ,Photobleaching ,Superresolution ,Mitochondria ,Molecular Imaging ,Microscopy, Fluorescence ,Acquisition time ,0210 nano-technology ,business ,HeLa Cells - Abstract
Super-resolution optical fluctuation imaging (SOFI) provides an elegant way of overcoming the diffraction limit in all three spatial dimensions by computing higher-order cumulants of image sequences of blinking fluorophores acquired with a classical widefield microscope. Previously, three-dimensional (3D) SOFI has been demonstrated by sequential imaging of multiple depth positions. Here we introduce a multiplexed imaging scheme for the simultaneous acquisition of multiple focal planes. Using 3D cross-cumulants, we show that the depth sampling can be increased. The simultaneous acquisition of multiple focal planes significantly reduces the acquisition time and thus the photobleaching. We demonstrate multiplane 3D SOFI by imaging fluorescently labelled cells over an imaged volume of up to 65 × 65 × 3.5 μm3 without depth scanning. In particular, we image the 3D network of mitochondria in fixed C2C12 cells immunostained with Alexa 647 fluorophores and the 3D vimentin structure in living Hela cells expressing the fluorescent protein Dreiklang., Super-resolution optical fluctuation imaging provides 3D images of biological specimens via blinking fluorophores. Geissbuehler et al. present a multiplexed version of this method that captures images at multiple focal planes simultaneously, reducing the acquisition time compared with standard approaches.
- Published
- 2014
11. Coordinate-targeted and coordinate-stochastic super-resolution microscopy with the reversibly switchable fluorescent protein Dreiklang
- Author
-
Stefan Jakobs, Tanja Brakemann, Flavie Lavoie-Cardinal, Nickels A. Jensen, Stefan W. Hell, Johann G. Danzl, and Katrin I. Willig
- Subjects
Diffraction ,Stochastic Processes ,Super-resolution microscopy ,Chemistry ,business.industry ,Resolution (electron density) ,Chromophore ,Fluorescence ,Atomic and Molecular Physics, and Optics ,Luminescent Proteins ,Dark state ,Optics ,Microscopy, Fluorescence ,Molecule ,Humans ,Stimulated emission ,Physical and Theoretical Chemistry ,Biological system ,business ,HeLa Cells - Abstract
Diffraction-unlimited far-field super-resolution fluorescence (nanoscopy) methods typically rely on transiently transferring fluorophores between two states, whereby this transfer is usually laid out as a switch. However, depending on whether this is induced in a spatially controlled manner using a pattern of light (coordinate-targeted) or stochastically on a single-molecule basis, specific requirements on the fluorophores are imposed. Therefore, the fluorophores are usually utilized just for one class of methods only. In this study we demonstrate that the reversibly switchable fluorescent protein Dreiklang enables live-cell recordings in both spatially controlled and stochastic modes. We show that the Dreiklang chromophore entails three different light-induced switching mechanisms, namely a reversible photochemical one, off-switching by stimulated emission, and a reversible transfer to a long-lived dark state from the S1 state, all of which can be utilized to overcome the diffraction barrier. We also find that for the single-molecule-based stochastic GSDIM approach (ground-state depletion followed by individual molecule return), Dreiklang provides a larger number of on-off localization events as compared to its progenitor Citrine. Altogether, Dreiklang is a versatile probe for essentially all popular forms of live-cell fluorescence nanoscopy.
- Published
- 2013
12. Establishment of a high content assay for the identification and characterisation of bioactivities in crude bacterial extracts that interfere with the eukaryotic cell cycle
- Author
-
Nickels A. Jensen, Karsten Niehaus, Klaus Gerth, D. Kapp, Tim Grotjohann, and Matthias Keck
- Subjects
Bisbenzimide ,Cytochalasin D ,ScanR ,Paclitaxel ,Cell ,Bioengineering ,Complex Mixtures ,Applied Microbiology and Biotechnology ,Cell Line ,Microbiology ,chemistry.chemical_compound ,Myxobacteria ,Image Processing, Computer-Assisted ,medicine ,Animals ,Cytochalasin ,Sf9 ,Myxococcales ,Propidium iodide ,Coloring Agents ,Cell Proliferation ,Fluorescent Dyes ,Sorangium cellulosum ,Microscopy ,Ploidies ,biology ,Drug discovery ,Cell Cycle ,High content screening microscopy ,General Medicine ,Cell cycle ,Cytostatic Agents ,biology.organism_classification ,Pyrrolidinones ,medicine.anatomical_structure ,Biochemistry ,chemistry ,Cell culture ,cells ,Macrolides ,Colchicine ,Biotechnology - Abstract
High content microscopy as a screening tool to identify bioactive agents has provided researchers with the ability to characterise biological activities at the level of single cells. Here. we describe the development and the application of a high content screening assay for the identification and characterisation of cytostatic bioactivities from Myxobacteria extracts. In an automated microscopy assay Sf9 insect cells were visualised utilising the stains bisbenzimide Hoechst 33342, calcein AM, and propidium iodide. Imaging data were processed by the ScanR Analysis software to determine the ploidy and vitality of each cell and to quantify cell populations. More than 98% of the Sf9 cells were viable and the culture consisted of diploid (similar to 30%), tetraploid (similar to 60%), polyploidic(
- Published
- 2009
13. An incremental approach to automated protein localisation
- Author
-
Nickels A. Jensen, Franz Kummert, and Marko Tscherepanow
- Subjects
Proteomics ,Intracellular Space ,Protein localisation ,Spodoptera ,Biology ,lcsh:Computer applications to medicine. Medical informatics ,Biochemistry ,Cell Line ,Image (mathematics) ,Structural Biology ,Image Processing, Computer-Assisted ,Animals ,Set (psychology) ,Cluster analysis ,lcsh:QH301-705.5 ,Molecular Biology ,Organelles ,Artificial neural network ,business.industry ,Applied Mathematics ,Cell Membrane ,Supervised learning ,Computational Biology ,Proteins ,Pattern recognition ,Computer Science Applications ,Cell biology ,Identification (information) ,ComputingMethodologies_PATTERNRECOGNITION ,lcsh:Biology (General) ,Microscopy, Fluorescence ,Molecular Probes ,lcsh:R858-859.7 ,Artificial intelligence ,DNA microarray ,business ,Algorithms ,Research Article - Abstract
Background The subcellular localisation of proteins in intact living cells is an important means for gaining information about protein functions. Even dynamic processes can be captured, which can barely be predicted based on amino acid sequences. Besides increasing our knowledge about intracellular processes, this information facilitates the development of innovative therapies and new diagnostic methods. In order to perform such a localisation, the proteins under analysis are usually fused with a fluorescent protein. So, they can be observed by means of a fluorescence microscope and analysed. In recent years, several automated methods have been proposed for performing such analyses. Here, two different types of approaches can be distinguished: techniques which enable the recognition of a fixed set of protein locations and methods that identify new ones. To our knowledge, a combination of both approaches – i.e. a technique, which enables supervised learning using a known set of protein locations and is able to identify and incorporate new protein locations afterwards – has not been presented yet. Furthermore, associated problems, e.g. the recognition of cells to be analysed, have usually been neglected. Results We introduce a novel approach to automated protein localisation in living cells. In contrast to well-known techniques, the protein localisation technique presented in this article aims at combining the two types of approaches described above: After an automatic identification of unknown protein locations, a potential user is enabled to incorporate them into the pre-trained system. An incremental neural network allows the classification of a fixed set of protein location as well as the detection, clustering and incorporation of additional patterns that occur during an experiment. Here, the proposed technique achieves promising results with respect to both tasks. In addition, the protein localisation procedure has been adapted to an existing cell recognition approach. Therefore, it is especially well-suited for high-throughput investigations where user interactions have to be avoided. Conclusion We have shown that several aspects required for developing an automatic protein localisation technique – namely the recognition of cells, the classification of protein distribution patterns into a set of learnt protein locations, and the detection and learning of new locations – can be combined successfully. So, the proposed method constitutes a crucial step to render image-based protein localisation techniques amenable to large-scale experiments.
- Published
- 2008
14. Fuzzy Logic Based Segmentation of Microcalcification in Breast Using Digital Mammograms Considering Multiresolution
- Author
-
Nickels A. Jensen, Marko Tscherepanow, and Franz Kummert
- Subjects
Active contour model ,Microscope ,business.industry ,Fuzzy set ,ComputingMethodologies_IMAGEPROCESSINGANDCOMPUTERVISION ,Wavelet transform ,Image segmentation ,Biology ,law.invention ,Set (abstract data type) ,law ,Genetic algorithm ,Segmentation ,Computer vision ,Artificial intelligence ,business - Abstract
In order to localise tagged proteins in living cells, the surrounding cells must be recognised first. Based on previous work regarding cell recognition in bright-field images, we propose an approach to the automated recognition of unstained live Drosophila cells, which are of high biological relevance. In order to achieve this goal, the original methods were extended to enable the additional application of an alternative microscopy technique, since the exclusive usage of bright-field images does not allow for an accurate segmentation of the considered cells. In order to cope with the increased number of parameters to be set, a genetic algorithm is applied. Furthermore, the employed segmentation and classification techniques needed to be adapted to the new cell characteristics. Therefore, a modified active contour approach and an enhanced feature set, allowing for a more detailed description of the obtained segments, are introduced.
- Published
- 2007
15. Cover Picture: Two-Color RESOLFT Nanoscopy with Green and Red Fluorescent Photochromic Proteins (ChemPhysChem 4/2014)
- Author
-
Ilaria Testa, Jakob Bierwagen, Stefan Jakobs, Nickels A. Jensen, Andre C. Stiel, Volker Westphal, Flavie Lavoie-Cardinal, Stefan W. Hell, and Andriy Chmyrov
- Subjects
Photochromism ,Chemistry ,RESOLFT ,Nanotechnology ,Cover (algebra) ,Physical and Theoretical Chemistry ,Fluorescence ,Atomic and Molecular Physics, and Optics - Published
- 2014
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.