9 results on '"Reto Fiolka"'
Search Results
2. Light sheet fluorescence microscopy
- Author
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Frederic Strobl, Friedrich Preusser, Reto Fiolka, Bo-Jui Chang, Katie McDole, Stephan Preibisch, and Ernst H. K. Stelzer
- Subjects
Microscope ,Materials science ,Optical sectioning ,business.industry ,Resolution (electron density) ,General Medicine ,Laser ,Photobleaching ,General Biochemistry, Genetics and Molecular Biology ,law.invention ,Biological specimen ,Optics ,law ,Light sheet fluorescence microscopy ,business ,Penetration depth - Abstract
Light sheet fluorescence microscopy (LSFM) uses a thin sheet of light to excite only fluorophores within the focal volume. Light sheet microscopes (LSMs) have a true optical sectioning capability and, hence, provide axial resolution, restrict photobleaching and phototoxicity to a fraction of the sample and use cameras to record tens to thousands of images per second. LSMs are used for in-depth analyses of large, optically cleared samples and long-term three-dimensional (3D) observations of live biological specimens at high spatio-temporal resolution. The independently operated illumination and detection trains and the canonical implementations, selective/single plane illumination microscope (SPIM) and digital scanned laser microscope (DSLM), are the basis for many LSM designs. In this Primer, we discuss various applications of LSFM for imaging multicellular specimens, developing vertebrate and invertebrate embryos, brain and heart function, 3D cell culture models, single cells, tissue sections, plants, organismic interaction and entire cleared brains. Further, we describe the combination of LSFM with other imaging approaches to allow for super-resolution or increased penetration depth and the use of sophisticated spatio-temporal manipulations to allow for observations along multiple directions. Finally, we anticipate developments of the field in the near future. Light sheet fluorescence microscopy (LSFM) is a technique that uses a thin sheet of light for illumination, allowing optical sectioning of the sample. In this Primer, Stelzer et al. outline the fundamental concepts behind LSFM, discuss the different experimental set-ups for light sheet microscopes and detail steps for processing LSFM images. The Primer also describes the range of applications for this technique across the biological sciences and concludes by discussing advances for enhancing imaging depth and resolution.
- Published
- 2021
3. Robust and automated detection of subcellular morphological motifs in 3D microscopy images
- Author
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Andrew R. Jamieson, Erik S. Welf, Reto Fiolka, Gaudenz Danuser, Meghan Driscoll, Tadamoto Isogai, and Kevin M. Dean
- Subjects
Morphogenesis ,Cell morphology ,Biochemistry ,Article ,Machine Learning ,03 medical and health sciences ,Imaging, Three-Dimensional ,Cell Line, Tumor ,Computer Graphics ,Humans ,Bleb (cell biology) ,Cell Shape ,Molecular Biology ,030304 developmental biology ,Microscopy ,0303 health sciences ,Cell morphogenesis ,Chemistry ,Cell Biology ,Cell biology ,Lamellipodium ,Signal transduction ,Filopodia ,Intracellular ,Signal Transduction ,Subcellular Fractions ,Biotechnology - Abstract
Rapid developments in live-cell three-dimensional (3D) microscopy enable imaging of cell morphology and signaling with unprecedented detail. However, tools to systematically measure and visualize the intricate relationships between intracellular signaling, cytoskeletal organization and downstream cell morphological outputs do not exist. Here, we introduce u-shape3D, a computer graphics and machine-learning pipeline to probe molecular mechanisms underlying 3D cell morphogenesis and to test the intriguing possibility that morphogenesis itself affects intracellular signaling. We demonstrate a generic morphological motif detector that automatically finds lamellipodia, filopodia, blebs and other motifs. Combining motif detection with molecular localization, we measure the differential association of PIP2 and KrasV12 with blebs. Both signals associate with bleb edges, as expected for membrane-localized proteins, but only PIP2 is enhanced on blebs. This indicates that subcellular signaling processes are differentially modulated by local morphological motifs. Overall, our computational workflow enables the objective, 3D analysis of the coupling of cell shape and signaling.
- Published
- 2019
4. Nuclear positioning facilitates amoeboid migration along the path of least resistance
- Author
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Gaudenz Danuser, Jörg Renkawitz, Robert Hauschild, Aglaja Kopf, Jack Merrin, Michael Sixt, Ingrid de Vries, Julian Stopp, Meghan Driscoll, Reto Fiolka, and Erik S. Welf
- Subjects
Male ,Cellular polarity ,Biology ,Microtubules ,Article ,Cell Line ,Extracellular matrix ,03 medical and health sciences ,0302 clinical medicine ,Cell Movement ,Microtubule ,medicine ,Animals ,Humans ,Compartment (development) ,Cells, Cultured ,030304 developmental biology ,Cell Nucleus ,0303 health sciences ,Multidisciplinary ,Chemotaxis ,Cell Polarity ,Cell biology ,Mice, Inbred C57BL ,medicine.anatomical_structure ,Cell culture ,Cytoplasm ,Female ,Porosity ,Nucleus ,Microtubule-Organizing Center ,030217 neurology & neurosurgery - Abstract
During metazoan development, immune surveillance and cancer dissemination, cells migrate in complex three-dimensional microenvironments(1–3). These spaces are crowded by cells and extracellular matrix, generating mazes with differently sized gaps that are typically smaller than the diameter of the migrating cell(4,5). Most mesenchymal and epithelial cells and some, but not all, cancer cells actively generate their migratory path using pericellular tissue proteolysis(6). By contrast, amoeboid cells such as leukocytes use non-destructive strategies of locomotion(7), raising the question how these extremely fast cells navigate through dense tissues. Here we reveal that leukocytes sample their immediate vicinity for large pore sizes, and are thereby able to choose the path of least resistance. This allows them to circumnavigate local obstacles while effectively following global directional cues such as chemotactic gradients. Pore-size discrimination is facilitated by frontward positioning of the nucleus, which enables the cells to use their bulkiest compartment as a mechanical gauge. Once the nucleus and the closely associated microtubule organizing centre pass the largest pore, cytoplasmic protrusions still lingering in smaller pores are retracted. These retractions are coordinated by dynamic microtubules; when microtubules are disrupted, migrating cells lose coherence and frequently fragment into migratory cytoplasmic pieces. As nuclear positioning in front of the microtubule organizing centre is a typical feature of amoeboid migration, our findings link the fundamental organization of cellular polarity to the strategy of locomotion.
- Published
- 2019
5. Light-sheet microscopy at high resolution
- Author
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Reto Fiolka
- Subjects
Imaging, Three-Dimensional ,Optics ,Materials science ,Microscopy, Fluorescence ,business.industry ,Light sheet fluorescence microscopy ,Biomedical Engineering ,Molecular Medicine ,High resolution ,Bioengineering ,business ,Applied Microbiology and Biotechnology ,Biotechnology - Published
- 2021
6. A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo
- Author
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Michael Z. Lin, Younghee Oh, Ho Leung Ng, Michelle A. Baird, Douglas S. Kim, Niloufar Ataie, Mark A. Kay, Jun Chu, Reto Fiolka, Erik S. Welf, Benjamin Kim, Ryohei Yasuda, Alex Sens, Clement Tran Tang, Michael W. Davidson, Feijie Zhang, Michelle Hu, Kevin M. Dean, Tal Laviv, John J. Macklin, and Hod Dana
- Subjects
0301 basic medicine ,Fluorescence-lifetime imaging microscopy ,Biomedical Engineering ,Bioengineering ,02 engineering and technology ,Applied Microbiology and Biotechnology ,Article ,Green fluorescent protein ,03 medical and health sciences ,Microscopy ,Bioluminescence ,Bioluminescence imaging ,Photoactivated localization microscopy ,Lighting ,Fluorescent Dyes ,Staining and Labeling ,Chemistry ,021001 nanoscience & nanotechnology ,Molecular Imaging ,Luminescent Proteins ,Microscopy, Fluorescence, Multiphoton ,030104 developmental biology ,Light sheet fluorescence microscopy ,Luminescent Measurements ,Biophysics ,Molecular Medicine ,Molecular imaging ,0210 nano-technology ,Biotechnology - Abstract
Orange-red fluorescent proteins (FPs) are widely used in biomedical research for multiplexed epifluorescence microscopy with GFP-based probes, but their different excitation requirements make multiplexing with new advanced microscopy methods difficult. Separately, orange-red FPs are useful for deep-tissue imaging in mammals owing to the relative tissue transmissibility of orange-red light, but their dependence on illumination limits their sensitivity as reporters in deep tissues. Here we describe CyOFP1, a bright, engineered, orange-red FP that is excitable by cyan light. We show that CyOFP1 enables single-excitation multiplexed imaging with GFP-based probes in single-photon and two-photon microscopy, including time-lapse imaging in light-sheet systems. CyOFP1 also serves as an efficient acceptor for resonance energy transfer from the highly catalytic blue-emitting luciferase NanoLuc. An optimized fusion of CyOFP1 and NanoLuc, called Antares, functions as a highly sensitive bioluminescent reporter in vivo, producing substantially brighter signals from deep tissues than firefly luciferase and other bioluminescent proteins.
- Published
- 2016
7. Resolution upgrades for light-sheet microscopy
- Author
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Reto Fiolka
- Subjects
0303 health sciences ,Materials science ,Microscope ,Light ,business.industry ,Optical Imaging ,Resolution (electron density) ,Cell Biology ,Biochemistry ,Fluorescence ,law.invention ,03 medical and health sciences ,Single objective ,Optics ,Microscopy, Fluorescence ,law ,Light sheet fluorescence microscopy ,Microscopy ,Image Processing, Computer-Assisted ,Animals ,Humans ,business ,Molecular Biology ,030304 developmental biology ,Biotechnology - Abstract
Single objective light-sheet fluorescence microscopes combine the convenience of conventional sample mounting with sensitive subcellular and super-resolution imaging of cells and tissues.
- Published
- 2019
8. Author Correction: Resolution upgrades for light-sheet microscopy
- Author
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Reto Fiolka
- Subjects
Materials science ,Optics ,business.industry ,Light sheet fluorescence microscopy ,Resolution (electron density) ,Cell Biology ,business ,Molecular Biology ,Biochemistry ,Biotechnology - Published
- 2019
9. Virtual slit scanning microscopy
- Author
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Andreas Stemmer, Reto Fiolka, and Yury Belyaev
- Subjects
Histology ,Materials science ,Microscope ,Aperture ,Confocal ,Analytical chemistry ,Physics::Optics ,Sensitivity and Specificity ,law.invention ,User-Computer Interface ,Optics ,Confocal microscopy ,law ,Microscopy ,Humans ,Image sensor ,Molecular Biology ,Microscopy, Confocal ,business.industry ,Scanning microscopy ,Astrophysics::Instrumentation and Methods for Astrophysics ,Scanning confocal electron microscopy ,Equipment Design ,Cell Biology ,Slit ,eye diseases ,Medical Laboratory Technology ,Microscopy, Electron, Scanning ,sense organs ,business ,HeLa Cells - Abstract
Histochemistry and Cell Biology, 128 (6), ISSN:0948-6143, ISSN:1432-119X
- Published
- 2007
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