Overview
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Publications
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(2023): Towards Widefield based Three-Dimensional Nuclear Nanostructure Analysis in the 1 Nanometer Resolution Range. Poster. Focus on Microscopy. Porto, 4. April, 2023
Abstract: The spatial nanostructure of the nuclear genome of higher organisms has emerged as a main topic of cell biology1. So far, a variety of methods have been applied for its elucidation by super-resolution approaches2,3. Here, we discuss application perspectives for a further improvement of nuclear nanostructure analysis by a combination of Localization Microscopy with laterally (SIM: Structured Illumination Microscopy) and axially patterned (SMI: Spatially Modulated Illumination)4. Numerical simulations indicate that a three-dimensional localization precision around 1nm may become feasible using fluorescence yields typical for single molecule localization microscopy (SMLM) applications. Together with the nanosizing capability of SMI, this may eventually be used to analyse macromolecular nuclear complexes and other nanostructures with a topological resolution approaching cryoelectron microscopy.
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(2023): Lens Free Ring Array Illumination microscopy. Combination of Super-Resolution with Large Field of View Imaging and long Working Distances. Poster. Focus on Microscopy. Porto, 4. April, 2023
Abstract: Using low NA objective lenses, light microscopy allows to "screen" large object areas to identify fluorescently labeled or autofluorescent structures of interest at relatively modest optical resolution; using separate instrumentation, selected sites can then be further analyzed at enhanced resolution, such as ultrastructural microscopy, or State-of-the-art super-resolution microscopy (SRM). This second step, however, typically requires high NA objective lenses with working distances in the submillimeter range. Here we present a novel concept which enables a direct integration of SRM (STED, SMLM, MINFLUX) into microscopy systems with working distances up to the multicentimeter range while still allowing nanometer scale resolution. This becomes possible by a "lens free" illumination mode with multiple coherent excitation beams positioned in a „ring-array“ arrangement around the low NA objective lens1. The feasibility of such a direct correlative microscopy method was validated by extensive numerical simulations; on the basis of these calculations, experimental implementation options shall be discussed. Such "Ring Array" illumination modes may be useful also for other correlative microscopy methods, such as a direct combination of light and electron microscopy in the same device (dCLEM).
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(2023): Ring Array Illumination Microscopy. Combination of Super-Resolution with Large Field of View Imaging and long Working Distances. In: bioRxiv. DOI: 10.1101/2023.09.05.555896
DOI: RemoteUri Abstract: Here we present a novel fluorescence microscopy concept which enables a direct integration of Super-Resolution Microscopy (SRM) approaches (SIM/Nanosizing, STED, SMLM, MINFLUX, SIMFLUX) into microscopy systems with working distances (WD) up to the multicentimeter range while still allowing nanometer scale resolution at selected sites. This becomes possible by a "leftsynthetic aperture"right illumination mode with multiple, constructively interfering excitation beams positioned in a "leftRing-Array"right arrangement around a beam free interior zone containing instrumentation involved in complementary imaging modes. The feasibility of such a direct correlative microscopy method is validated by extensive numerical simulations; on the basis of these calculations, experimental implementation options are discussed. Such "leftRing Array"right illumination modes may be useful for various correlative microscopy methods, such as a direct combination of correlative light and electron microscopy in the same device (dCLEM); or a direct combination of low NA/large field-of-view widefield microscopy and super-resolution of selected sites in the same device (direct Correlative Opical Microscopy/dCOLM). Ring-Array supported correlative microscopy modes will open novel imaging perspectives in a variety of disciplines, from material sciences to biomedical applications.
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(2022): Advanced Door Area Monitoring. Precision Photonic Systems. OST. Ostschweizer Fachhochschule. Institut für Mikrotechnik und Photonik. Dornbirn, 1. Dezember, 2022
Abstract: Door Area Monitoring is an important focus of automated door manufacturers. Presently available door area analytics can not only detect a person wishing to pass through the door, but also discriminate the speed and direction of travel. We use a miniaturized door automation system to study such systems far beyond the conventional detection capabilities required for opening and closing the door: Doors are only partially opened where needed, and at a higher level, the results of image and ranging sensor data evaluation such as gender, age, customer identification etc. can be fed into smart building surveillance.
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(2022) : Online Teaching. Überlegungen zu digitalem Lehren und Lernen In: Forster, Michael; Alt, Sharon; Hanselmann, Marcel; Deflorin, Patricia (Hg.): Digitale Transformation an der Fachhochschule Graubünden: Case Studies aus Forschung und Lehre: Chur: FH Graubünden Verlag, S. 87-100. Available online at https://www.fhgr.ch/fh-graubuenden/ueber-die-fh-graubuenden/wofuer-stehen-wir/digitalisierung/digitalisierungswissen-fuer-graubuenden/#c15147, last checked on 20.01.2023
Abstract: Digitale Lehre bietet vielfältige, medienspezifische Möglichkeiten, um Lehrinhalte zu vermitteln. Allerdings ist die didaktische und technische Umsetzung oftmals eine Herausforderung. Wir diskutieren Vor- und Nachteile der digitalen Lehre anhand von transdisziplinären Beispielen aus der Lehrpraxis.
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(2022): Advanced Door Area Monitoring (Precision Photonic Systems, Dornbirn, 13. Dezember). Available online at https://doi.org/10.5281/zenodo.7432706, last checked on 20.01.2023
Abstract: Door Area Monitoring is an important focus of automated door manufacturers. Presently available door area analytics can not only detect a person wishing to pass through the door, but also discriminate the speed and direction of travel. We use a miniaturized door automation system to study such systems far beyond the conventional detection capabilities required for opening and closing the door: Doors are only partially opened where needed, and at a higher level, the results of image and ranging sensor data evaluation such as gender, age, customer identification etc. can be fed into smart building surveillance.
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(2022): Spatially modulated illumination microscopy. Application perspectives in nuclear nanostructure analysis. In: Philosophical transactions. Series A. Mathematical, physical, and engineering sciences 380. Available online at https://doi.org/10.1098/rsta.2021.0152, last checked on 17.02.2022
Abstract: Thousands of genes and the complex biochemical networks for their transcription are packed in the micrometer sized cell nucleus. To control biochemical processes, spatial organization plays a key role. Hence the structure of the cell nucleus of higher organisms has emerged as a main topic of advanced light microscopy. So far, a variety of methods have been applied for this, including confocal laser scanning fluorescence microscopy, 4Pi-, STED- and localization microscopy approaches, as well as (laterally) structured illumination microscopy (SIM). Here, we summarize the state of the art and discuss application perspectives for nuclear nanostructure analysis of spatially modulated illumination (SMI). SMI is a widefield-based approach to using axially structured illumination patterns to determine the axial extension (size) of small, optically isolated fluorescent objects between less than or equal to 200 nm and greater than or equal to 40 nm diameter with a precision down to the few nm range; in addition, it allows the axial positioning of such structures down to the 1 nm scale. Combined with SIM, a three-dimensional localization precision of less than or equal to 1 nm is expected to become feasible using fluorescence yields typical for single molecule localization microscopy applications. Together with its nanosizing capability, this may eventually be used to analyse macromolecular complexes and other nanostructures with a topological resolution, further narrowing the gap to Cryoelectron microscopy. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 2)'.
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(2021): Deep Learning in Microscopy. Imaging ONEWORLD series. Royal Microscopial Society. Online, 19. Juli, 2021
Abstract: Recent advancements in microscopic image analysis have made use of the tremendous progress that artificial intelligence has seen in the evaluation of digital images. The application of deep learning techniques to microscopic image analysis ranges from image restoration and denoising to automated cellular and subcellular profiling to generation of augmented reconstructed image data, encompassing e.g. super-resolution images from single molecule localization microscopy data, virtual refocussing, content aware neuronal reconstructions and many more. However, deep learning methods are generally based on the availability of large amounts of reliable ground truth data. In the talk I will highlight some of the recent advances in deep learning applied to microscopic data with a focus on applications for super-resolution microscopy.
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(2021) : Chess recognition using 3D patterned illumination camera In: Osten, Wolfgang; Nikolaev, Dmitry P.; Zhou, Jianhong (Hg.): Thirteenth International Conference on Machine Vision (ICMV 2020): Proceedings: International Conference on Machine Vision (ICMV): Rom, 2. - 6. November. SPIE The international society for optics and photonics (Proceedings of SPIE), S. 520-527
DOI: https://doi.org/10.1117/12.2587054 Abstract: Computer Vision has been applied to augment traditional board games such as Chess for a number of reasons. While augmented reality enhances the gaming experience, the required additional hardware (e.g. head gear) is still not widely accepted in everyday leisure activities, and therefore, camera based methods have been developed to interface the computer with the real-life chess board. However, traditional 2D camera approaches suffer from ill-defined environmental conditions (lighting, viewing angle) and are therefore severely limited in their application. To answer this issue, we have incorporated a consumer-grade depth camera based on patterned illumination. We could show that in combination with traditional 2D color images, the recognition of chess pieces is made easier, which allows seamless integration of the real-life chess pieces with the computer program. Our method uses a fusion approach from depth and RGB camera data and is suitable for two distant players to play against each other, using two physical sets of chess.
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(2020): High-resolution deep view microscopy of cells and tissues. In: Quantum Electronics 50, S. 2-8. Available online at http://dx.doi.org/10.1070/QEL17204, last checked on 31.01.2020
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(2019): Bildverarbeitung und Maschinelles Sehen für Studierende der Ingenieurwissenschaften. Poster. 120. Jahrestagung der deutschen Gesellschaft für angewandte Optik (DGaO). Deutsche Gesellschaft für angewandte Optik. Darmstadt, 12.-15. Juni, 2019. Available online at https://www.dgao-proceedings.de/abstract/abstract_only.php?id=2447, last checked on 14.02.2020
Abstract: Der Einsatz photonischer Technologien setzt sich in den kommenden Jahren verstärkt fort. Bildverarbeitung spielt bei diesen Entwicklungen eine zentrale Rolle. Mit Beiträgen aus der Industrie wurde ein Bachelor-Curriculum mit Schwerpunkt «Bildverarbeitung» für Studierende der Ingenieurwissenschaften konzipiert. Ein wichtiges Ziel des Kurses war es, theoretische und praktische Erfahrungen in Automatisierungstechnologien zu vermitteln, die für zukünftige Ingenieure von größter Bedeutung sein werden. In diesem Beitrag werden wichtige Konzepte unseres Bildverarbeitungskurses vorgestellt, und praktische Geräte und Laborausstattung beschrieben, die für diesen Kurs bereitgestellt wurden. Der dreigeteilte Kurs besteht aus Präsenzsequenzen, aus Übungssequenzen mit Programmieraufgaben, sowie aus einer Phase Problemorientierten Lernens (POL), in welcher die Studierenden ihre eigenen Bildverarbeitungsprojekte realisieren. Es werden einige Beispielprojekte aus dem Frühjahrs- bzw. Herbstsemester 2018 vorgestellt. Kursinhalt und -materialien wurden hinsichtlich des technischen Gehalts und der Lerneffektivität bewertet. Die Ergebnisse dieser Bewertung werden in diesem Beitrag vorgestellt.
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(2019): Holzwerkstoffklassifizierung mit Machine Vision unter Verwendung einer Acht-Kanal-Farbbeleuchtung. 120. Jahrestagung der deutschen Gesellschaft für angewandte Optik (DGaO). Deutsche Gesellschaft für angewandte Optik. Darmstadt, 12. Juni, 2019. Available online at https://www.dgao-proceedings.de/archiv/120_titel_d.php, last checked on 31.01.2020
Abstract: Die Unterscheidung zwischen Echtholz und Nachbildungen ist wichtig für die Bewertung von Möbeln und Wohnraum. Moderne Oberflächenfertigungsverfahren machen es dem menschlichen Auge praktisch unmöglich, die verwendeten Baumaterialien richtig zu identifizieren, ohne das Objekt zu beschädigen. Wir präsentieren eine rein optische Technik zur Klassifizierung verschiedener Eichenparkettmaterialien anhand eines Beleuchtungsschemas mit acht verschiedenen Spektren von UV bis IR. Das System diente der Unterscheidung zwischen Vinyl-Replik und fünf verschiedenen Echtholz-Eichendekors, wobei eine Klassifizierungsgenauigkeit von 97% erreicht wurde. Weitere Anwendungsmöglichkeiten sind die Suche nach passenden Ersatzdekors beim Austausch schadhafter Panelen, z.B. nach Reparaturen.
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(2019): Wenn der Körper das Passwort ist. In: Polyscope 2019, S. 30-31. Available online at https://www.polyscope.ch/archiv/2019/19/wenn-der-koerper-das-passwort-ist/
Abstract: Gesichtserkennung ist ein aktuelles Forschungsthema, nicht nur in der Neurowissenschaft. Mit modernen lichtbasierten Technologien kann man Gesichtserkennung auch auf elektronischen Geräten wie Handys realisieren. Zusammen mit dem Fingerabdruckscanner betreten wir mit der Gesichtserkennung ein neues Zeitalter der digitalen Sicherheit: Entweder ich benutze ein Passwort, oder mein Körper ist das Passwort.
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(2019): Lens Free Super-Resolution Microscopy. Distributed Aperture Illumination Allows Large Working Distances. In: Imaging & Microscopy 2019, S. 23-25. Available online at https://www.imaging-git.com/magazine/imaging-microscopy-issue-42019
Abstract: High-resolution microscopy methods typically use objective lenses with large numerical apertures, i.e. low working distances (WD) in the range of 0.2 mm. This impedes its application to thick transparent specimens, or to objects with large topographical differences. To extend high-resolution microscopy also to whole tissues and to the material sciences, lens free microscope systems allowing WDs up to the multicentimeter range and an optical resolution down to the nanometer range may be constructed.
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(2019): Superauflösende Mikroskopie mit sehr großem Arbeitsabstand durch verteilte Aperturbeleuchtung. Poster. 120. Jahrestagung der deutschen Gesellschaft für angewandte Optik (DGaO). Deutsche Gesellschaft für angewandte Optik. Darmstadt, 12.-15. Juni, 2019. Available online at https://www.dgao-proceedings.de/abstract/abstract_only.php?id=2449, last checked on 14.02.2020
Abstract: Die Grenzen der konventionellen Lichtmikroskopie („Abbe-Limit“) hängen entscheidend von der numerischen Apertur (NA) der Objektivlinse ab. Die Bildgebung bei großen Arbeitsabständen oder einem großen Sichtfeld erfordert typischerweise Objektive mit niedriger NA, wodurch die optische Auflösung auf den Bereich von mehreren Mikrometern reduziert wird. Basierend auf numerischen Simulationen der Intensitätsfeldverteilung stellen wir ein Beleuchtungssystem für ein hochauflösendes Mikroskop vor, das eine dreidimensionale (3D) optische Auflösung um 150 nm für Arbeitsabstände bis zum Zentimeterbereich ermöglicht. Grundsätzlich erlaubt das System eine große Flexibilität, da das Beleuchtungskonzept zur Annäherung an die Punktspreizfunktion herkömmlicher Mikroskopoptiken mit dem zusätzlichen Vorteil einer anpassbaren Pupillenfunktion verwendet werden kann. Verglichen mit dem Abbe-Limit unter Verwendung einer Objektivlinse mit einem so großen Arbeitsabstand wird ein Volumenauflösungspotenzial in der Größenordnung von 104 geschätzt.
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(2019) : Schlaue Helfer dank Embedded Vision . Blog (FHGR Blog) . Available online at https://blog.fhgr.ch/blog/schlaue-helfer-dank-embedded-vision/ , last checked on 05.03.2021
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(2019): Nanoscale distribution of TLR4 on primary human macrophages stimulated with LPS and ATI. In: Nanoscale 11, S. 9769-9779. Available online at http://dx.doi.org/10.1039/C9NR00943D, last checked on 20.02.2020
Abstract: Toll-like receptor 4 (TLR4) plays a crucial role in the recognition of invading pathogens. Upon activation by lipopolysaccharides (LPS), TLR4 is recruited into specific membrane domains and dimerizes. In addition to LPS, TLR4 can be stimulated by wheat amylase-trypsin inhibitors (ATI). ATI are proteins associated with gluten containing grains, whose ingestion promotes intestinal and extraintestinal inflammation. However, the effect of ATI vs. LPS on the membrane distribution of TLR4 at the nanoscale has not been analyzed. In this study, we investigated the effect of LPS and ATI stimulation on the membrane distribution of TLR4 in primary human macrophages using single molecule localization microscopy (SMLM). We found that in unstimulated macrophages the majority of TLR4 molecules are located in clusters, but with donor-dependent variations from ∼51% to ∼75%. Depending on pre-clustering, we found pronounced variations in the fraction of clustered molecules and density of clusters on the membrane upon LPS and ATI stimulation. Although clustering differed greatly among the human donors, we found an almost constant cluster diameter of ∼44 nm for all donors, independent of treatment. Together, our results show donor-dependent but comparable effects between ATI and LPS stimulation on the membrane distribution of TLR4. This may indicate a general mechanism of TLR4 activation in primary human macrophages. Furthermore, our methodology visualizes TLR4 receptor clustering and underlines its functional role as a signaling platform.
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(2018): Benutzerpositionsabhängige 3D-Daten-Anzeige ohne stereoskopische Hardware und ohne Headse. Poster. 119. Jahrestagung der deutschen Gesellschaft für angewandte Optik (DGaO). Deutsche Gesellschaft für angewandte Optik. Darmstadt, 23.-26. Mai, 2018. Available online at https://www.dgao-proceedings.de/abstract/abstract_only.php?id=2277, last checked on 21.02.2020
Abstract: Stereoskopische Bildverarbeitungsmodule haben aufgrund der benötigten Hardware (Bildaufnahme, 3D-Brille) nur begrenzten Erfolg in der Technik- und Konsumwelt. In den letzten Jahren hat vor allem der Spielesektor von spezialisierten VR-Brillen profitiert. Viele andere industrielle und biomedizinische Anwendungen, wie z.B. Tomografieverfahren oder CAD-Konstruktion, haben die erhöhten 3D-Renderingfähigkeiten heutiger Computerhardware bisher nicht voll ausgenutzt. Wir verwenden Standardhardware (Monitor und Webcam), um benutzerpositionsabhängige Projektionen von 3D-Daten ohne zusätzliche Spezialbrillen anzuzeigen. Der Ansatz bietet viele Anwendungsmöglichkeiten von der medizinischen Bildgebung über Konstruktion und CAD-Design, Architektur bis hin zu Kunst und Performances. Abhängig von dem Ort des Nutzers, werden die 3D-Daten für den jeweiligen Benutzersichtwinkel (Zoom) und die Blickrichtung unterschiedlich gerendert. Wenn sich der Benutzer vor dem Monitor bewegt, werden verschiedene Bereiche des gerenderten Objekts sichtbar. Wenn der Benutzer sich dem Bildschirm nähert, verringert sich der Ansichtswinkel, was durch eine vergrößerte Version des gerenderten Objekts visualisiert wird.
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(2018) : User-position aware adaptive display of 3D data without additional stereoscopic hardware In: Verikas, Antanas; Nikolaev, Dmitry P.; Radeva, Petia; Zhou, Jianhong (Hg.): Eleventh International Conference on Machine Vision (ICMV 2018): Proceedings: International Conference on Machine Vision (ICMV): München, 1. - 3. November. SPIE The international society for optics and photonics (Proceedings of SPIE). Available online at https://doi.org/10.1117/12.2522678, last checked on 14.02.2020
Abstract: Stereoscopic vision modules have seen limited success in both engineering and consumer world, due to the required additional hardware (image acquisition, Virtual Reality headsets, 3D glasses). In the last years, especially the gaming and education sectors have benefited from such specialized headgear, providing virtual or augmented reality. However, many other industrial and biomedical applications such as e.g. computer aided design (CAD) or tomographic data display, so far have not fully exploited the increased 3D rendering capabilities of present-day computer hardware. We present an approach to use standard desktop PC hardware (monitor and webcam) to display user-position aware projections of 3D data without additional headgear. The user position is detected from webcam images, and the rendered 3D data (i.e. the view) is adjusted to match the corresponding user position, resulting in a quasi virtual reality rendering, albeit without the 3D effect of proper 3D head-gear. The approach has many applications from medical imaging, to construction and CAD, to architecture, to exhibitions, arts and performances. Depending on the user location, i.e. the detected head position, the data is rendered differently to attribute for the user view angle (zoom) and direction. As the user moves his or her head in front of the monitor, different features of the rendered object become visible. As the user moves closer to the screen, the view angle of the rendered data is decreased, resulting in a zoomed-in version of the rendered object.
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(2018) : Der Pin hat ausgedient: Heute gilt die Gesichtserkennung . Blog (FHGR Blog) . Available online at https://blog.fhgr.ch/blog/der-pin-hat-ausgedient-heute-gilt-die-gesichtserkennung/ , last checked on 19.03.2021
Abstract: Gesichtserkennung ist ein brandaktuelles Forschungsthema, nicht nur in den Neurowissenschaften. Mit modernen lichtbasierten Technologien ist es gelungen, Gesichtserkennung neuerdings auch auf elektronischen Geräten wie z.B. Smartphones möglich zu machen. Zusammen mit dem Fingerabdruckscanner betreten wir mit der Gesichtserkennung ein neues Zeitalter der digitalen Sicherheit: Entweder ich benutze ein Passwort, oder ich bzw. mein Körper ist das Passwort.
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(2018): Der Zellkern - eine Stadt in der Zelle, Teil 2. In: Biologie in unserer Zeit 48, S. 45-53. Available online at https://doi.org/10.1002/biuz.201810640, last checked on 07.05.2023
Abstract: Im zweiten Teil des Beitrags zur Zellkernarchitektur beschreiben wir die bahnbrechenden Möglichkeiten der superauflösenden Fluoreszenzmikroskopie (Nanoskopie) und neuer molekularbiologischer Verfahren zur Erforschung der Struktur von Chromatindomänen im Nanometer-Maßstab und ihrer räumlichen Anordnung zu größeren Aggregaten. Neue experimentelle Daten und Modelle sprechen für eine entscheidende Rolle dieser Strukturen für die Genregulation und andere Funktionen des Zellkerns.
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(2018): Super-resolution binding activated localization microscopy through reversible change of DNA conformation. In: Nucleus 9, S. 182-189. Available online at https://doi.org/10.1080/19491034.2017.1419846
Abstract: Methods of super-resolving light microscopy (SRM) have found an exponentially growing range of applications in cell biology, including nuclear structure analyses. Recent developments have proven that Single Molecule Localization Microscopy (SMLM), a type of SRM, is particularly useful for enhanced spatial analysis of the cell nucleus due to its highest resolving capability combined with very specific fluorescent labeling. In this commentary we offer a brief review of the latest methodological development in the field of SMLM of chromatin designated DNA Structure Fluctuation Assisted Binding Activated Localization Microscopy (abbreviated as fBALM) as well as its potential future applications in biology and medicine.
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(2017): Super-resolved linear fluorescence localization microscopy using photostable fluorophores. A virtual microscopy study. In: Optics Communications, S. 42-50. Available online at https://doi.org/10.1016/j.optcom.2017.06.078, last checked on 06.05.2023
Abstract: Current approaches to overcome the conventional limit of the resolution potential of light microscopy (of about 200 nm for visible light), often suffer from non-linear effects, which render the quantification of the image intensities in the reconstructions difficult, and also affect the quantification of the biological structure under investigation. As an attempt to face these difficulties, we discuss a particular method of localization microscopy which is based on photostable fluorescent dyes. The proposed method can potentially be implemented as a fast alternative for quantitative localization microscopy, circumventing the need for the acquisition of thousands of image frames and complex, highly dye-specific imaging buffers. Although the need for calibration remains in order to extract quantitative data (such as the number of emitters), multispectral approaches are largely facilitated due to the much less stringent requirements on imaging buffers. Furthermore, multispectral acquisitions can be readily obtained using commercial instrumentation such as e.g. the conventional confocal laser scanning microscope.
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(2017): Super-Resolution Microscopy. A Practical Guide. Weinheim, Germany: Wiley-VCH
Abstract: This unique book on super-resolution microscopy techniques presents comparative, in-depth analyses of the strengths and weaknesses of the individual approaches. It was written for non-experts who need to understand the principles of super-resolution or who wish to use recently commercialized instruments as well as for professionals who plan to realize novel microscopic devices. Explaining the practical requirements in terms of hardware, software and sample preparation, the book offers a wealth of hands-on tips and practical tricks to get a setup running, provides invaluable help and support for successful data acquisition and specific advice in the context of data analysis and visualization. Furthermore, it addresses a wide array of transdisciplinary fields of applications. The author begins by outlining the joint efforts that have led to achieving super-resolution microscopy combining advances in single-molecule photo-physics, fluorophore design and fluorescent labeling, instrument design and software development. The following chapters depict and compare current main standard techniques such as structured illumination microscopy, single-molecule localization, stimulated emission depletion microscopy and multi-scale imaging including light-sheet and expansion microscopy. For each individual approach the experimental setups are introduced, the imaging protocols are provided and the various applications illustrated. The book concludes with a discussion of future challenges addressing issues of routine applications and further commercialization of the available methods. Guiding users in how to make choices for the design of their own experiments from scratch to promising application, this one-stop resource is intended for researchers in the applied sciences, from chemistry to biology and medicine to physics and engineering.
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(2017): Super-resolution microscopy with very large working distance by means of distributed aperture illumination. In: Scientific reports 7. Available online at https://doi.org/10.1038/s41598-017-03743-4, last checked on 06.05.2023
Abstract: The limits of conventional light microscopy ("Abbe-Limit") depend critically on the numerical aperture (NA) of the objective lens. Imaging at large working distances or a large field-of-view typically requires low NA objectives, thereby reducing the optical resolution to the multi micrometer range. Based on numerical simulations of the intensity field distribution, we present an illumination concept for a super-resolution microscope which allows a three dimensional (3D) optical resolution around 150 nm for working distances up to the centimeter regime. In principle, the system allows great flexibility, because the illumination concept can be used to approximate the point-spread-function of conventional microscope optics, with the additional benefit of a customizable pupil function. Compared with the Abbe-limit using an objective lens with such a large working distance, a volume resolution enhancement potential in the order of 104 is estimated.
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(2017) : Super-Resolution Microscopy. Interference and Pattern Techniques In: Kubitscheck, Ulrich: Fluorescence Microscopy: From Principles to Biological Applications: Weinheim: Wiley-VCH, S. 291-319
Abstract: Fluorescence microscopy techniques using patterned illumination light offer the opportunity to extract high-resolution object information beyond the conventional resolution limit. This chapter describes in detail two widefield methods that apply interference of the excitation light to make high-resolution object information accessible. These are structured illumination microscopy (SIM) (also referred to as patterned excitation microscopy, PEM) and spatially modulated illumination (SMI). In SIM, the object is illuminated with a periodic illumination pattern. This pattern is used in order to manipulate the object's spatial frequencies. Two methods to spatially modulate the excitation intensity in SIM are common: two-beam interference, also called fringe projection, and three-beam interference, the so-called grid projection. The secondary focus of this chapter is on the less common method of SMI, where two opposing objective lenses are used to generate a high-frequency interference pattern along the optical axis. The SMI method is used to measure the size of nanostructures with great precision.
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(2017): Super-resolution microscopy approaches to nuclear nanostructure imaging. In: Methods, S. 11-32. Available online at https://doi.org/10.1016/j.ymeth.2017.03.019, last checked on 06.05.2023
Abstract: The human genome has been decoded, but we are still far from understanding the regulation of all gene activities. A largely unexplained role in these regulatory mechanisms is played by the spatial organization of the genome in the cell nucleus which has far-reaching functional consequences for gene regulation. Until recently, it appeared to be impossible to study this problem on the nanoscale by light microscopy. However, novel developments in optical imaging technology have radically surpassed the limited resolution of conventional far-field fluorescence microscopy (ca. 200nm). After a brief review of available super-resolution microscopy (SRM) methods, we focus on a specific SRM approach to study nuclear genome structure at the single cell/single molecule level, Spectral Precision Distance/Position Determination Microscopy (SPDM). SPDM, a variant of localization microscopy, makes use of conventional fluorescent proteins or single standard organic fluorophores in combination with standard (or only slightly modified) specimen preparation conditions; in its actual realization mode, the same laser frequency can be used for both photoswitching and fluorescence read out. Presently, the SPDM method allows us to image nuclear genome organization in individual cells down to few tens of nanometer (nm) of structural resolution, and to perform quantitative analyses of individual small chromatin domains; of the nanoscale distribution of histones, chromatin remodeling proteins, and transcription, splicing and repair related factors. As a biomedical research application, using dual-color SPDM, it became possible to monitor in mouse cardiomyocyte cells quantitatively the effects of ischemia conditions on the chromatin nanostructure (DNA). These novel "molecular optics" approaches open an avenue to study the nuclear landscape directly in individual cells down to the single molecule level and thus to test models of functional genome architecture at unprecedented resolution.
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(2017): Imaging chromatin nanostructure with binding-activated localization microscopy based on DNA structure fluctuations. In: Nucleic Acids Research 45. Available online at https://doi.org/10.1093/nar/gkw1301, last checked on 06.05.2023
Abstract: Advanced light microscopy is an important tool for nanostructure analysis of chromatin. In this report we present a general concept for Single Molecule localization Microscopy (SMLM) super-resolved imaging of DNA-binding dyes based on modifying the properties of DNA and the dye. By careful adjustment of the chemical environment leading to local, reversible DNA melting and hybridization control over the fluorescence signal of the DNA-binding dye molecules can be introduced. We postulate a transient binding as the basis for our variation of binding-activated localization microscopy (BALM). We demonstrate that several intercalating and minor-groove binding DNA dyes can be used to register (optically isolate) only a few DNA-binding dye signals at a time. To highlight this DNA structure fluctuation-assisted BALM (fBALM), we applied it to measure, for the first time, nanoscale differences in nuclear architecture in model ischemia with an anticipated structural resolution of approximately 50 nm. Our data suggest that this approach may open an avenue for the enhanced microscopic analysis of chromatin nano-architecture and hence the microscopic analysis of nuclear structure aberrations occurring in various pathological conditions. It may also become possible to analyse nuclear nanostructure differences in different cell types, stages of development or environmental stress conditions.
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(2016): Perspectives in Super-Resolved Fluorescence Microscopy. What Comes Next?. In: Frontiers in Physics 4. Available online at https://doi.org/10.3389/fphy.2016.00011, last checked on 06.05.2023
Abstract: The Nobel Prize in Chemistry 2014 has been awarded to three scientists involved in the development of STED and PALM super-resolution fluorescence microscopy (SRM) methods. They have proven that it is possible to overcome the 100 year old theoretical limit for the resolution potential of light microscopy (of about 200 nm for visible light), which for decades has precluded a direct glimpse of the molecular machinery of life. None of the present-day super-resolution techniques have invalidated the Abbe limit for light optical detection; however, they have found clever ways around it. In this report, we discuss some of the challenges still to be resolved before arising SRM approaches will be fit to bring about the revolution in Biology and Medicine envisaged. Some of the challenges discussed are the applicability to image live and/or large samples, the further enhancement of resolution, future developments of labels, and multi-spectral approaches.
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(2016): Chronic Inflammation Under the Microscope. In: Microscopy Today 24, S. 38-45. Available online at https://doi.org/10.1017/S1551929516000894, last checked on 23.05.2023
Abstract: In the course of chronic inflammation certain body areas are recurrently inflamed. This goes along with many human diseases. With the help of widefield light microscopy, the underlying processes can be examined from a cellular level to whole organisms. This article presents several widefield microscopy applications such as immunofluorescence, live-cell imaging, histology, and ratiometric analysis to get insight into the development of chronic inflammation, the related diseases, and their treatment.
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(2016): Single Molecule Localization Microscopy of Mammalian Cell Nuclei on the Nanoscale. In: Frontiers in Genetics 7. Available online at https://doi.org/10.3389/fgene.2016.00114, last checked on 06.05.2023
Abstract: Nuclear texture analysis is a well-established method of cellular pathology. It is hampered, however, by the limits of conventional light microscopy (ca. 200 nm). These limits have been overcome by a variety of super-resolution approaches. An especially promising approach to chromatin texture analysis is single molecule localization microscopy (SMLM) as it provides the highest resolution using fluorescent based methods. At the present state of the art, using fixed whole cell samples and standard DNA dyes, a structural resolution of chromatin in the 50-100 nm range is obtained using SMLM. We highlight how the combination of localization microscopy with standard fluorophores opens the avenue to a plethora of studies including the spatial distribution of DNA and associated proteins in eukaryotic cell nuclei with the potential to elucidate the functional organization of chromatin. These views are based on our experience as well as on recently published research in this field.
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(2016): Quantitative super-resolution localization microscopy of DNA in situ using Vybrant® DyeCycle™ Violet fluorescent probe. In: Data in Brief, S. 157-171. Available online at https://doi.org/10.1016/j.dib.2016.01.041, last checked on 06.05.2023
Abstract: Single Molecule Localization Microscopy (SMLM) is a recently emerged optical imaging method that was shown to achieve a resolution in the order of tens of nanometers in intact cells. Novel high resolution imaging methods might be crucial for understanding of how the chromatin, a complex of DNA and proteins, is arranged in the eukaryotic cell nucleus. Such an approach utilizing switching of a fluorescent, DNA-binding dye Vybrant® DyeCycle™ Violet has been previously demonstrated by us (Żurek-Biesiada et al., 2015) [1]. Here we provide quantitative information on the influence of the chemical environment on the behavior of the dye, discuss the variability in the DNA-associated signal density, and demonstrate direct proof of enhanced structural resolution. Furthermore, we compare different visualization approaches. Finally, we describe various opportunities of multicolor DNA/SMLM imaging in eukaryotic cell nuclei.
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(2016): Localization microscopy of DNA in situ using Vybrant(®) DyeCycle™ Violet fluorescent probe. A new approach to study nuclear nanostructure at single molecule resolution. In: Experimental Cell Research 343, S. 97-106. Available online at https://doi.org/10.1016/j.yexcr.2015.08.020, last checked on 06.05.2023
Abstract: Higher order chromatin structure is not only required to compact and spatially arrange long chromatids within a nucleus, but have also important functional roles, including control of gene expression and DNA processing. However, studies of chromatin nanostructures cannot be performed using conventional widefield and confocal microscopy because of the limited optical resolution. Various methods of superresolution microscopy have been described to overcome this difficulty, like structured illumination and single molecule localization microscopy. We report here that the standard DNA dye Vybrant(®) DyeCycle™ Violet can be used to provide single molecule localization microscopy (SMLM) images of DNA in nuclei of fixed mammalian cells. This SMLM method enabled optical isolation and localization of large numbers of DNA-bound molecules, usually in excess of 10(6) signals in one cell nucleus. The technique yielded high-quality images of nuclear DNA density, revealing subdiffraction chromatin structures of the size in the order of 100nm; the interchromatin compartment was visualized at unprecedented optical resolution. The approach offers several advantages over previously described high resolution DNA imaging methods, including high specificity, an ability to record images using a single wavelength excitation, and a higher density of single molecule signals than reported in previous SMLM studies. The method is compatible with DNA/multicolor SMLM imaging which employs simple staining methods suited also for conventional optical microscopy.
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(2015): A transient ischemic environment induces reversible compaction of chromatin. In: Genome Biology. Available online at https://doi.org/10.1186/s13059-015-0802-2, last checked on 06.05.2023
Abstract: BACKGROUND Cells detect and adapt to hypoxic and nutritional stress through immediate transcriptional, translational and metabolic responses. The environmental effects of ischemia on chromatin nanostructure were investigated using single molecule localization microscopy of DNA binding dyes and of acetylated histones, by the sensitivity of chromatin to digestion with DNAseI, and by fluorescence recovery after photobleaching (FRAP) of core and linker histones. RESULTS Short-term oxygen and nutrient deprivation of the cardiomyocyte cell line HL-1 induces a previously undescribed chromatin architecture, consisting of large, chromatin-sparse voids interspersed between DNA-dense hollow helicoid structures 40-700 nm in dimension. The chromatin compaction is reversible, and upon restitution of normoxia and nutrients, chromatin transiently adopts a more open structure than in untreated cells. The compacted state of chromatin reduces transcription, while the open chromatin structure induced upon recovery provokes a transitory increase in transcription. Digestion of chromatin with DNAseI confirms that oxygen and nutrient deprivation induces compaction of chromatin. Chromatin compaction is associated with depletion of ATP and redistribution of the polyamine pool into the nucleus. FRAP demonstrates that core histones are not displaced from compacted chromatin; however, the mobility of linker histone H1 is considerably reduced, to an extent that far exceeds the difference in histone H1 mobility between heterochromatin and euchromatin. CONCLUSIONS These studies exemplify the dynamic capacity of chromatin architecture to physically respond to environmental conditions, directly link cellular energy status to chromatin compaction and provide insight into the effect ischemia has on the nuclear architecture of cells.
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(2015): Single-Molecule Localization Microscopy allows for the analysis of cancer metastasis-specific miRNA distribution on the nanoscale. In: Oncotarget 6, S. 44745-44757. Available online at https://doi.org/10.18632/oncotarget.6297, last checked on 06.05.2023
Abstract: We describe a novel approach for the detection of small non-coding RNAs in single cells by Single-Molecule Localization Microscopy (SMLM). We used a modified SMLM-setup and applied this instrument in a first proof-of-principle concept to human cancer cell lines. Our method is able to visualize single microRNA (miR)-molecules in fixed cells with a localization accuracy of 10-15 nm, and is able to quantify and analyse clustering and localization in particular subcellular sites, including exosomes. We compared the metastasis-site derived (SW620) and primary site derived (SW480) human colorectal cancer (CRC) cell lines, and (as a proof of principle) evaluated the metastasis relevant miR-31 as a first example. We observed that the subcellular distribution of miR-31 molecules in both cell lines was very heterogeneous with the largest subpopulation of optically acquired weakly metastatic cells characterized by a low number of miR-31 molecules, as opposed to a significantly higher number in the majority of the highly metastatic cells. Furthermore, the highly metastatic cells had significantly more miR-31-molecules in the extracellular space, which were visualized to co-localize with exosomes in significantly higher numbers. From this study, we conclude that miRs are not only aberrantly expressed and regulated, but also differentially compartmentalized in cells with different metastatic potential. Taken together, this novel approach, by providing single molecule images of miRNAs in cellulo can be used as a powerful supplementary tool in the analysis of miRNA function and behaviour and has far reaching potential in defining metastasis-critical subpopulations within a given heterogeneous cancer cell population.