Questo lavoro è stato finanziato dalla Max Planck Society.

<ol>
	<li>Axelrod, D., Thompson, N. L., Burghardt, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+internal+inflection+fluorescent+microscopy.">Total internal inflection fluorescent microscopy.</a> J. Microsc. 129, 19-28 (1983).</li><li>Axelrod, D., Omann, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+microscopy.">Combinatorial microscopy.</a> Nat. Rev. Mol. Cell Biol. 7, 944-952 (2006).</li><li>Axelrod, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+imaging+of+surface+fluorescence+with+very+high+aperture+microscope+objectives.">Selective imaging of surface fluorescence with very high aperture microscope objectives.</a> J. Biomed. Opt. 6, 6-13 (2001).</li><li>Dominguez-Escobar, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processive+movement+of+MreB-associated+cell+wall+biosynthetic+complexes+in+bacteria.">Processive movement of MreB-associated cell wall biosynthetic complexes in bacteria.</a> Science. 333, 225-228 (2011).</li><li>Sparkes, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bleach+it,+switch+it,+bounce+it,+pull+it:+using+lasers+to+reveal+plant+cell+dynamics.">Bleach it, switch it, bounce it, pull it: using lasers to reveal plant cell dynamics.</a> J. Exp. Bot. 62, 1-7 (2011).</li><li>Uchida, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+synthesis+and+absolute+configuration+of+avenolide,+extracellular+factor+in+Streptomyces+avermitilis.">Total synthesis and absolute configuration of avenolide, extracellular factor in Streptomyces avermitilis.</a> J. Antibiot. (Tokyo). , (2011).</li><li>Yu, H., Wedlich-Soldner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+actin+dynamics:+Generating+randomness+by+formin(g)+and+moving.">Cortical actin dynamics: Generating randomness by formin(g) and moving.</a> Bioarchitecture. 1, 165-168 (2011).</li><li>Yu, J. H., Crevenna, A. H., Bettenbuhl, M., Freisinger, T., Wedlich-Soldner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+actin+dynamics+driven+by+formins+and+myosin+V.">Cortical actin dynamics driven by formins and myosin V.</a> J. Cell. Sci. 124, 1533-1541 (2011).</li><li>Berchtold, D., Walther, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TORC2+plasma+membrane+localization+is+essential+for+cell+viability+and+restricted+to+a+distinct+domain.">TORC2 plasma membrane localization is essential for cell viability and restricted to a distinct domain.</a> Mol. Biol. Cell. 20, 1565-1575 (2009).</li><li>Vizcay-Barrena, G., Webb, S. E., Martin-Fernandez, M. L., Wilson, Z. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+and+single-molecule+imaging+of+plant+fluorescent+proteins+using+total+internal+reflection+fluorescence+microscopy+(TIRFM).">Subcellular and single-molecule imaging of plant fluorescent proteins using total internal reflection fluorescence microscopy (TIRFM).</a> J. Exp. Bot. 62, 5419-5428 (2011).</li></ol>

Non ci sono conflitti di interesse dichiarati.

Microscopia TIRF è la tecnica di scelta per proteine ​​di immagine periferici. La profondità bassa penetrazione del campo evanescente minimizza di luce fuori fuoco, che porta ad un ottimo segnale rispetto al rumore e consente di acquisizione dati con un frame rate elevati, o immagini di proteine ​​espresse molto debolmente. La combinazione di un elevato contrasto e frame rate elevati microscopia TIRF rende uno strumento ideale per immagini dinamiche spazio-temporali di proteine ​​localizzate corticale. È ...

<table border="1"><tbody><tr><td> Nome dello strumento / reagente </td><td> Tipo </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> Orbital Shaker </td><td> Strumento </td><td> UniEquip </td><td> UNITWIST 3-D ROCKER SHAKER </td></tr><tr><td> TIRF microscopio </td><td> Fino a </td><td></td><td> Configurazione personalizzata </td></tr><tr><td> Contenitore di vetro </td><td> Strumento </td><td> Vitlab </td><td> 340-232880353 </td></tr><tr><td> Rastrello di colorazione Ceramic </td><td> Strumento </td><td> Sci Thomas. </td><td> 8542E40 </td></tr><tr><td> Concanavalina A </td><td> Reagente </td><td> Sigma </td><td> L7647 </td></tr><tr><td> Coprivetrini # 1 (18 x 18 mm) </td><td> Microscopio </td><td> Menzel Gläser </td><td> BB018018A1 </td></tr><tr><td> Vetrini </td><td> Microscopio </td><td> Menzel Glaser </td><td> AA00000102E </td></tr><tr><td> Olio di immersione </td><td> Microscopio </td><td> Zeiss </td><td> Immersol 518F </td></tr><tr><td> Agarosio </td><td> Reagente </td><td> Invitrogen </td><td> 16500-500 </td></tr><tr><td> FluoSpheres </td><td> Reagente </td><td> Invitrogen </td><td> F8795 </td></tr></tbody></table>

visualization of cortex organization and dynamics in microorganisms, using total internal reflection fluorescence microscopy

Microscopia TIRF è emerso come una tecnologia di imaging potente per studiare spazio-temporali dinamica delle molecole fluorescenti in vitro e in cellule viventi 1. Il fenomeno ottico di riflessione interna totale si verifica quando la luce passa da un mezzo con alto indice di rifrazione in un mezzo con indice di rifrazione basso ad un angolo maggiore di un angolo caratteristica critica (cioè più vicino ad essere parallelo al confine). Sebbene tutta la luce viene riflessa in tali condizioni, una onda evanescente viene creato attraverso e che si propaga lungo il confine, che decade esponenzialmente con la distanza, e penetra solo aree campione che sono 100-200 nm prossimità dell&#39;interfaccia 2. Oltre ad assicurare un&#39;elevata risoluzione assiale, l&#39;eccitazione ridotta di fluorofori fuoco crea un segnale molto alti rapporti di rumore e minimizza gli effetti dannosi di photobleaching 2,3. Essendo una tecnica widefield, TIRF permette anche più veloce di immagini acquisizione rispetto alla maggior parte di scansione in base configurazioni confocale. A prima vista, la profondità di penetrazione basso TIRF sembra essere incompatibile con imaging di cellule batteriche e fungine, spesso circondati da pareti cellulari spesse. Al contrario, abbiamo scoperto che le pareti cellulari del lievito e cellule batteriche effettivamente migliorare la utilizzabilità di TIRF e aumentare la gamma di strutture osservabili 4-8. Molti processi cellulari possono quindi accedere direttamente TIRF in piccoli microrganismi unicellulari, che spesso offrono potenti tecniche di manipolazione genetica. Questo ci permette di eseguire esperimenti in biochimica in vivo, dove la cinetica delle interazioni proteiche e delle attività può essere valutato direttamente in cellule viventi. Descriviamo qui le singole fasi necessarie per ottenere immagini di alta qualità per TIRF Saccharomyces cerevisiae o cellule di Bacillus subtilis. Segnaliamo vari problemi che possono Affect visualizzazione TIRF di sonde fluorescenti in cellule ed illustrare la procedura con esempi di applicazione diversi. Infine, abbiamo dimostrato come le immagini TIRF può essere ulteriormente migliorata utilizzando consolidate tecniche di restauro delle immagini.

Watch this Scientific Journal Video about Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy at JoVE.com

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

Total Internal Reflection Fluorescence (TIRF) microscopia è un approccio efficace per osservare le strutture in prossimità della superficie cellulare ad elevato contrasto e risoluzione temporale. Abbiamo dimostrato come TIRF può essere utilizzato per studiare dinamica della proteina alla corteccia di cellule di cellule batteriche e fungine parete chiusi.

Visualizzazione di Cortex Organizzazione e Dynamics in microrganismi, utilizzando Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

visualization-cortex-organization-dynamics-microorganisms-using-total

Research

JoVE Journal

Bioengineering

11.4K Views.  Max Planck Institute of Biochemistry. Total Internal Reflection Fluorescence (TIRF) microscopia è un approccio efficace per osservare le strutture in prossimità della superficie cellulare ad elevato contrasto e risoluzione temporale. Abbiamo dimostrato come TIRF può essere utilizzato per studiare dinamica della proteina alla corteccia di cellule di cellule batteriche e fungine parete chiusi.

Video: Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On the end of the microscope's cantilever is a nano-scale probe, which traverses image areas ranging from nanometers to micrometers, measuring the elevation of macromolecules resting on the substrate surface at any given point. Electrostatic forces cause proteins, lipids, and nucleic acids to loosely attach to the substrate in random orientations and permit imaging. The generated data resemble a topographical map, where the macromolecules resolve as three-dimensional particles of discrete sizes (Figure 1) 1,2. Tapping mode AFM involves the repeated oscillation of the cantilever, which permits imaging of relatively soft biomaterials such as DNA and proteins. One of the notable benefits of AFM over other nanoscale microscopy techniques is its relative adaptability to visualize individual proteins and macromolecular complexes in aqueous buffers, including near-physiologic buffered conditions, in real-time, and without staining or coating the sample to be imaged. 
The method presented here describes the imaging of DNA and an immunoadsorbed transcription factor (i.e. the glucocorticoid receptor, GR) in buffered solution (Figure 2). Immunoadsorbed proteins and protein complexes can be separated from the immunoadsorbing antibody-bead pellet by competition with the antibody epitope and then imaged (Figure 2A). This allows for biochemical manipulation of the biomolecules of interest prior to imaging. Once purified, DNA and proteins can be mixed and the resultant interacting complex can be imaged as well. Binding of DNA to mica requires a divalent cation 3,such as Ni2+ or Mg2+, which can be added to sample buffers yet maintain protein activity. Using a similar approach, AFM has been utilized to visualize individual enzymes, including RNA polymerase 4 and a repair enzyme 5, bound to individual DNA strands. These experiments provide significant insight into the protein-protein and DNA-protein biophysical interactions taking place at the molecular level. Imaging individual macromolecular particles with AFM can be useful for determining particle homogeneity and for identifying the physical arrangement of constituent components of the imaged particles. While the present method was developed for visualization of GR-chaperone protein complexes 1,2 and DNA strands to which the GR can bind, it can be applied broadly to imaging DNA and protein samples from a variety of sources.

A tapping mode atomic force microscope (AFM) method for the visualization of plasmid DNA, cytoplasmic proteins, and DNA-protein complexes is described. The method includes alternate approaches for preparing samples for AFM imaging following biochemical manipulation. DNA containing specific protein interacting regions are observed in near-physiologic buffer conditions.

Visualizzazione del DNA ricombinante e Complessi proteina usando la microscopia a forza atomica

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On ...

Ricerca

Bioingegneria

Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM)

Surface topology, e.g. of cells growing on a substrate, is determined with nanometer precision by Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM). Cells are cultivated on transparent slides and incubated with a fluorescent marker homogeneously distributed in their plasma membrane. Illumination occurs by a parallel laser beam under variable angles of total internal reflection (TIR) with different penetration depths of the evanescent electromagnetic field. Recording of fluorescence images upon irradiation at about 10 different angles permits to calculate cell-substrate distances with a precision of a few nanometers. Differences of adhesion between various cell lines, e.g. cancer cells and less malignant cells, are thus determined. In addition, possible changes of cell adhesion upon chemical or photodynamic treatment can be examined. In comparison with other methods of super-resolution microscopy light exposure is kept very small, and no damage of living cells is expected to occur.

Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy VA-TIRFM

Topology of cell adhesion on a substrate is measured with nanometre precision by variable-angle total internal reflection fluorescence microscopy (VA-TIRFM).

Nanotopology dell&#39;adesione cellulare su angolo di inclinazione variabile Microscopia Total Internal Reflection Fluorescence (VA-TIRFM)

Surface topology, e.g. of cells growing on a substrate, is determined with nanometer precision by Variable-Angle Total Internal Reflection Fluorescence ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

Ad alta risoluzione Spatiotemporal Analisi del recettore Dynamics per singola molecola di microscopia a fluorescenza

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

3D Orbital Inseguimento in una Modified microscopio a due fotoni: un&#39;applicazione per il monitoraggio di intracellulare vescicole

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

Da Veloce imaging di fluorescenza di Molecular legge Diffusione su membrane cellulare dal vivo in un microscopio commerciale

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy (DHM)

Accurately detecting and counting sparse bacterial samples has many applications in the food, beverage, and pharmaceutical processing industries, in medical diagnostics, and for life detection by robotic missions to other planets and moons of the solar system. Currently, sparse bacterial samples are counted by culture plating or epifluorescence microscopy. Culture plates require long incubation times (days to weeks), and epifluorescence microscopy requires extensive staining and concentration of the sample. Here, we demonstrate how to use off-axis digital holographic microscopy (DHM) to enumerate bacteria in very dilute cultures (100-104 cells/mL). First, the construction of the custom DHM is discussed, along with detailed instructions on building a low-cost instrument. The principles of holography are discussed, and a statistical model is used to estimate how long videos should be to detect cells, based on the optical performance characteristics of the instrument and the concentration of the bacterial solution (Table 2). Video detection of cells at 105, 104, 103, and 100 cells/mL is demonstrated in real time using un-reconstructed holograms. Reconstruction of amplitude and phase images is demonstrated using an open-source software package.

Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy DHM

Digital holographic microscopy (DHM) is a volumetric technique that allows imaging samples 50-100X thicker than brightfield microscopy at comparable resolution, with focusing performed post-processing. Here DHM is used for identifying, counting, and tracking microorganisms at very low densities and compared with optical density measurements, plate count, and direct count.

Quantificare i microrganismi alle concentrazioni basse usando microscopia digitale oleografica (DHM)

Accurately detecting and counting sparse bacterial samples has many applications in the food, beverage, and pharmaceutical processing industries, in ...

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic significance. Possible reasons for the lack of exploration are the experimental limitations and technical challenges that render the study of cell division in 3D culture inefficient. Here, we describe an imaging-based method to efficiently study mammalian cell division and cell-matrix interactions in 3D collagen matrices. Cells labeled with fluorescent H2B are synchronized using the combination of thymidine blocking and nocodazole treatment, followed by a mechanical shake-off technique. Synchronized cells are then embedded into a 3D collagen matrix. Cell division is monitored using live-cell microscopy. The deformation of collagen fibers during and after cell division, which is an indicator of cell-matrix interaction, can be monitored and quantified using quantitative confocal reflection microscopy. The method provides an efficient and general approach to study mammalian cell division and cell-matrix interactions in a physiologically relevant 3D environment. This approach not only provides novel insights into the molecular basis of the development of normal tissue and diseases, but also allows for the design of novel diagnostic and therapeutic approaches.

This protocol efficiently studies mammalian cell division in 3D collagen matrices by integrating synchronization of cell division, monitoring of division events in 3D matrices using live-cell imaging technique, time-resolved confocal reflection microscopy and quantitative imaging analysis.

Divisione delle cellule dei mammiferi in matrici 3D tramite microscopia confocale quantitativa di riflessione

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately ten&#160;to twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.

Here we present a practical guide of building an integrated microscopy system, which merges conventional epi-fluorescent imaging, single-molecule detection-based super-resolution imaging, and multi-color single-molecule detection, including single-molecule fluorescence resonance energy transfer imaging, into one set-up in a cost-efficient way.

Lo svolgimento di diverse modalità di Imaging con un microscopio a fluorescenza

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

Visualizzazione di formazione di aderenze in cellule per mezzo di avanzati filatura microscopia di fluorescenza di riflessione interna totale disco

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence microscopy. Super-resolution three-dimensional Structured Illumination Microscopy (3D-SIM) is a promising tool to overcome this hurdle and reveal the molecular details of the process of germination of Bacillus subtilis (B. subtilis) spores. Here, we describe the use of a modified SIMcheck (ImageJ)-assistant 3D imaging process and fluorescent reporter proteins for SIM microscopy of B. subtilis spores&#8217; germinosomes, cluster(s) of germination proteins. We also present a (standard)3D-SIM imaging procedure for FM4-64 staining of B. subtilis spore membranes. By using these procedures, we obtained unsurpassed resolution for germinosome localization and show that &#62;80% of B. subtilis KGB80 dormant spores obtained after sporulation on defined minimal MOPS medium have one or two GerD-GFP and GerKB-mCherry foci. Bright foci were also observed in FM4-64 stained spores&#8217; 3D-SIM images suggesting that inner membrane lipid domains of different fluidity likely exist. Further studies that use double labeling procedures with membrane dyes and germinosome reporter proteins to assess co-localization and thus get an optimal overview of the organization of Bacillus germination proteins in the inner spore membrane are possible.

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

Germinant receptor proteins cluster in &#8216;germinosomes&#8217; in the inner membrane of Bacillus subtilis spores. We describe a protocol using super resolution microscopy and fluorescent reporter proteins to visualize germinosomes. The protocol also identifies spore inner membrane domains that are preferentially stained with the membrane dye FM4-64.

Visualizzazione di Germinosomes e la membrana interna in Bacillus subtilis spore

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence ...