Name: Visualizzazione di Cortex Organizzazione e Dynamics in microrganismi, utilizzando Total Internal Reflection Fluorescence Microscopy (Video) | JoVE Journal
Uploaded: 2012-05-01T12:03:00.000+00:00
Duration: 14 min 14 s
Description: Scopri la microscopia TIRF per studiare la dinamica delle proteine al cortice cellulare in microrganismi come lieviti e batteri. Questa tecnica offre alta risoluzione e contrasto per analisi in vivo.

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This work was funded by the Max Planck society.

<ol><li>Axelrod, D., Thompson, N. L., Burghardt, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Total+internal+inflection+fluorescent+microscopy%5BTitle%5D)+AND+%22J.+Microsc%22%5BJournal%5D)">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/pubmed?term=((Combinatorial+microscopy%5BTitle%5D)+AND+%22Nat.+Rev.+Mol.+Cell+Biol%22%5BJournal%5D)">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/pubmed?term=((Selective+imaging+of+surface+fluorescence+with+very+high+aperture+microscope+objectives%5BTitle%5D)+AND+%22J.+Biomed.+Opt%22%5BJournal%5D)">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/pubmed?term=((Processive+movement+of+MreB-associated+cell+wall+biosynthetic+complexes+in+bacteria%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((Bleach+it%2C+switch+it%2C+bounce+it%2C+pull+it%3A+using+lasers+to+reveal+plant+cell+dynamics%5BTitle%5D)+AND+%22J.+Exp.+Bot%22%5BJournal%5D)">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/pubmed?term=((Total+synthesis+and+absolute+configuration+of+avenolide%2C+extracellular+factor+in+Streptomyces+avermitilis%5BTitle%5D)+AND+%22J.+Antibiot.+%28Tokyo%29%22%5BJournal%5D)">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/pubmed?term=((Cortical+actin+dynamics%3A+Generating+randomness+by+formin%28g%29+and+moving%5BTitle%5D)+AND+%22Bioarchitecture%22%5BJournal%5D)">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/pubmed?term=((Cortical+actin+dynamics+driven+by+formins+and+myosin+V%5BTitle%5D)+AND+%22J.+Cell.+Sci%22%5BJournal%5D)">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/pubmed?term=((TORC2+plasma+membrane+localization+is+essential+for+cell+viability+and+restricted+to+a+distinct+domain%5BTitle%5D)+AND+%22Mol.+Biol.+Cell%22%5BJournal%5D)">TORC2 plasma membrane localization is essential for cell viability and restricted to a distinct domain.</a> Mol. Biol. Cell. 20, 1565-1575 (2009).</li>
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No conflicts of interest declared.

TIRF microscopy is the technique of choice to image peripheral proteins. The low penetration depth of the evanescent field minimizes of out of focus light, which leads to a very good signal to noise ratio and allows data acquisition with high frame rates, or imaging of very weakly expressed proteins. The combination of high contrast and high frame rates makes TIRF microscopy a perfect tool for imaging spatio-temporal dynamics of cortically localized proteins. Interestingly the thick cell wall surrounding many microorgani...

<table><tbody><tr><td>Orbital Shaker</td><td>UniEquip</td><td>UNITWIST 3-D ROCKER SHAKER</td><td></td></tr><tr><td>TIRF microscope</td><td></td><td>Customized setup</td><td></td></tr><tr><td>Glass container </td><td>Vitlab</td><td>340-232880353</td><td></td></tr><tr><td>Ceramic staining rack</td><td>Thomas Scientific</td><td>8542E40</td><td></td></tr><tr><td>Concanavalin A</td><td>Sigma-Aldrich</td><td>L7647</td><td></td></tr><tr><td>Coverslips #1(18 x 18 mm)</td><td>Menzel-Glaser</td><td>BB018018A1</td><td></td></tr><tr><td>Microscope Slides</td><td>Menzel-Glaser</td><td>AA00000102E</td><td></td></tr><tr><td>Immersion Oil</td><td>Carl Zeiss, Inc.</td><td>Immersol 518F</td><td></td></tr><tr><td>Agarose</td><td>Invitrogen</td><td>16500-500</td><td></td></tr><tr><td>FluoSpheres</td><td>Invitrogen</td><td>F8795</td><td></td></tr></tbody></table>

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

Visualization of Cortex Organization and Dynamics in Microorganisms, using 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. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.
At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.
We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

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

The overall goal of this procedure is to acquire high quality, total internal reflection, fluorescence or turf images of cell wall bearing microorganisms. This is accomplished by first preparing cover slips and cells. The second step is to precisely align the microscope setup for optimal image quality.Next, the correct incidents, angles, and imaging conditions are chosen for acquisition of images or movies. The final step is post imaging processing of the acquired images. Ultimately, turf microscopy is used to study the dynamics of protein localization at the cell cortex.The main advantage of this technique over exceeding metals like conventional or confocal for microscopy, is that the image contrast int microscopy it's very high, allowing fast times and low light. As such, This method can help answer key questions in the field of membrane research, such as the mechanisms of lateral protein segregation, or the mobility of membrane proteins. Cover slips for turf microscopy should be cleaned from dust particles prior to use.Since turf is sensitive to background signals arising from dust on a dirty cover slip. A cleaned cover slip with less background is required to begin the procedur

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

Research

JoVE Journal

Bioengineering

Here we demonstrate the protocols for performing single-molecule fluorescence microscopy on living bacterial cells to enable functional molecular complexes to be detected, tracked and quantified.

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

This video describes TIRF, a total internal reflection microscopy-based technique to visualize actin and microtubule polymerization dynamics. The method allows the high-resolution imaging of actin and microtubule coupling dynamics in real-time, which is essential for understanding cellular crosstalks.

TIRF Microscopy to Visualize Actin and Microtubule Coupling Dynamics

Source: Daniele, F., et. al TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis. J. Vis. Exp. (2015).This video demonstrates the imaging of human neuroblastoma cells using a total internal reflection fluorescence (TIRF) microscope to visualize fluorophore-tagged synaptic vesicles. The cells are first focused in epifluorescence mode. Adjustments are then made to achieve total internal reflection,...

Cell Imaging Using Total Internal Reflection Fluorescence Microscopy

Source: Winkle, C. C., et al. Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis. J. Vis. Exp. (2016)This video demonstrates total internal reflection fluorescence (TIRF) microscopy for visualizing exocytic events in cortical neurons. A pH-sensitive green fluorescent protein (GFP) marker fluoresces upon vesicle fusion, with selective TIRF excitation capturing membrane-proximal exocytosis in time-lapse imaging.

Total Internal Reflection Fluorescence Microscopy for Visualization of Exocytic Events 

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.

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

This protocol is a guide for implementing interference reflection microscopy on a standard fluorescence microscope for label-free, high-contrast, high-speed imaging of microtubules using in vitro surfaces assays.

Implementation of Interference Reflection Microscopy for Label-free, High-speed Imaging of Microtubules

We describe an imaging approach for the determination of the average oligomeric state of mEGFP-tagged-receptor oligomers induced by ligand binding in the plasma membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&amp;B) analysis.

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis

Here, a TIRF microscopy-based in vitro reconstitution assay is presented to simultaneously quantify and compare the dynamics of two microtubule populations. A method is described to simultaneously view the collective activity of multiple microtubule-associated proteins on crosslinked microtubule bundles and single microtubules.

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

We present a protocol for implementing interference-reflection microscopy and total-internal-reflection-fluorescence microscopy for the simultaneous imaging of dynamic microtubules and fluorescently labeled microtubule-associated proteins.

Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic Microtubules and Associated Proteins

This protocol is a guide for visualizing dynamic actin and microtubules using an in vitro total internal fluorescence (TIRF) microscopy assay.

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy

Scopri la microscopia TIRF per studiare la dinamica delle proteine al cortice cellulare in microrganismi come lieviti e batteri. Questa tecnica offre alta risoluzione e contrasto per analisi in vivo.

Visualizzazione di Cortex Organizzazione e Dynamics in microrganismi, utilizzando Total Internal Reflection Fluorescence Microscopy (Video) | JoVE Journal

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

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