Name: visualization of cortex organization and dynamics in microorganisms, using total internal reflection fluorescence microscopy
Uploaded: 2012-05-01T12:03:00
Description: Watch this Scientific Journal Video about Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy at JoVE.com

This work was funded by the Max Planck society.

1. Sample Preparation
<ol class="lalpha">
 <li>Preparing cover slips
 Coverslips should be cleaned from dust particles as TIRF is sensitive to background signals arising from the coverslip (Fig. 1A, Movie 1).
 <ol>
 <li>Using forceps place cover slips in a ceramic holder with lid.</li>
 <li>Fill glass container with 1 M NaOH (can be reused multiple times).</li>
 <li>Incubate for 2 h under slow continuous rotation (orbital shaker). Excessive inc...

<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>

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 border="1">
 <tbody>
 <tr>
 <td>Name of the tool/reagent</td>
 <td>Type</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Orbital Shaker</td>
 <td>Tool</td>
 <td>UniEquip</td>
 <td>UNITWIST 3-D ROCKER SHAKER</td>
 </tr>
 <tr>
 <td>TIRF microscope</td>
 <td>Till</td>
 <td>&nbsp;</td>
 <td>Customized setup</td>
 </tr>
 <tr>
 <td>Glass container </td>
 <td>Tool</td>
 <td>Vitlab</td>
 <td>340-232880353</td>
 </tr>
 <tr>
 <td>Ceramic staining rack</td>
 <td>Tool</td>
 <td>Thomas Sci.</td>
 <td>8542E40</td>
 </tr>
 <tr>
 <td>Concanavalin A</td>
 <td>Reagent</td>
 <td>Sigma</td>
 <td>L7647</td>
 </tr>
 <tr>
 <td>Coverslips #1(18 x 18 mm)</td>
 <td>Microscope</td>
 <td>Menzel Gl&auml;ser</td>
 <td>BB018018A1</td>
 </tr>
 <tr>
 <td>Microscope Slides</td>
 <td>Microscope</td>
 <td>Menzel Gl&auml;ser</td>
 <td>AA00000102E</td>
 </tr>
 <tr>
 <td>Immersion Oil</td>
 <td>Microscope</td>
 <td>Zeiss</td>
 <td>Immersol 518F</td>
 </tr>
 <tr>
 <td>Agarose</td>
 <td>Reagent</td>
 <td>Invitrogen</td>
 <td>16500-500</td>
 </tr>
 <tr>
 <td>FluoSpheres</td>
 <td>Reagent</td>
 <td>Invitrogen</td>
 <td>F8795</td>
 </tr>
 </tbody>
</table>

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.

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) 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. ...

Research

JoVE Journal

Bioengineering

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 ...

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

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 ...

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., ...

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 ...

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 ...

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

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 ...

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 ...

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 ...

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 ...

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

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 ...