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

इस काम के मैक्स प्लैंक सोसायटी द्वारा वित्त पोषित किया गया था.

1. नमूना तैयार करना <ol class="lalpha"><li> कवर निकल जाता है की तैयारी Coverslips धूल के कणों से साफ किया जाना चाहिए के रूप में TIRF पृष्ठभूमि coverslip (छवि 1 ए, मूवी 1) से उत्पन्न होने वाले संकेतों के प्रति संवेदनशील है. <ol><li> ढक्...

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	<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 माइक्रोस्कोपी छवि परिधीय प्रोटीन के लिए पसंद की तकनीक है. क्षणभंगुर क्षेत्र की कम गहराई में प्रवेश ध्यान प्रकाश है, जो शोर अनुपात करने के लिए एक बहुत अच्छा संकेत होता है और उच्च फ्रेम दर, या बहुत दुर्?...

<table border="1"><tbody><tr><td> उपकरण / अभिकर्मक के नाम </td><td> टाइप </td><td> कंपनी </td><td> सूचीपत्र संख्या </td></tr><tr><td> कक्षीय शेखर </td><td> उपकरण </td><td> UniEquip </td><td> UNITWIST 3 - डी घुमाव शेखर </td></tr><tr><td> TIRF खुर्दबीन </td><td> तक </td><td></td><td> अनुकूलित सेटअप </td></tr><tr><td> ग्लास कंटेनर </td><td> उपकरण </td><td> Vitlab </td><td> 340-232,880,353 </td></tr><tr><td> सिरेमिक धुंधला रैक </td><td> उपकरण </td><td> थॉमस विज्ञान. </td><td> 8542E40 </td></tr><tr><td> Concanavalin एक </td><td> अभिकर्मक </td><td> सिग्मा </td><td> L7647 </td></tr><tr><td> # 1 (18 x 18 मिमी) coverslips </td><td> माइक्रोस्कोप </td><td> मेंजेल Glaser </td><td> BB018018A1 </td></tr><tr><td> खुर्दबीन स्लाइड </td><td> माइक्रोस्कोप </td><td> मेंजेल जीलेज़र </td><td> AA00000102E </td></tr><tr><td> विसर्जन तेल </td><td> माइक्रोस्कोप </td><td> Zeiss </td><td> Immersol 518F </td></tr><tr><td> Agarose </td><td> अभिकर्मक </td><td> Invitrogen </td><td> 16500-500 </td></tr><tr><td> FluoSpheres </td><td> अभिकर्मक </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

कुल आंतरिक प्रतिबिंब प्रतिदीप्ति माइक्रोस्कोपी (TIRF) संरचनाओं उच्च विपरीत और लौकिक संकल्प में कोशिका की सतह के करीब निरीक्षण के लिए एक शक्तिशाली तरीका है. हम प्रदर्शन कैसे TIRF दीवार संलग्न सेल बैक्टीरियल और फंगल कोशिकाओं के प्रांतस्था में प्रोटीन की गतिशीलता का अध्ययन करने के लिए नियोजित किया जा सकता है.

प्रांतस्था संगठन और गतिशीलता के सूक्ष्मजीवों में दृश्य, कुल आंतरिक प्रतिबिंब प्रतिदीप्ति माइक्रोस्कोपी का उपयोग

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

पुनः संयोजक डीएनए और प्रोटीन परिसरों परमाणु शक्ति माइक्रोस्कोपी का प्रयोग का विज़ुअलाइज़ेशन

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

कोशिका आसंजन की चर कोण कुल आंतरिक प्रतिबिंब प्रतिदीप्ति माइक्रोस्कोपी (VA-TIRFM) पर Nanotopology

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

एकल अणु प्रतिदीप्ति माइक्रोस्कोपी द्वारा रिसेप्टर गतिशीलता के उच्च संकल्प Spatiotemporal विश्लेषण

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

एक संशोधित दो photon माइक्रोस्कोप में 3 डी कक्षीय ट्रैकिंग: Intracellular vesicles की ट्रैकिंग के लिए एक आवेदन

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

एक वाणिज्यिक माइक्रोस्कोप में लाइव कोशिका झिल्ली पर आण्विक प्रसार कानून को फास्ट प्रतिदीप्ति इमेजिंग से

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

कम सांद्रता पर सूक्ष्मजीवों को बढ़ाता डिजिटल होलोग्राम माइक्रोस्कोपी (DHM) का उपयोग

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

मात्रात्मक फोकल प्रतिबिंब माइक्रोस्कोपी के माध्यम से 3 डी मैट्रिक्स में स्तनधारी सेल डिवीजन

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

एक प्रतिदीप्ति माइक्रोस्कोप के साथ कई इमेजिंग मोड का आयोजन

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

उन्नत कताई डिस्क के माध्यम से कोशिकाओं में आसंजन गठन visualizing-कुल आंतरिक प्रतिबिंब प्रतिदीप्ति माइक्रोस्कोपी

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

बसिलस सबटिल् स बीजाणु में अंकुरोसोम और भीतरी झिल्ली का दृश्यावलोकन

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