| Fluorescence imaging for monitoring the colocalization of two single molecules in living cells. | |
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MedLine Citation:
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PMID: 15596511 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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The interaction, binding, and colocalization of two or more molecules in living cells are essential aspects of many biological molecular processes, and single-molecule technologies for investigating these processes in live cells, if successfully developed, would become very powerful tools. Here, we developed simultaneous, dual-color, single fluorescent molecule colocalization imaging, to quantitatively detect the colocalization of two species of individual molecules. We first established a method for spatially correcting the two full images synchronously obtained in two different colors, and then for overlaying them with an accuracy of 13 nm. By further assessing the precision of the position determination, and the signal/noise and signal/background ratios, we found that two single molecules in dual color can be colocalized to within 64-100 nm (68-90% detectability) in the membrane of cells for GFP and Alexa633. The detectability of true colocalization at the molecular level and the erroneous inclusion of incidental approaches of two molecules as colocalization have to be compromised at different levels in each experiment, depending on its purpose. This technique was successfully demonstrated in living cells in culture, monitoring colocalization of single molecules of E-cadherin fused with GFP diffusing in the plasma membrane with single molecules of Alexa633 conjugated to anti-E-cadherin Fab externally added to the culture medium. This work established a benchmark for monitoring the colocalization of two single molecules, which can be applied to wide ranges of studies for molecular interactions, both at the levels of single molecules and collections of molecules. |
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Authors:
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Ikuko Koyama-Honda; Ken Ritchie; Takahiro Fujiwara; Ryota Iino; Hideji Murakoshi; Rinshi S Kasai; Akihiro Kusumi |
Publication Detail:
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Type: Evaluation Studies; Journal Article Date: 2004-12-13 |
Journal Detail:
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Title: Biophysical journal Volume: 88 ISSN: 0006-3495 ISO Abbreviation: Biophys. J. Publication Date: 2005 Mar |
Date Detail:
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Created Date: 2005-03-01 Completed Date: 2005-07-06 Revised Date: 2009-11-18 |
Medline Journal Info:
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Nlm Unique ID: 0370626 Medline TA: Biophys J Country: United States |
Other Details:
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Languages: eng Pagination: 2126-36 Citation Subset: IM |
Affiliation:
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Kusumi Membrane Organizer Project, Exploratory Research for Advanced Technology Organization (ERATO/SORST), Japan. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Fluorescent Dyes
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analysis*,
metabolism* Green Fluorescent Proteins / metabolism, ultrastructure Image Enhancement / methods Image Interpretation, Computer-Assisted / methods* Membrane Proteins / metabolism*, ultrastructure* Microscopy, Fluorescence, Multiphoton / methods* Microscopy, Video / methods* Protein Interaction Mapping / methods* |
| Chemical | |
Reg. No./Substance:
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0/Fluorescent Dyes; 0/Membrane Proteins; 147336-22-9/Green Fluorescent Proteins |
| Comments/Corrections | |
Comment In:
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Biophys J. 2009 Aug 19;97(4):L5-7
[PMID:
19686638
]
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From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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