Document Detail

Diffusion measurements inside biofilms by image-based fluorescence recovery after photobleaching (FRAP) analysis with a commercial confocal laser scanning microscope.
MedLine Citation:
PMID:  20639359     Owner:  NLM     Status:  MEDLINE    
Research about the reactional and structural dynamics of biofilms at the molecular level has made great strides, owing to efficient fluorescence imaging methods in terms of spatial resolution and fast acquisition time but also to noninvasive conditions of observation consistent with in situ biofilm studies. In addition to conventional fluorescence intensity imaging, the fluorescence recovery after photobleaching (FRAP) module can now be routinely implemented on commercial confocal laser scanning microscopes (CLSMs). This method allows measuring of local diffusion coefficients in biofilms and could become an alternative to fluorescence correlation spectroscopy (FCS). We present here an image-based FRAP protocol to improve the accuracy of FRAP measurements inside "live" biofilms and the corresponding analysis. An original kymogram representation allows control of the absence of perturbing bacterial movement during image acquisition. FRAP data analysis takes into account molecular diffusion during the bleach phase and uses the image information to extract molecular diffusion coefficients. The fluorescence spatial intensity profile analysis used here for the first time with biofilms is supported both by our own mathematical model and by a previously published one. This approach was validated to FRAP experiments on fluorescent-dextran diffusion inside Lactococcus lactis and Stenotrophomonas maltophilia biofilms, and the results were compared to previously published FCS measurements.
François Waharte; Karine Steenkeste; Romain Briandet; Marie-Pierre Fontaine-Aupart
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Validation Studies     Date:  2010-07-16
Journal Detail:
Title:  Applied and environmental microbiology     Volume:  76     ISSN:  1098-5336     ISO Abbreviation:  Appl. Environ. Microbiol.     Publication Date:  2010 Sep 
Date Detail:
Created Date:  2010-08-25     Completed Date:  2010-11-30     Revised Date:  2011-07-25    
Medline Journal Info:
Nlm Unique ID:  7605801     Medline TA:  Appl Environ Microbiol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  5860-9     Citation Subset:  IM    
Cell and Tissue Imaging Facility (PICT-IBiSA), CNRS-Institut Curie, Centre de Recherche, Paris, France.
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MeSH Terms
Biofilms / growth & development*
Fluorescence Recovery After Photobleaching / methods*
Image Processing, Computer-Assisted / methods
Lactococcus lactis / growth & development,  metabolism,  physiology*
Microscopy, Confocal / methods*
Stenotrophomonas maltophilia / growth & development,  metabolism,  physiology*

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