Diffusion measurements inside biofilms by image-based fluorescence recovery after photobleaching (FRAP) analysis with a commercial confocal laser scanning microscope.

摘要: Research about the reactional and structural dynamics of biofilms at molecular level has made great strides, owing to efficient fluorescence imaging methods in terms spatial resolution fast acquisition time but also noninvasive conditions observation consistent with situ biofilm studies. In addition conventional intensity imaging, recovery after photobleaching (FRAP) module can now be routinely implemented on commercial confocal laser scanning microscopes (CLSMs). This method allows measuring local diffusion coefficients could become an alternative correlation spectroscopy (FCS). We present here image-based FRAP protocol improve accuracy measurements inside "live" corresponding analysis. An original kymogram representation control absence perturbing bacterial movement during image acquisition. data analysis takes into account bleach phase uses information extract coefficients. The profile used for first is supported both by our own mathematical model a previously published one. approach was validated experiments fluorescent-dextran Lactococcus lactis Stenotrophomonas maltophilia biofilms, results were compared FCS measurements.