METHODS – Biofilms can be cultivated in
vitro by inoculating laboratory flow cells. Staining methods such as
commercially available probes or green fluorescence protein (GFP) expression are
used for fluorescence image acquisition using CSLM. The choice of staining
method is system specific and done according to the individual characteristics
and the purpose of the analysis. Thin optical cross sections of the biofilm are
imaged using CSLM. The entire three-dimensional biofilm structure is recorded by
scanning along the biofilm depth, and the stacks of cross sections are stored as
digital images. The software tools for quantitative analysis use a stack of
optical cross sections by converting it into a matrix of data points, or
three-dimensional image.
Prior to quantitative image processing, the raw images from CSLM undergo a
series of preprocessing steps which include
Correction of flow cell tilting
Solid substratum surface detection
Contrast calibration (specially relevant for binary image processing)