Background: Currently the combination of molecular tools, imaging techniques and\nanalysis software offer the possibility of studying gene activity through the use of\nfluorescent reporters and infer its distribution within complex biological threedimensional\nstructures. For example, the use of Confocal Scanning Laser Microscopy\n(CSLM) is a regularly-used approach to visually inspect the spatial distribution of a\nfluorescent signal. Although a plethora of generalist imaging software is available to\nanalyze experimental pictures, the development of tailor-made software for every\nspecific problem is still the most straightforward approach to perform the best\npossible image analysis. In this manuscript, we focused on developing a simple\nmethodology to satisfy one particular need: automated processing and analysis of\nCSLM image stacks to generate 3D fluorescence profiles showing the average\ndistribution detected in bacterial colonies grown in different experimental conditions\nfor comparison purposes.\nResults: The presented method processes batches of CSLM stacks containing threedimensional\nimages of an arbitrary number of colonies. Quasi-circular colonies are\nidentified, filtered and projected onto a normalized orthogonal coordinate system,\nwhere a numerical interpolation is performed to obtain fluorescence values within a\nspatially fixed grid. A statistically representative three-dimensional fluorescent pattern\nis then generated from this data, allowing for standardized fluorescence analysis\nregardless of variability in colony size. The proposed methodology was evaluated by\nanalyzing fluorescence from GFP expression subject to regulation by a stressinducible\npromoter.
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