Computational protocol: Analysis of a marine phototrophic biofilm by confocal laser scanning microscopy using the new image quantification software PHLIP

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[…] Interfacial biofilms constitute an important reservoir of microbial life in aquatic systems. The main focus of biofilm research has been on the examination of bacterial biofilms (reviewed in []), which cause widespread problems in industrial fluid processing applications (reviewed in []) and play a major role in human infection diseases []. In general, growth of biofilms is initiated by the attachment of cells to a conditioned substratum, after which in situ growth of micro-organisms and production of extracellular matrix components build up the biofilm structure []. Many studies have investigated the physiology and structure of bacterial biofilms in order to understand the underlying processes of attachment, detachment and growth (reviewed in []).In contrast to medical and industrial settings where biofilms generally develop in the dark, those present in freshwater and marine aquatic ecosystems are often exposed to sunlight. In these environments, biofilms are diverse species communities typically dominated by micro-algal consortia [-]. Phototrophic biofilms in aquatic environments represent an important carbon source for other trophic levels [,] and affect mass transfer processes at the ecosystem scale [].Confocal laser scanning microscopy (CLSM) is an important method for the study of biofilm structure. Since its first application [], CLSM has become widely used to improve the understanding of the biofilm architecture []. CLSM is a non-destructive and non-invasive method with the capability to provide time-resolved three-dimensional images of biofilms. In addition, multiple fluorescent channels can be recorded simultaneously, which offers the possibility to directly observe the development of individual biofilm components []. Multi-channel observations of phototrophic biofilms take advantage of autofluorescence of the micro-algae or record the fluorescence signal of biofilm components labeled with specific markers. Analysis of CLSM images has shown that biofilm communities form highly structured microbial assemblies [,]. Studies using CLSM have further confirmed that the development of biofilms depends on various factors including mass transport properties [] and have shown the importance of metabolic interactions within the microbial communities themselves [].In many studies, the analysis of CLSM data has been of rather qualitative than quantitative nature and consisted entirely of a visual image inspection [,]. This approach is however subjective and not feasible when large quantities of data have to be analyzed, which is often necessary to ensure the significance of the outcome of the analyses []. For quantitative analysis of CLSM data, computer software with different functionalities ranging from cell number counting [] to the classification of bacteria morphotypes [] are currently available and are increasingly being used. In order to address the necessity of a more directed morphological quantification of biofilm data, image analysis programs such as COMSTAT [] and ISA [] were developed to quantitatively analyze single channel 3D CLSM data of biofilm imaging by determining a set of morphological parameters. Despite the wide use of the available image analysis software in morphological investigations of bacterial biofilms [,,,], their application is restricted to the analysis of single-channel CLSM data at a time. This single-channel limitation falls short of the capabilities of modern CLSM devices and is a significant disadvantage since multi-channel images have to be quantified in separate analysis sessions and multiple channel analysis distinguishing various biofilm components cannot be approached comprehensively.This communication describes a new image quantification package, PHLIP (PHobia Laser scanning microscopy Imaging Processor), which enables analysis of multi-channel CLSM data. PHLIP was developed in the scope of the EU/FP5 funded project PHOBIA (Phototrophic Biofilms and Its potential Applications []) and extends previous work on automated CLSM image analysis [,] by including a new set of tools to automatically quantify CLSM imaging in biofilm systems, necessary to produce statistically meaningful results. PHLIP is available as an open source project (see Materials and Methods) and encourages users to extend the program's capabilities. The potential of PHLIP is illustrated here in a study of the temporal development of a natural phototrophic biofilm. [...] One and two way nested analysis of variance (ANOVA) designs were used to test the effects of days and sampling slides on the biofilm parameters obtained from the PHLIP analysis. For one way ANOVA, slides were nested within days. For two way ANOVA, slides were nested within the interaction between days and channels. Analyses were performed on box-cox transformed data []. In addition, regression analysis was performed on the all-channel parameters. Either linear or exponential decay functions were used to fit the datasets. Analyses of variance were performed using Statistica 6.1, while regression analysis was performed using the software package Origin 6.0. […]

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