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

Lukas N Mueller12*, Jody FC de Brouwer3, Jonas S Almeida24, Lucas J Stal5 and João B Xavier6

Author Affiliations

1 Institute for Molecular Systems Biology, ETH Hönggerberg, CH-8093 Zürich, Switzerland

2 Instituto de Tecnologia de Quimíca e Biológica, Universidade Nova de Lisboa, 2781-901 Oeiras, Portugal

3 Scottish Association for Marine Science, Oban, Argyll, Scotland, PA37 1QA, UK

4 Department of Biostatistics, Bioinformatics and Epidemiology, Medical University of South Carolina, Charleston SC 29425, USA

5 Center for Estuarine and Marine Ecology, Netherlands Institute of Ecology (NIOO-KNAW), PO Box 140, 440 0 AC Yerseke, The Netherlands

6 Department of Biotechnology, Delft University of Technology, 2628 BC Delft, The Netherlands

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BMC Ecology 2006, 6:1  doi:10.1186/1472-6785-6-1

Published: 16 January 2006



Confocal laser scanning microscopy (CLSM) is the method of choice to study interfacial biofilms and acquires time-resolved three-dimensional data of the biofilm structure. CLSM can be used in a multi-channel modus where the different channels map individual biofilm components. This communication presents a novel image quantification tool, PHLIP, for the quantitative analysis of large amounts of multichannel CLSM data in an automated way. PHLIP can be freely downloaded from webcite


PHLIP is an open source public license Matlab toolbox that includes functions for CLSM imaging data handling and ten image analysis operations describing various aspects of biofilm morphology. The use of PHLIP is here demonstrated by a study of the development of a natural marine phototrophic biofilm. It is shown how the examination of the individual biofilm components using the multi-channel capability of PHLIP allowed the description of the dynamic spatial and temporal separation of diatoms, bacteria and organic and inorganic matter during the shift from a bacteria-dominated to a diatom-dominated phototrophic biofilm. Reflection images and weight measurements complementing the PHLIP analyses suggest that a large part of the biofilm mass consisted of inorganic mineral material.


The presented case study reveals new insight into the temporal development of a phototrophic biofilm where multi-channel imaging allowed to parallel monitor the dynamics of the individual biofilm components over time. This application of PHLIP presents the power of biofilm image analysis by multi-channel CLSM software and demonstrates the importance of PHLIP for the scientific community as a flexible and extendable image analysis platform for automated image processing.