PHLIPquantitative biofilm imaging

Why another imaging software?

modern confocal microscopes record fluorescent signals in parallel for multiple channels

automated image analysis required to process large datasets

a software package which is extensible and flexible

Group of Prof. J. S. Almeida, ITQB, Lisbon

How PHLIP started…

previous work of Joao Xavier in image analysis- Xavier, J. B et al. 2001. Objective threshold selection procedure (OTS) for segmentation of scanning laser confocal microscope images. J Microbiol Methods 47:169-80. - Xavier, J. B et al . 2003. Automated biofilm morphology quantification from confocal laserscanning microscopy imaging. Water Sci Technol 47:31-7.

2003: EU-Project PHOBIA multi-channel CLSM software: PHOBIA Laser scanning Image Processor

Joao unifies his image analysis work in a MATLABpackage; version 0.1 of PHLIP

2004: start in PHOBIA and continue developing PHLIP,current release is version 0.4.1

Group of Prof. J. S. Almeida, ITQB, Lisbon

What is PHLIP?

MATLAB package, no additional toolboxes needed

modular architecture, can be easily modified orextended by new imaging function

features a new XML data structure PHLIP-ML

detailed information on: http://www.phlip.org

freely available under an open source license (http://sourceforge.net/projects/phlip)

Group of Prof. J. S. Almeida, ITQB, Lisbon

Program flow of PHLIP

Matlab package, no additional toolboxes needed

freely available under an open source license: http://sourceforge.net/projects/phlip

supports different microscope Leica formats

features a new XML data structure, PHLIP-ML

Group of Prof. J. S. Almeida, ITQB, Lisbon

1. Data Input

support for different Leica formats easily extendable with other formats PHLIP-ML, a new vendor neutral CLSM format

Group of Prof. J. S. Almeida, ITQB, Lisbon

2. Image preprocessing

Several function to preprocess the image data:

Group of Prof. J. S. Almeida, ITQB, Lisbon

3. Thresholding and GUI

Segmentation of the images can be done by: - automatic threshold determination - manually with help of GUI

Group of Prof. J. S. Almeida, ITQB, Lisbon

thresholdedthreshold & original

4. Morphological description

- Single channel: most popular parameters (Yang et al., 2000)

- Two channel: comparative analysis (Xavier et al., 2003)

- All channel: overall description of fluorescent channels

Group of Prof. J. S. Almeida, ITQB, Lisbon

5. Data output

Save results in HTML formatted file…

…or export in PHLIP-ML data format:

Group of Prof. J. S. Almeida, ITQB, Lisbon

Application of PHLIP

PHLIP was presented at the annual IWA Biofilm Conference, October 2004.

publications: Mueller et al., 2004, Proceedings IWA Biofilm symposium. Las Vegas, Montana. GrayMerod et al., 2004, Proceedings IWA Biofilm symposium. Las Vegas, Montana. Mueller et al., submitted.

some research groups working with PHLIP: Prof. S Wuertz, UC Davis, USA Dr. JFC de Brouwer, Yerseke, NL Dr. T Neu, Magdeburg, D

Group of Prof. J. S. Almeida, ITQB, Lisbon

Application of PHLIP

Example application of de Brouwer (unpublished):-> development of phototrophic biofilmat different light intensities-> biovolume analysis of 3 channels:bacteria / extracel. matrix / algae's

Group of Prof. J. S. Almeida, ITQB, Lisbon

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Outlook

new morphological parameters as for exampleFractal Dimension in 3D

support for CLSM data from cryosections

function for X / Y - rotation of shifted image stacks

Group of Prof. J. S. Almeida, ITQB, Lisbon

Acknowledgements

Thanks to…

Jonas Almeida

Joao Xavier

Jody de Brouwer

All members of the Biomath Group in Lisbon

Group of Prof. J. S. Almeida, ITQB, Lisbon