phlip quantitative biofilm imaging. why another imaging software? modern confocal microscopes...
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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
all channels
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Bacteria
Algae
Extracel. matrix
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
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