AQUAN: A Macro for Automated and QUantitative ANalysis of Fluorescence in situ Hybridization Images
Summary:
AQUAN is developed to automatically and quantitatively analyze fluorescence in situ hybridization (FISH) images with Visilog software (version 6, Noésis, Les Ulis, France). Visilog is a image processing package developed by Noesis and is available commercially for MS-Windows and UNIX with X-Windows. Specifically, AQUAN is designed to identify individual objects in 4′, 6-diamidino-2-phenylindole (DAPI) images and recover two types of data from the corresponding FISH images, those describing the individual objects and those describing the background. For each object identified in the DAPI mask, the area, perimeter, shape, length, width, location, maximum intensity, mean intensity, total intensity, and mean background were retrieved automatically. Parameters for each cell are exported to a data file with tab-delimited text format for analysis with standard spreadsheet software. These data are further analyzed with cluster analysis (fuzzy k-means) to differentiate cells as positive and negative. Details about this macro and it application can be found in our published paper (Automated Image Analysis for Quantitative Fluorescence In Situ Hybridization with Environmental Samples) in Applied and Environmental Microbiology, May 2007, p. 2956-2962, Vol. 73, No. 9. free full text through PubMed
The AQUAN macro can be downloaded from the following link (FISH_monocamera_single.vba). In case of questions, please contact Dr. Julie L. Zilles (jzilles at illinois.edu).
User Instruction
This macro is used for the analysis of images from single labeling FISH experiments. Images should be captured from DAPI-stained cells and FISH probe hybridized cells, and saved in the format of tif.
- Make a new folder (C:\imagana) under C drive and copy all images to this folder.
- Rename DAPI images as “d01.tif, d02.tif… “, and rename corresponding FISH images as “f_d01.tif, f_d02.tif…”.
- Make an index file (listtif.txt) containing the names of DAPI images, each in a separate line, and save it in folder C:\imagana.
- Load the AQUAN macro and run the analysis; the result file (result.xls) will be generated in folder C:\imagana
To load this VBA code, download AQUAN (FISH_monocamera_single.vba) and paste it to the folder “C:\Program Files\Noesis\Visilog62\vba\”, then open “vba” menu in the title bar of Visilog, and click “open project”, finally choose “FISH_monocamera_single.vba” and the code will be loaded in the VBA editor of Visilog.
To use VBA macro, simply click “vba” menu in the title bar, then click “macros (Alt+F8″)”, choose the macro in “macro in”, finally click “run”.
Copyright
This program is copyrighted by Zhi Zhou, Marie Noelle Pons, Lutgarde Raskin, and Julie L. Zilles, and distributed under the following conditions:
You may make copies of AQUAN for your own use and modify those copies. All copies of AQUANN must retain our name and copyright notice. You may not sell AQUAN or distribute AQUAN in conjunction with a commercial product or service without the expressed written consent of the authors. Furthermore, because AQUAN is a derived work, Visilog’s copyright still applies.
THIS PROGRAM IS PROVIDED “AS IS”‘ AND WITHOUT ANY EXPRESS OR IMPLIED WARRANTIES, INCLUDING, WITHOUT LIMITATION, THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE.
Acknowledgements
This material is based upon work supported by the UIUC-CNRS (Centre National de Recherche Scientifique) Cooperation Program, the U.S. Department of Agriculture under cooperative agreement AG 58-3620-1-179, and the National Pork Board project #05-068.