Bioinformatics Advance Access published online on June 9, 2005
Bioinformatics, doi:10.1093/bioinformatics/bti531
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1 DVP/OVRR/CBER/FDA, Bethesda, MD 20892, United States
* To whom correspondence should be addressed.
Motivation: Recent advances in confocal microscopy have allowed scientists to assess the expression and to some extent the interaction/colocalization of multiple molecules within cells and tissues. In some instances, accurately quantifying the colocalization of two or more proteins may be critical. This can require the acquisition of multiple z-plane images (z stacks) throughout a specimen and as such we report here the successful development of a freeware, open-source image analysis tool, IMAJIN_COLOC, developed in PERL (v. 5.8, build 806) using the PERLMagick libraries (ImageMagick). Using a pixel-by-pixel analysis algorithm, IMAJIN_COLOC can analyze images for antigen expression (any number of colors) and can measure all possible combinations of colocalization for up to three colors by analyzing a z stack gallery acquired for each sample. The simultaneous (i.e., in a single pass) analysis of three-color colocalization, and batch analysis capabilities are distinctive features of this program. Results: A control image, containing known individual and colocalized pixel counts, was used to validate the accuracy of IMAJIN_COLOC. As further validation, pixel counts and colocalization values from the control image were compared to those obtained with the software packaged with the Zeiss laser-scanning microscope (LSM AIM, version 3.2). The values from both programs were found to be identical. To demonstrate the applicability of this program in addressing novel biological questions, we examined the role of neurons in eliciting an immune reaction in response to viral infection. Specifically, we successfully examined expression of the chemokine RANTES in measles virus (MV) infected hippocampal neurons and quantified changes in RANTES production throughout the entire disease period. The resultant quantitative data were also evaluated visually using a gif image created during the analysis. Availability: PERL (ActivePerl, version 5.8) is available at activestate.com; the PERLMagick libraries are available at imagemagick.org, and IMAJIN_COLOC, the source code and user documentation can be downloaded from http://www.fda.gov/cber/research/imaging/imageanalysis.htm.
Received September 15, 2004
Revised April 27, 2005
Accepted June 6, 2005
Article
A quantitative determination of multi-protein interactions by the analysis of confocal images using a pixel-by-pixel assessment algorithm
2 Laboratory of Experimental Carcinogenesis, National Cancer Institute, Bethesda, MD 20892, United States
3 DVP/OVRR/CBER/FDA, Bethesda, MD 20892, United States; Department of Psychiatry, Johns Hopkins University, Baltimore, MD, United States; Department of Medicine, Johns Hopkins University, Baltimore, MD, United States
T. H. Malik, E-mail: Malik{at}cber.fda.gov
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