Bioinformatics Advance Access originally published online on December 3, 2008
Bioinformatics 2009 25(3):346-352; doi:10.1093/bioinformatics/btn620
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Estimation of errors introduced by confocal imaging into the data on segmentation gene expression in Drosophila
1St Petersburg State Polytechnic University, St Petersburg, 195251, Russia and 2Stony Brook University, New York, 11794-3600, USA
*To whom correspondence should be addressed.
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Abstract |
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Motivation: Currently the confocal scanning microscopy of fluorescently tagged molecules is extensively employed to acquire quantitative data on gene expression at cellular resolution. Following this approach, we generated a large dataset on the expression of segmentation genes in the Drosophila blastoderm, that is widely used in systems biology studies. As data accuracy is of critical importance for the success of studies in this field, we took a shot to evaluate possible errors introduced in the data by acquisition and processing methods. This article deals with errors introduced by confocal microscope.
Results: In confocal imaging, the inevitable photon noise is commonly reduced by the averaging of multiple frames. The averaging may introduce errors into the data, if single frames are clipped by microscope hardware. A method based on censoring technique is used to estimate and correct this type of errors. Additional source of errors is the quantification of blurred images. To estimate and correct these errors, the Richardson–Lucy deconvolution method was modified to provide the higher accuracy of data read off from blurred images of the Drosophila blastoderm. We have found that the sizes of errors introduced by confocal imaging make up 
5–7% of the mean intensity values and do not disguise the dynamic behavior and characteristic features of gene expression patterns. We also defined a range of microscope parameters for the acquisition of sufficiently accurate data.
Availability: http://urchin.spbcas.ru/downloads/step/step.htm
Contact: myasnikova{at}spbcas.ru
Supplementary information: Supplementary data are available at Bioinformatics online.
Associate Editor: Olga Troyanskaya
Received on July 18, 2008; revised on November 25, 2008; accepted on November 27, 2008
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