Estimation of errors introduced by confocal imaging into the data on segmentation gene expression in Drosophila.
BACKGROUND
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 approximately 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.


BACKGROUND
http://urchin.spbcas.ru/downloads/step/step.htm
Myasnikova
Ekaterina
E
St Petersburg State Polytechnic University, St Petersburg, 195251, Russia. myasnikova@spbcas.ru
Surkova
Svetlana
S
Panok
Lena
L
Samsonova
Maria
M
Reinitz
John
J
D000818:Animals; D001756:Blastoderm / Q000378:metabolism; D004330:Drosophila / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D020869:Gene Expression Profiling / Q000379:methods; D017344:Genes, Insect; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D018613:Microscopy, Confocal / Q000379:methods*; D012044:Regression Analysis