Data reproducibility in fluorescence image analysis.
Fluorescence image analysis provides quantitative data on fluorescence in situ hybridization signals (FISH), immunofluorescence labelings, Green Fluorescent Protein (GFP) expression and microarrays. It is a valuable tool for decision making in the fields of biology and medicine. The aim of this study was to evaluate the reproducibility of fluorescence intensity measurements and standardization when acquisitions are performed under various but well defined conditions. Fluorescent intensity of standard beads (Inspeck series, Molecular Probes) was repeatedly measured using an image analyzer and automated procedures. Images were acquired using several integration times and neutral filter sets. A standardization procedure was used for expressing the data in a same unit: data were multiplied by the light attenuation factor and were divided by the CCD integration times. Results show that 1) standardization is possible 2) accurate and reliable fluorescence measurements can be obtained and 3) specimens showing large differences in fluorescence intensity can be objectively compared. Moreover fluorescent test slides including fluorochrome solutions and altuglas slides were tested for shading correction and as overall test systems.
Souchier
Catherine
C
INSERM U309 - DyOGen, Institut Albert Bonniot, La Tronche, France. Catherine.Souchier@ujf-grenoble.fr
Brisson
Christine
C
Batteux
Bernadette
B
Robert-Nicoud
Michel
M
Bryon
Paul-André
PA
D002138:Calibration / Q000592:standards; D007091:Image Processing, Computer-Assisted / Q000592:standards*; D008856:Microscopy, Fluorescence / Q000379:methods / Q000592:standards*; D012015:Reference Standards; D015203:Reproducibility of Results; D012984:Software