Presentation: P02010

All optical systems are more efficient in transmission of light along their optical axis than their periphery. The systematic error, termed vignetting, is caused by greater aperture blockage of off-axis light rays. This results in uneven sample illumination and fluorescence collection, yielding images that appear brighter at center, and darker at edges. To correct the error, one employs flat-field calibration (FFC): imaging of a uniform reference standard followed by software processing of sample images. Accurate FFC requires standards that closely mimic the optical properties of actual cell samples. This work discusses why confocal systems can employ a simple fluorescent solution as standard, while accurate FFC in wide-field microscopy requires optically thin standards, such as propriety reference plates developed by GE Healthcare. A standard should also be bleach resistant and long-term stable. We report results that establish the validity of the uniform reference standards and apply them to cellular imaging on IN Cell Analyzer platforms.

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