The art of capturing biomolecular images is, at its heart, about harvesting photons. Collecting more photons from a sample results in sharper images and detection of faint protein bands which otherwise could have been missed. More photons also leads to a greater signal-to-noise (S/N) ratio and increased quantitation confidence.
The present generation of ECL reagents for chemiluminescence-based detection, such as ECL Prime, uses the enzymatic horseradish peroxidase (HRP) luminol reaction to deliver a stable, high signal. In fluorescence-based detection, CyDye fluorophores are the industry standard and now span across the entire visible range, with Amersham CyDye 700 and 800 near infrared (NIR)- labeled antibodies recently taking center stage.
Modern imagers have light sources, emission filters, and lenses designed to maximize detection of emitted sample photons. If more detected photons are better, when in the imaging process should we stop collecting photons and how can we decide the best exposure time? Image capture is usually allowed to continue to obtain maximum signal, with the limiting factor being saturation of bands of interest.
Grayscale images in .TIF format, composed exclusively of gray shades or levels, are used for quantitation in applications like Western blotting. In a 16-bit .TIF file generated by the imager, there are 65 535 gray levels. When surplus photons are collected, the maximum image pixel value remains at 65 535, regardless of the exposure time, and results in saturation. Yet, we might still want to collect more photons to see details in the images and detect weak bands. So, how can we overcome this challenge?
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