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Enhanced Imaging of Chemiluminescence
Sponsored,vendor-submitted protocol   Sponsored by Bio-Rad Laboratories    Published in BioTechniques Protocol Guide 2008 (p.15)

Signal Detection by Direct Digital Imaging vs. X-ray Film

Imaging chemiluminescent blots with charged-coupled device (CCD) cameras is rapid, and the easiest, most accurate method. Traditionally, the chemiluminescent signal from blots has been detected by X-ray film. The popularity of chemiluminescence has led to development of special films with enhanced sensitivity to the blue wavelengths of light emitted by chemiluminescent substrates. If quantitative data is desired, film exposed to chemiluminescent signals is imaged on a densitometer, and the digital image analyzed to produce the final data. With recent advances in technology and reduction in the cost of computers and CCD cameras, direct digital acquisition of chemiluminescent signals has become practical. A CCD camera requires only that a wrapped blot be placed on the imaging surface, and the integration (exposure) time set. Then the signal is analyzed.

Film is a sensitive medium for capturing signals but an inherent drawback is its sigmoidal response to light, which results in a narrow linear response region, thus limiting its dynamic range. In contrast, a 16 bit CCD camera such as that on the Molecular Imager® VersaDoc™ MP 5000 System has a nonsaturated linear response over the same range of samples (Figure 1).


(Click to enlarge)


Image Quality Perception

A common misperception when comparing images from CCD cameras to X-ray film is that the CCD image is not as good as the film image. A high quality image is one without saturated features, and the data throughout the entire sample range is linear. Film reaches saturation quickly, and does not indicate the degree to which the exposure is beyond saturation. Therefore, a broad range of signals appears similar, preventing direct quantitative comparison (Figure 2, upper panel). A CCD image, on the other hand, with its greater dynamic range, will typically have data distributed throughout the sample range (Figure 2, lower panel). An image closer in appearance to the more familiar X-ray film can be achieved by transforming the representation of the CCD image to express the data from mid range and higher at gray levels that are close to black.


Fig 2. (Click to enlarge)


Summary

Digital acquisition of chemiluminescent signals has become a practical technology that is easy to use. Compared to film, CCD imaging has a greater dynamic range, which can be easily displayed by adjusting the data expression.

BioMax and Kodak are trademarks of Eastman Kodak Company.

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