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Determining the Signal-to-Noise Ratio and Optimal Photomultiplier gain setting in the GenePix 4000B

the ability to resolve true signal from background noise. For fluorescent array imaging, SNR is commonly calculated as:

Signal Background
Standard Deviation of Background

A higher SNR indicates higher signal over background noise; a signal-to-noise ratio of 3 is commonly considered the lower limit for accurate detection. Signal may be detected below this value, but the accuracy of quantitative measurements decreases significantly. Referring to the equation above, the SNR can be maximized by increasing signal, decreasing background, or decreasing the noise (i.e., standard deviation of the background pixels).

In fluorescent arrays, the primary source of background signal is non-specific hybridization in the same plane of focus as the sample. The most effective way to increase the numerator in the SNR equation is to optimize hybridization and stringency wash conditions to minimize non-specific hybridization.

The denominator in the equation is determined by the evenness of the background signal. Contributing sources include biochemical factors such as probe purity and hybridization uniformity, as well as instrument factors such as stray photons and electronic noise inherent to all PMTs. Axon Instruments core expertise is in ultra-low-noise signal amplification, and the GenePix 4000 Array Scanners are designed to minimize all sources of instrument noise.

Reflecting the importance of the SNR, GenePix Pro has a built-in script that automatically calculates the SNR for the scanned microarray, and draws a histogram of the SNR for each wavelength. The Signal-to- Noise ratio may also be calcul
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