Siobhan Pickett, M.S., Sean Carriedo, Ph.D. and Chang Wang, Ph.D.
Axon Instruments, Inc. 3280 Whipple Road, Union City, CA 94587
Last updated: September 13, 2001
Dark Current noise: Even in the absence of light input, the thermal emissions from any photon-detecting device can produce a low amount of noise (measured in electrons per second) that has been termed Dark Current noise. In the case of PMTs, Dark Current noise can originate from the photocathode and/or from current leaking through the dynodes of the PMT. The GenePix 4000B has been designed to reduce dark current noise to negligible levels through two approaches: 1) the PMTs chosen for use in the GenePix 4000B show very low dark current levels, and 2) the dwell time of the lasers on each pixel is very short resulting in a negligible number of dark current electrons generated for each pixel.
Shot noise: Shot or statistical noise is the only significant type of noise that needs to be considered in the GenePix 4000B. It is generated by the input of light and results from the variable nature of photons. With shot noise, it is important to realize that as the signal intensity increases the shot noise increases as the square root of the signal collected, so the SNR actually decreases as signal intensity increases.
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 calculated manually as outlined below. Either method is useful for comparing scanners from the same manufacturer or from different manufacturers.
1. Select a slide that is representative of your typical data slides.
The type of PMT used in the GenePix 4000 Array Scanners has a wide dynamic range, so it detects and amplifies both strong specific signal on arrayed spots, and low-level background fluorescence, which is primarily due to non-specific hybridization. Simply increasing the gain to the maximum setting may not produce the optimum SNR. Thus, the optimal PMT setting must be determined empirically. The first step is to make a dilution series of the probes to be used. Next, the relationship between the SNR and PMT gain is determined. From these data the optimal PMT settings for the fluorescent probes is obtained. Data presented below illustrates the relationship between a wide range of PMT voltages and the concentration for the commonly used Cy3 and Cy5 probes.
1. SNR as a Function of PMT Gain for a Single Dye Concentration.
Figure 1: SNR for both the Cy5 (Red line) and Cy3 (Green line) was measured over the full range of PMT voltages for a single spot in the middle of a dilution series (A, below saturation at 1000 V), and near the detection limit (B, defined as SNR = 3).
Figure 2: SNR over a full range of PMT voltages for both the Cy3 (A) and Cy5 (B) was measured for a dilution series spanning six orders of magnitude.