The development of chemiluminescence as an alternative to radioactivity for documentation of electrophoresis blots (eg Westerns) has necessitated the development of a new generation of documentation systems with exceptionally high sensitivity and dynamic range.
These systems typically use 12-bit, cooled CCD cameras in place of the 8-bit non-cooled cameras normally used in gel documentation. This article aims to illustrate the importance of these specifications in achieving the higher level of performance for the system. The illustrations are images acquired using the UVIchemi chemiluminescence documentation system (UVItec, UK) together with the TD216 test device (Biostep, Germany) which employs a set of LEDs to emit a broad range of signal intensities from 60 photons/ms up to 4.8 x 106 photons/ms.
The number of bits associated with the camera determines the number of grey levels of the image. For an 8-bit camera this number is 256 whereas for a 12-bit camera it is 4096. Therefore, a faint signal detected by a 12-bit camera, would incorporate 16x more grey-levels than the same signal with an 8-bit camera, making measurement of the signal more precise. A 12-bit dynamic range also allows a broader range of signal intensities to be simultaneously detected and measured.
The effect of cooling a CCD camera is to reduce the amount of anoise' generated by the system. Specifically, cooling reduces a type of noise called the adark current', which is a temperature dependent asignal' generated by the camera sensors even when there is no light to detect. CCD cameras are often cooled to below 0oC to reduce dark current noise to a minimum. In the UVIchemi system, the sensor chip is cooled directly by a two-stage Peltier device. In addition, the camera compartment is also cooled by another Peltier device. In some systems, cooling may be less efficient, with the camera merely being externally cooled by a single Peltier. The benefit of low noise in an imaging system is that it increase sensitivity by minimising amasking' of faint signals by excessive noise and increases dynamic range by maximising the useable portion of the total 12-bit range.
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UVitec is based in Cambridge, UK. www.uvitec.co.uk
