A unique charge-coupled device/xenon arc lamp based imaging system for theaccurate detection and quantitation of multicolour fluorescence

In recent years the use of fluorescent dyes in biological applications has dramatically increased. The continual improvement in the capabilities of th ese fluorescent dyes demands increasingly sensitive detection systems that provide accurate quantitation over a wide linear dynamic range. In the fiel d of proteomics, the detection, quantitation and identification of very low abundance proteins are of extreme importance in understanding cellular pro cesses. Therefore, the instrumentation used to acquire an image of such sam ples, for spot picking and identification by mass spectrometry, must be sen sitive enough to be able, not only to maximise the sensitivity and dynamic range of the staining dyes but, as importantly, adapt to the ever changing portfolio of fluorescent dyes as they become available. Just as the availab le fluorescent probes are improving and evolving so are the users applicati on requirements. Therefore, the instrumentation chosen must be flexible to address and adapt to those changing needs. As a result, a highly competitiv e market for the supply and production of such dyes and the instrumentation for their detection and quantitation have emerged. The instrumentation cur rently available is based on either laser/photomultiplier tube (PMT) scanni ng or lamp/charge-coupled device (CCD) based mechanisms. This review briefl y discusses the advantages and disadvantages of both System types for fluor escence imaging, gives a technical overview of CCD technology and describes in detail a unique xenon/are lamp CCD based instrument, from PerkinElmer L ife Sciences. The Wallac-1442 ARTHUR (TM) is unique in its ability to scan both large areas at high resolution and give accurate selectable excitation over the whole of the UV/visible range. It operates by filtering both the excitation and emission wavelengths, providing optimal and accurate measure ment and quantitation of virtually any available dye and allows excellent s pectral resolution between different fluorophores. This flexibility and exc itation accuracy is key to multicolour applications and future adaptation o f the instrument to address the application requirements and newly emerging dyes.