Performance validation of an improved Xenon-arc lamp-based CCD camera system for multispectral imaging in proteomics

Abstract

Advances in gel-based nonradioactive protein expression and PTM detection using fluorophores has served as the impetus for developing analytical instrumentation with improved imaging capabilities. We describe a CCD camera-based imaging instrument, equipped with both a high-pressure Xenon arc lamp and a UV transilluminator, which provides broad-band wavelength coverage (380-700 nm and UV). With six-position filter wheels, both excitation and emission wavelengths may be selected, providing optimal measurement and quantitation of virtually any dye and allowing excellent spectral resolution among different fluorophores. While spatial resolution of conventional fixed CCD camera imaging systems is typically inferior to laser scanners, this problem is circumvented with the new instrument by mechanically scanning the CCD camera over the sample and collecting multiple images that are subsequently automatically reconstructed into a complete high-resolution image. By acquiring images in succession, as many as four different fluorophores may be evaluated from a gel. The imaging platform is suitable for analysis of the wide range of dyes and tags commonly encountered in proteomics investigations. The instrument is unique in its capabilities of scanning large areas at high resolution and providing accurate selectable illumination over the UV/visible spectral range, thus maximizing the efficiency of dye multiplexing protocols.