Exciting Immunofluorescence at the Violet End: AlexaFluors and Quantum Dots
Francis L. Battye
The Walter & Eliza Hall Institute
The early expansion of flow cytometric immunofluorochromes proceeded in the long wavelength direction, first with the introduction of red wavelength lasers and then with the construction of an array of tandem dyes that took emissions to the edge of the infrared, while less development occurred at the violet end of the visible spectrum. The advent of short wavelength solid-state lasers has stimulated renewed interest in that spectral region.
This paper reports on a series of practical tests of the usefulness of several ultra-violet and violet-excited immunofluorochromes.
The primary test system hardware was a MoFlo cell sorter, on which was installed a solid-state 405nm laser to be used as a selectable alternative to an existing 351-364nm UV beam. Modifications to the laser steering tower and optics were required. Some additional analyses were performed on an LSR-I analyser. The biological system was mouse lymphoid cells stained with biotinilated B220 and avidin conjugated fluorochrome.
The fluorochromes tested were the conventional Alexa Fluors 350 and 405 and the more avant-garde Quantum Dots of the Streptavidin Sampler Kit with emissions at 525, 565, 585, 605, 655, 705 and 800nm. All were compared for signal to background with standard Phycoerythrin.streptavidin.