The LightCyclerTM: A Microvolume Multisample Fluorimeter with Rapid Temperature Control

Abstract

Experimental and commercial microvolume fluorimeters with rapid temperature control are described. Fluorescence optics adopted from flow cytometry were used to interrogate 1 – 10 – μL samples in glass capillaries. Homogeneous temperature control and rapid change of sample temperatures (10°C/s) were obtained by a circulating air vortex. A prototype 2-color, 32-sample version was constructed with a xenon arc for excitation, separate excitation and emission paths, and photomultiplier tubes for detection. The commercial LightCyclerTM, a 3-color, 24-sample instrument, uses a blue light-emitting diode for excitation, paraxial epi-illumination through the capillary tip and photodiodes for detection. Applications include analyte quantification and nucleic acid melting curves with fluorescent dyes, enzyme assays with fluorescent substrates and techniques that use fluorescence resonance energy transfer. Microvolume capability allows analysis of very small or expensive samples. As an example of one application, rapid cycle DNA amplification was continuously monitored by three different fluorescence techniques, which included using the double-stranded DNA dye SYBR® Green I, a dual-labeled 5′-exonuclease hydrolysis probe, and adjacent fluorescein and Cy5TM-labeled hybridization probes. Complete amplification and analysis requires only 10–15 min.