Background-free cytometry using rare earth complex bioprobes

In the analytical fields of microbiology, disease diagnosis, and antibioterrorism, there are increasing demands for rapid yet inexpensive quantification of rare cells. This has proven to be challenging by the conventional spectral discrimination of using traditional fluorescent probes, since the strong autofluorescence from background cells or particles overlaps spectrally with the probe fluorescence. This is particularly true when the target cell occurs at very low frequency (one in more than 100,000 background cells) representing a needle-in-a-haystack problem. This chapter describes a low-cost solution to overcome this problem by employing a novel detection technology, namely the use of rare-earth (lanthanide) complex bioprobes with luminescence lifetimes in the hundreds of microseconds. Due to this long persistence in lifetime, microsecond duration luminescence can be detected under conditions where fluorescent backgrounds would overwhelm the emission of conventional fluorochromes. The nanosecond duration autofluorescence associated with cells can be suppressed by time-gated detection, allowing detection of long lifetime lanthanide-based bioprobes with minimal background interference. This technology is applicable to a broad range of detection technologies in both cytometry and imaging. In this chapter, we highlight a typical application in the monitoring of the rare microbial pathogens Cryptosporidium parvum and Giardia lamblia against the complex background of concentrated drinking water. We also describe recent nanotechnological developments in the production of rare-earth nanoparticle bioprobes required for this technology. Other applications of rare-earth bioprobes and time-gated flow cytometry will also be discussed.