Fluorescence lifetime detection with particle counting devices

Fluorescence-based single particle counting devices have become very powerful tools for human health-related applications such as the detection of blood-borne pathogens. Instead of passing the sample fluid through a thin tube or microfluidic chip, as it is commonly practiced in flow cytometers and sorter devices, single particle counters scan the fluid volume by rotation and translation of the sample container. Hence, single particle counters are not limited by the fluid flow friction and can scan a large volume in a short timeframe while maintaining high sensitivity. A single particle can be detected in a milliliter of the fluid sample within minutes, and diagnostics are being developed using this principle. Until now, signal detection with particle counters has been based on signal intensity and signal separation into multiple wavelength bands coupled with multiple detectors, which limits the number of species that can be resolved. In this paper, we applied fluorescence lifetime detection to single particle counting to increase specificity and enable multiplexing with a single detector. We demonstrate how this principle can be used for diagnostic assays based on fluorescence quenching.