01/02/2019
Principle of extension of the parameter space by lifetime-based multiplexing in flow cytometry

Principle of extension of the parameter space by lifetime-based multiplexing in flow cytometry

Source: BAM, K. Hoffmann, FB 1.2

Flow cytometry is a valuable tool in basic research, environmental and food analysis, microbiology, clinical diagnosis, and other fields. Immuno-analytical platforms (bead-based immunoassays) consisting of flow cytometers and fluorescent microbeads allow for high throughput methods and gain popularity in, e.g., determination of pharmaceuticals, anthropogenic markers and bacteria in wastewaters. Our article in Scientific Reports describes an alternative method to the commonly used intensity-based encoding for spectral multiplexing in Flow Cytometry. As shown, luminescence lifetime (LT) as an additional encoding parameter can significantly increase the number of accessible and distinguishable analytes.

In close cooperation with two small and medium-sized enterprises (SMEs), a robust platform for lifetime flow cytometry (LT-FCM) consisting of an LT-FCM instrument and micrometer-sized polymer beads with luminophores showing different luminescence lifetimes was developed. For this proof-ofconcept research, we evaluated the impact of measurement parameters and instrument settings such as integration time range and data bin width, as well as measurement conditions (photon count number, signal-to-background ratio) on the classification of LT-codes. The current performance level of this newly developed, compact and low-cost LT-FCM using a single excitation wavelength and a single detection channel is five codes involving organic and inorganic encoding luminophores with LTs in the range of nanoseconds. LT-encoding in FCM can find broad applications ranging from alternative staining strategies for cell studies to the use of luminescent reporters whose emission properties are either sensitive to their microenvironment or respond to bioanalytical binding events by changing their luminescence decay kinetics. Moreover, label-free techniques based on intrinsic luminescence lifetime differences (autofluorescence) are conceivable. LT-FCM in routine analysis can help to increase the information density, e.g. from bioassays, by read-out of multiple analytes or targets in a single measurement through parallel and efficient detection.

Luminescence lifetime encoding in time-domain flow cytometry
Daniel Kage, Katrin Hoffmann, M. Wittkamp, J. Ameskamp, W. Göhde, Ute Resch-Genger
Scientific Reports, 2018
BAM Department Analytical Chemistry; Reference Materials