(131g) Single Cell Analysis Using Time-Resolved Spectroscopic Measurements in Flow Cytometry

Flow cytometry is a fluidic-based bioanalytical tool in which single cells are observed for the total amounts of fluorescence and Raleigh scatter light that are emitted subsequent to laser excitation.  As cells flow past the laser excitation source, the spectral-based measurements are rapidly made and interpreted to indicate new cellular phenomena and responses to treatments.  Transient cell movement in flow cytometry allows high-throughput measurements but also typically limits the ability to carefully discriminate the distinct spectral details elicited from any given endogenous or exogenous fluorophore.  In this contribution, we present a new time-resolved cytometry approach based on a time-domain spectroscopic approach.   A rapidly scanning laser beam is controlled by an acousto-optic deflector, to interact with a cell multiple times as it rapidly moves at a laminar flow rate through a flow channel.   Using deconvolution and other analysis approaches, the fluorescence decay is extracted.  Fluorophores with different lifetime values ranging approximately from 3 ns to 25 ns have been examined with this new type of flow cytometer.  The lifetime decay profiles appropriately reflect if the fluorophore has a longer fluorescence lifetime (~20ns) by having a longer “tail” or a shorter lifetime (~3 ns) by a shorter “tail.”   The instrument is further characterized by introducing multiple fluorescence lifetimes so as to observe multiple-fluorescence decays.   This new concept of flow cytometry can be largely impacting in that it could lead to in-depth single cell analysis for rare events (i.e. circulating tumor cells), and alleviate widespread cytometry issues with spectral overlap.