Eliminating Interference from Autofluorescence in Flow Cytometry - SUMMARY
Flow cytometry is a powerful cell analysis tool, providing information on cell count, shape, size, DNA content,
redox state, membrane permeability, and surface receptors, among other features. It can be used on both live
and fixed cells; and can be used to non-destructively sort cells based on these characteristics. Flow cytometry
has utility in research and clinical fields as diverse and important as tumor biology research, cancer and AIDS
diagnostics, stem cell therapy, immunophenotyping, and cancer immunotherapy.
Flow cytometry relies extensively on fluorescent labeling cells; with the emission light collected from the
interrogation region divided spectrally by filters and beam splitters, and directed to detectors that measure
intensity. One common problem derives from the fact that the exogenously added fluorophores are not the only
source of fluorescence in the sample. So-called cellular autofluorescence can be, in some circumstances, a
major contributor to the signal present in each of the detection channels. A number of solutions have been
proposed, but they often involve complex experimental protocols, or complex processing of data. This leaves
the field wanting for a solution to the nagging problem of interference from cellular autofluorescence.
Here we propose to use fluorescence lifetime as a second discriminating parameter in order to distinguish
autofluorescence interference from the desired signal. We propose to build a simple system using a single
pulsed laser, which will be sufficient to demonstrate feasibility of our method. The system will include a high-
speed pulsed 375-nm laser capable of 0.5-ns pulses and the required fast electronics and 3 emission
channels, which span the range of the broad cellular autofluorescence.
We will leverage the deep expertise of Kinetic River in designing and building complex, innovative cytometry
instruments; implementing novel hardware solutions to enable lifetime measurements in flow cytometry; and
innovative, proprietary, and patent-protected techniques for rapid analysis of complex lifetime decay curves.
We will be assisted by consultants and collaborators with deep experience in fluorescence lifetime
measurement, autofluorescence signals, cell-based assays and algorithm development. To demonstrate the
feasibility of our approach, we are pursuing the following strategy: (1) build prototype instrument and optimize
system hardware; (2) characterize instrument performance; and (3) validate using relevant biological samples.
Successful completion of this work will pave the way for further development in Phase II, which will see the
addition of other laser lines with lifetime capabilities to create a general purpose instrument. This instrument
will be similar in cost to similarly equipped cytometers, but will have the advantage of automated elimination of
cellular autofluorescence. Such an instrument will have wide-ranging utility, increased ease of use, and will
allow for clearer interpretation of experimental results.
