Compensation-Free, Highly Multiplexed Flow Cytometer - PROJECT SUMMARY
Flow cytometry is a workhorse technique in research and development as well as in clinical laboratories for
diagnosis and monitoring of disease. It is particularly useful in distinguishing between populations of immune
cells based on their expressed cell surface antigens. Standard flow cytometers use fluorescent tags, often
conjugated to monoclonal antibodies, to give qualitative and quantitative information about specific molecules
in the cell. This molecular specificity, coupled with the fact that information is obtained on a cell-by-cell basis
with very high throughput (up to 30,000 cells per second), make this a powerful technique. The ability to
multiplex (measure a variety of different molecular species in a single cell) further adds to its utility and to the
complexity of the scientific questions that can be addressed using this technique.
However, the level of multiplexing currently has limitations. Flow cytometry analysis typically relies solely on
spectral information of the fluorescent tags and is thus limited by the spectral overlap of fluorophore emissions.
Currently, employing even moderate levels of multiplexing requires complex instrumentation and careful
experimental design, execution and analysis to compensate for spectral spillover of signal into multiple
channels. This severely limits the range of scientific questions that can be addressed using current
technologies, deters novices in the technique from attempting more complex yet scientifically relevant
experiments, and is widely regarded as the major bottleneck in the field.
To overcome this limitation, we propose to build on our results from Phase I where we demonstrated feasibility
for an innovative approach that uses fluorescence lifetime as a separate, additional discriminating
measurement parameter. Our scheme for using fluorescent lifetime for multiplexing is simple, scalable, and
supported by preliminary data from our prototype instrument. Here we propose to upgrade our Phase I
instrument to increase multiplexing capability, challenge that instrument with a battery of verification tests, and
validate using a relevant biological assay, benchmarking results against a conventional flow cytometer.
The result will be a system enabling compensation-free flow cytometry experiments of 12 colors, while
requiring fewer lasers and detectors than similarly equipped commercial systems. Such a system would
serve a large segment of the market, including clinical cytometry, and is expected to see broad adoption. This
would pave the way for further development of an ultra-high (30+) parameter instrument suitable for
immunophenotyping, yet requiring significantly less compensation than current systems, and pushing the
boundaries of experimental complexity. Given flow cytometry’s wide-spread use and importance, this project
will have a high impact in many biomedical and clinical applications. Several large instrumentation companies
have already indicated an eagerness to engage in strategic partnerships aimed at commercialization.
