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
and that very high throughput is possible (30,000 cells per second can be analyzed), 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. Typically, flow cytometry analysis 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 for the simultaneous interrogation of multiple
parameters within a cell requires high levels of complexity in instrumentation and analysis, and careful design
and execution of experiments. The related "compensation problem" (compensating for spillover of signal from
a fluorophore into multiple channels—due to the broad spectrum of most fluorophores) also causes significant
instrument complexity, cumbersome workflow, and inaccurate results. These factors put severe limits on the
range of scientific questions that can be addressed using current technologies, deter novices in the technique
from attempting more complex yet scientifically relevant experiments, and collectively are widely regarded as
the major current bottleneck in flow cytometry.
To overcome this limitation, we have developed 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. The
proposed project will establish the feasibility of lifetime-based multiplexing by modifying our experimental
platform with key hardware and algorithm improvements, challenging the resulting prototype with a
comprehensive set of verification and validation tests of increasing complexity, and culminating with a
comparison of our technology to existing technology in a standard four-color cell-based assay.
A successful outcome will lay the foundation for our planned development of commercial instruments (both
analyzers and sorters) that offer two major benefits to end users: (a) simple, turnkey, compensation-free
operation for instruments with low-to-medium levels of multiplexing; and (b) high-end instruments with two to
three times the current maximum multiplexing capability.
