New Techniques for Time-resolved Flow Cytometry

Flow cytometry is a well-established and powerful high-throughput fluorescence measurement tool that allows for the sorting and enrichment of subpopulations of cells expressing unique fluorescence signatures. Flow cytometers measure total fluorescence from cells as well as the total amount of light that scatters off of each cell. Cytometry instruments cannot discriminate between different exogenous fluorophores that are tagged to cells and which spectrally overlap. Therefore cytometers are constantly improved and new versions are marketed that have expanded capabilities. New generations of cytometers for example, include systems that detect full spectral bandwidths, and microscopic-like images of fluorescence from single cells. Another unique enhancement to flow cytometers is adapting the cytometry hardware so that the device can measure a parameter called the fluorescence lifetime. The fluorescence lifetime is the average time each fluorophore spends in its excited state before decaying back to a ground level. Fluorescence lifetime measurements are useful in flow cytometry; this value is related to the relaxation kinetic profile (i.e. exponential) of excitable organic fluorophores, fluorescent proteins, and other inorganic species that fluoresce. A major advantage to measuring the fluorescence lifetime is that it is independent of fluorophore concentration and therefore not affected by non-linearity that can occur during fluorescence intensity measurements. Moreover the fluorescence lifetime is sensitive to biological events that influence fluorescence relaxation such as pH, temperature, and dark-state conversion (e.g. Forster resonance energy transfer (FRET)). These unique fluorescence lifetime characteristics make the lifetime an optimum photophysical parameter for flow cytometry.;At the first stage of this PhD dissertation project I designed, built, and optimized a new flow cytometry system and tested the ability to measure the fluorescence lifetime with specialty equipment we refer to as 'analog hardware.' I used values proportional to the fluorescence lifetime to sort cells and microspheres and prove sorting was possible with this new technique. The approach is known in the electro-optics field as 'homodyne detection' of high-frequency modulated optical signals. Using this theory I extracted the phase shift of fluorescence signals, which is a value proportional to the fluorescence lifetime. This technique is referred to here and in my published work as phase-filtering; it is presented, described and discussed in chapter 4.;In the second part of this dissertation project, I focused on the major issue of introducing florescence lifetime measurements onto a cytometer with minimal hardware modifications. Cytometry facility managers prefer to not make major changes to the cytometers in their core facilities. Therefore I sought to simplify how a cytometer can measure the fluorescence lifetime with very minimal modifications. I was able to do so by exploiting digital signal processing methods. I introduced a new parameter, referred to herein as the fluorescence-pulse-delay (FPD). The FPD can be measured from all cytometers, even those that were not originally designed to measure the fluorescence lifetime. I proved the FPD it is a valid representation of the average fluorescence lifetime and published this work in a premiere cytometry journal. A patent is also pending based on this idea. This is discussed in chapter 5.;In the last part of this dissertation project, I studied ways in which our data analysis can be improved to expand the resolution of the fluorescence lifetime measurement. I found that it is possible to take fluorescence lifetime data and extract more than just the average fluorescence lifetime. Until now no cytometry systems have proved able to measure more than average fluorescence lifetimes from single cells. Measuring a variety of lifetimes (in contrast to an average) is critical in biological sciences; multiple fluorescence lifetime components correlate to multiple intracellular events, micro environmental changes, and signaling pathways. One reason this approach has not been proved is because in cytometry cells pass through a finely focused laser beam in a very short time (e.g.10 micros). This short period of time limits the homodyne analysis methods. Therefore I proved how a data analysis method called polar plotting is possible and results in multiple fluorescence lifetime components. Polar plots help to visualize several participating fluorescent components and reveal multiple fluorescence lifetime component distributions. I demonstrate this by simultaneously combing values called phase shift and demodulation. This work will soon be submitted for publication pending collaborator comments. The details of this are discussed in chapter 6.