UC Irvine

Through fluorescence lifetime imaging microscopy (FLIM), traditional fluorescence detection capabilities are expanded to distinctly isolate multiple targets within the same spectral window based on intrinsic material differences in the lifetimes of probes. This work demonstrates the diversity of FLIM as a sensitive diagnostics and prognosis platform stemming from a tumorigenic background and broadening detection to utilizing immune markers with a commercially available set of fluorophores of unique lifetimes. Previous work in the Haun Lab has shown capabilities of detection of five differentiated lifetime species within the same spectral window with a focus in breast cancer applications. This work expands to six distinct probes within one spectral window, namely the green emission window, utilizing a cellular subtyping gating theory to maximize the information one pixel can contain based on applications within the temporal and spatial regimes. Clinically, having the ability to temporally target multiple molecular immune responses within one window, means that utilizing other color windows simultaneously will provide a level of sensitivity to one specimen where patient-specific diagnostics and prognosis capabilities become comprehensive and more achievable. Utilizing 6 probes in one color emission channel when multiplied by the approximate 10 windows achievable around the visible spectra makes FLIM capable of detecting a competitive multiplexing number of 60 plus molecular markers from a patient sample. The clinical potential of this work is demonstrated through initial phases with healthy patient samples of peripheral blood mononuclear cells (PBMCs) isolated from whole blood. Refining individualized patient treatment is an important goal since cancers often are heterogeneous in nature making a high achieving, versatile multiplexing FLIM platform ideal for visualizing cancer diagnosis and potential metastasis abnormalities by targeting known immune responses and emerging immune tumor markers that in parallel over multiple-colored spectra can deliver a powerful individualized patient platform. The FLIM field has also addressed many autofluorescence metabolism markers for mammalian work that can help identify the health, activity, and potential cancerous manifestations present within a cell linked to amplified signals from NADH and similar targets. This application is not yet well explored for plant health but is demonstrated as a promising direction for Protoplast development which attributes to understanding the health of crops, plant wound healing, and other viability factors. In summary, FLIM is a powerful analysis platform for various applications specifically in diagnostics and prognosis of various cell types and diseases.