Phasor Analysis of Fluorescence Lifetime Enables Quantitative Multiplexed Molecular Imaging of Three Probes

Abstract

The excited-state lifetime is an intrinsic property of fluorescent molecules that can be leveraged for multiplexed imaging. An advantage of fluorescence lifetime-based multiplexing is that signals from multiple probes can be gathered simultaneously, whereas traditional spectral fluorescence imaging typically requires multiple images at different excitation and emission wavelengths. Additionally, lifetime and spectra could both be utilized to expand the multiplexing capacity of fluorescence. However, resolving exogenous molecular probes based exclusively on the fluorescence lifetime has been limited by technical challenges in analyzing lifetime data. The phasor approach to lifetime analysis offers a simple, graphical solution that has increasingly been used to assess endogenous cellular autofluorescence to quantify metabolic factors. In this study, we employed the phasor analysis of FLIM to quantitatively resolve three exogenous, antibody-targeted fluorescent probes with similar spectral properties based on lifetime information alone. First, we demonstrated that three biomarkers that were spatially restricted to the cell membrane, cytosol, or nucleus could be accurately distinguished using FLIM and phasor analysis. Next, we successfully resolved and quantified three probes that were all targeted to cell surface biomarkers. Finally, we demonstrated that lifetime-based quantitation accuracy can be improved through intensity matching of various probe−biomarker combinations, which will expand the utility of this technique. Importantly, we reconstructed images for each individual probe, as well as an overlay of all three probes, from a single FLIM image. Our results demonstrate that FLIM and phasor analysis can be leveraged as a powerful tool for simultaneous detection of multiple biomarkers with high sensitivity and accuracy.

Navigating FRET Imaging Techniques for Biosensor Applications

“…Further explanation of phasor theory for TD-and FD-FLIM can be found in the ISS phasor tech note (89), in a paper on three-probe multiplexed lifetime phasor by Rahim et al (90), in an extensive paper by Ranjit et al (91) and in our own work on particle-based phasor analysis which includes a broad theoretical explanation (11). The phasor method is interesting as it allows the 'sorting' of FLIM pixels rather than applying a strenuous fitting algorithm.…”

Two-photon excitation fluorescence in ophthalmology: safety and improved imaging for functional diagnostics

“…FLIM extracts the lifetime from the fluorescence emission (10), allowing the separation of fluorophores that just by the intensity at a particular wavelength would not be distinguishable (10,(13)(14)(15)(16)(17)) (see Supplementary Material).…”

Particle-based phasor-FLIM-FRET resolves protein-protein interactions inside single viral particles

“…The phasor approach to FLIM attempts the unmixing of contributing factors in a graphical way but equally requires user input such as the instrumental referencing ( 29 ), characterization of the autofluorescence contribution, and correct location of pure species in the phasor plot ( 6 , 19 ). Nonetheless, it is generally accepted that phasor-FLIM allows for an easy identification of data clusters and species assignment combined with convenient data representation using the image-phasor reciprocity ( 30 ). Indeed, one can select pixels from within the image and analyze these in phasor space or, inversely, one can select a region of interest in phasor space, and false-color these in the image.…”