Ratiometric fluorescence polarization as a cytometric functional parameter: theory and practice

Abstract

The use of ratiometric fluorescence polarization (RFP) as a functional parameter in monitoring cellular activation is suggested, based on the physical phenomenon of fluorescence polarization dependency on emission wavelengths in multiple (at least binary) solutions. The theoretical basis of this dependency is thoroughly discussed and examined via simulation. For simulation, aimed to imitate a fluorophore-stained cell, real values of the fluorescence spectrum and polarization of different single fluorophore solutions were used. The simulation as well as the experimentally obtained values of RFP indicated the high sensitivity of this measure. Finally, the RFP parameter was utilized as a cytometric measure in three exemplary cellular bioassays. In the first, the apoptotic effect of oxLDL in a human Jurkat FDA-stained T cell line was monitored by RFP. In the second, the interaction between cell surface membrane receptors of human T lymphocyte cells was monitored by RFP measurements as a complementary means to the fluorescence resonance energy transfer (FRET) technique. In the third bioassay, cellular thiol level of FDA- and CMFDA-labelled Jurkat T cells was monitored via RFP.

Fluorescence anisotropy: from single molecules to live cells

“…FA has the added advantage of being able to operate in a ratiometric mode, with an interesting example being an application that studied cellular activation using cytometry. 31 Examples of the use of FA in the areas of receptor research and high-throughput drug discovery are provided in reviews by Leopoldo et al, 32 and Wu and Doberstein. 33 Various classes of binding agents can be identified, with a common distinction being categorization as either a protein/ peptide, or a nucleic acid.…”

The Immunosuppressive Effect of Methotrexate in Active Rheumatoid Arthritis Patients vs. its Stimulatory Effect in Nonactive Patients, as Indicated by Cytometric Measurements of CD4 + T Cell Subpopulations

“…This assumption is supported by our previous results (Herman et al, 2003), which demonstrate (by widely accepted techniques of apoptosis detection) that MTX apoptotic effect is found only in activated healthy mononuclear cells, i.e., blast lymphocytes, but not in resting lymphocytes. The monitored changes in fluorescence polarization reflect the intracellular plasma viscosity, during either lymphocyte stimulation Kaplan et al, 1997) or early apoptotic signal , thus providing a valuable indicator of cellular functionality (Cercek et al, 1974;Shapiro, 1995;Yishai et al, 2003). The assumption is also supported by the presently reported results of FDA hyperpolarization.…”

Extended dynamic range imaging for noise mitigation in fluorescence anisotropy imaging

“…Direct in vivo fluorescence intensity measurements are crucial for measuring fluorescence marker concentration or other intrinsic parameters, but they can be quite challenging due to the absence of an internal reference and the presence of various nonrelevant artifacts, e.g., tissue scattering and absorption, sample-detector path geometry, or fluctuations in the excitation source. 1,2 Ratiometric fluorescence indicators are better posed than the fluorescence intensity ones and have been used extensively to measure for example changes of calcium ions concentrations, 3 membrane potentials, 4,5 and other parameters. 6 Depending on the specific imaging modality, the fluorescence signal is typically measured via excitation or emission under different or the same conditions of polarizations and wavelengths, and the ratiometric quantity is directly or indirectly calculated by taking the ratio of the two measurements.…”

“…Direct in vivo fluorescence intensity measurements are crucial for measuring fluorescence marker concentration or other intrinsic parameters, but they can be quite challenging due to the absence of an internal reference and the presence of various nonrelevant artifacts, e.g., tissue scattering and absorption, sample–detector path geometry, or fluctuations in the excitation source. 1 , 2 …”