A frequency Domain Phase/Modulation Technique for Intracellular Multicomponent Fluorescence Analysis: Technical Approach and Pharmacological Applications

Abstract

A UV confocal laser microspectrofluorimeter prototype has been adapted for fluorescence lifetime measurements by using a frequency-domain phase/modulation method (modulation frequency 1 to 200 MHz, lifetime resolution: tenth of a ns). This technique enables excited state lifetimes of several fluorescent components to be resolved and determined. Through a global analysis, specific spectral contribution of each species can be monitored with no need to use model spectra. This approach is efficient to distinguish strongly overlapping components (e.g., intracellular multicomponent fluorescence signal) which otherwise cannot easily be discriminated from each other. Experimental set‒up is first described. Application dealing with an antisense oligonucleotide (a synthetic dT15oligomer analogue containing isopolar, non-isosteric, phosphonate [3'-O-P-CH2-O-5'] internucleotide linkages) bound to a fluorescent label (tetramethylrhodamine dye), in various solutions and interacting with living cells, is then presented. This is of a major interest for antisense and/or antigene strategies which have recently been developed as efficient ways to cure viral and/or malignant diseases. The frequency-domain phase/modulation technique enabled oligonucleotide stability inside the cells to be checked.

Intracellular uptake of modified oligonucleotide studied by two fluorescence techniques

“…The spectral maximum of the 1.4 ns component is at 584 nm while the emission maximum of the 4.9 ns component is shifted to 589 nm (estimated precision of the lifetime values is 0.3 ns and that of the emission maximum wavelength is 3 nm in this case). The latter component was already observed previously in the case of 3T3 cells incubated with TMRM (4.84 ns, 590 nm) 15. We interpret it, therefore, as a result of fluorophore binding to biomolecular targets in membranes and/or cytoplasm.…”

Cellular uptake of phosphorothioate oligonucleotide facilitated by cationic porphyrin: A microfluorescence study

“…Spectral dependence of the phase shift and the demodulation was recorded and a global analysis performed to decompose multicomponent fluorescence spectra to single lifetime spectral components. A more detailed description of the setup can be found elsewhere 10…”

Fluorophore spectroscopy in aqueous glycerol solution: the interactions of glycerol with the fluorophore

“…For testing the readiness of FLT measurement systems, Rhodamine-123 (τ = 4.2 ns) [36], Acridine Orange (τ = 2 ns) [37] and tetramethylrhodamine methyl ester (TMRM, τ = 2.4 ns) [38] were used as references. The measured values τ M = 4.158, 2.06 and 2.414 ns, respectively, are in good agreement with literature.…”