Frequency-domain lifetime measurements and sensing in highly scattering media

Abstract

For clinical chemistry, or for non-invasive sensing through skin, it is often necessary to obtain quantitative information in highly scattering media. We describe two simple methods for fluorescence lifetime measurements in highly scattering media, and in particular in an intralipid suspension. Lifetimes can be measured using an intensity decay law that accounts for the time delays and pulse-broadening effects of multiple light-scattering events in the intralipid. Alternatively, the phase and modulation measurements can be performed relative.to a reference fluorophore with a known lifetime. These approaches provide reliable lifetime data for a pH-sensitive fluorophore contained within a microeuvette 4 mm under the surface of the intralipid suspension. Fluorescence lifetime-based sensing is now recognized as a valuable methodology in clinical and analytical chemistry, and the possibility of lifetime-based sensing in turbid media has been demonstrated.

Keywords: Clinical chemistry; Fluorescence; Fluorescence spectroscopy; Frequency-domain fluorescence; Lifetime-based sensing; Non-invasive sensing; pH sensing; Photon migration; Time-resolved fluorescence.

Fig. 1.

Intuitive description of transdermal lifetime-based sensing.

Fig. 2.

Experimental geometry for lifetime-based sensing in turbid media.

Fig. 3.

Effect of multiple scattering events on time-domain (top) or phase-modulation (bottom) measurements of a fluorescence lifetime. Pulsed or modulated excitation (· · · · ·), time-delayed scattered light (—), and thefluorescence (- - -), which is further delayed by the lifetime τ, arc shown.

Fig. 4.

Absorption and emission spectra oftbe pH sensor Carboxy SNARF-6 (CF6) and the reference fluorophore Texas Red Hydrazide (TRH). The transmission profile (· · · · ·) of the 620 nm interference filter used to isolate the emission is also shown. Intralipid was not present.

Fig. 5.

Frequency response of TRH in the microeuvette at various depths below the intralipid surface.

Fig. 6.

Frequency response of CS6, in the microcuvette 4 nun below the intralipid surface, when measured relative to the scattered light (A), relative to the TRI-I standard (B), and without intralipid (C).

Fig. 7.

pH sensing at a 4 mm depth in 0.25% intralipid using CS6. Excitation wa the mode-locked output of an argon-ion laser at 514 nm. The emission was detected at 640 nm. Phase and modulation data were obtained without intralipid (○), in intralipid vs. the scattered fight from the surface (●) and in intralipid vs. the TRH standard (■).