In this work, we demonstrate the use of Two-Photon Fluorescence Lifetime Imaging Microscopy (TP-FLIM) for
intracellular calcium ([Ca2+]i) measurement with a calcium sensitive fluorescent dye in a neuronal cell model and brain
tissue. Calcium perturbations were induced via chemical stimulation and our results signify the potential of TP-FLIM
for quantitative ([Ca2+]i) measurement within the physiological range of intracellular [Ca2+].
The fluorescent decay kinetics of an exogenous fluorescent dye injected or immobilized in a polymer implant can be sensitive to the biochemical environment of the tissue and can provide quantitative in vivo biochemical information of the tissue site. In this research project, we develop the rationale for NIR fluorescence spectroscopy of biologically relevant dyes for the sensing of analyte concentration, such as Ca2+, pH, glucose, etc. In vivo analytical sensing using fluorescence spectroscopy is complicated not only by the scattering of tissue, but also by the fact that most sensing fluorophores exhibit more than one (multi-exponential) decay lifetime. The objective of this paper is to demonstrate the concept of fluorescence lifetime spectroscopy in scattering media using a pH sensing dye, Carboxy Seminaphthofluorescein-1 (C-SNAFL-1). Work to extrapolate our results to an analyte-sensing dye construct embedded in a polymer are also presented.
Supported by the National Institutes of Health (R01 CA67176).
The generation and propagation of fluorescence light through tissue offers the potential for biomedical diagnostics and clinical biosensing. Arising from an exogenous fluorescent dye injected as a contrast agent or immobilized in a polymer implant, the fluorescent decay kinetics can be sensitive to the biochemical environment of the tissue, providing quantitative in vivo information of the confined tissue site. To date, all studies of fluorescence in scattering media have been confined to dyes with single-exponential decay kinetics, yet most of sensing fluorophores exhibit multi-exponential decay. The goal of the study was to develop a time-dependent model describing the generation and propagation of fluorescent light emitted from dyes exhibiting multi-exponential decay kinetics. To experimentally investigate multi-exponential decays, we employed two dyes, 3,3-diethylthiatricarbocyanine iodide (DTTCI) and Indocynanine Green (ICG), which exhibit distinctly different lifetimes. The experimental study was divided into two parts. In the first part, fluorescence phase-modulation measurements were made as a function of modulation frequency at various concentration ratios of the two dyes in dilute nonscattering solutions. In the second part, frequency measurements of phase-modulation were made at similar concentration ratios as the first part, but in presence of scattering (2% intralipid). The multi-exponential decay parameters extracted from the phase-modulation data in the first part of the study were used to predict the phase-shift and amplitude-attenuation values in the presence of scattering. Using a diffusion model, the predictions were then compared to the second part of the experimental study.
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