A hyperspectral fluorescence lifetime imaging (FLIM) instrument is developed to study endogenous fluorophores in biological tissue as an optical biopsy tool. This instrument is able to spectrally, temporally, and spatially resolve fluorescence signal, thus providing multidimensional information to assist clinical tissue diagnosis. An acousto-optic tunable filter (AOTF) is used to realize rapid wavelength switch, and a photomultiplier tube and a high-speed digitizer are used to collect the time-resolved fluorescence decay at each wavelength in real time. The performance of this instrument has been characterized and validated on fluorescence tissue phantoms and fresh porcine skin specimens. This dual-arm AOTF design achieves high spectral throughput while allowing microsecond nonsequential, random wavelength switching, which is highly desirable for time-critical applications. In the results reported here, a motorized scanning stage is used to realize spatial scanning for two-dimensional images, while a rapid beam steering technique is feasible and being developed in an ongoing project.

Glioma itself accounts for 80% of all malignant primary brain tumors, and glioblastoma multiforme (GBM) accounts for 55% of such tumors. Diffuse reflectance and fluorescence spectroscopy have the potential to discriminate healthy tissues from abnormal tissues and therefore are promising noninvasive methods for improving the accuracy of brain tissue resection. Optical properties were retrieved using an experimentally evaluated inverse solution. On average, the scattering coefficient is 2.4 times higher in GBM than in low grade glioma (LGG), and the absorption coefficient is 48% higher. In addition, the ratio of fluorescence to diffuse reflectance at the emission peak of 460 nm is 2.6 times higher for LGG while reflectance at 650 nm is 2.7 times higher for GBM. The results reported also show that the combination of diffuse reflectance and fluorescence spectroscopy could achieve sensitivity of 100% and specificity of 90% in discriminating GBM from LGG during ex vivo measurements of 22 sites from seven glioma specimens. Therefore, the current technique might be a promising tool for aiding neurosurgeons in determining the extent of surgical resection of glioma and, thus, improving intraoperative tumor identification for guiding surgical intervention.

Photodynamic Therapy (PDT) provides an opportunity for treatment of various invasive tumors by the use of a cancer targeting photosensitizing agent and light of specific wavelengths. However, real-time monitoring of drug localization is desirable because the induction of the phototoxic effect relies on interplay between the dosage of localized drug and light. Fluorescence emission in PDT may be used to monitor the uptake process but fluorescence intensity is subject to variability due to scattering and absorption; the addition of fluorescence lifetime may be beneficial to probe site-specific drug-molecular interactions and cell damage. We investigated the fluorescence lifetime changes of Photofrin® at various intracellular components in the Mat-LyLu (MLL) cell line. The fluorescence decays were analyzed using a bi-exponential model, followed by segmentation analysis of lifetime parameters. When Photofrin® was localized at the cell membrane, the slow lifetime component was found to be significantly shorter (4.3 ± 0.5 ns) compared to those at other locations (cytoplasm: 7.3 ± 0.3 ns; mitochondria: 7.0 ± 0.2 ns, p < 0.05).

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The fluorescence of Intralipid and polystyrene microspheres with sphere diameter of 1 µm at a representative lipid and microsphere concentration for simulation of mucosal tissue scattering has not been a subject of extensive experimental study. In order to elucidate the quantitative relationship between lipid and microsphere concentration and the respective fluorescent intensity, the extrinsic fluorescence spectra between 360 nm and 650 nm (step size of 5 nm) were measured at different lipid concentrations (from 0.25% to 5%) and different microsphere concentrations (0.00364, 0.0073, 0.0131 spheres per cubic micrometer) using laser excitation at 355 nm with pulse energy of 2.8 µJ. Current findings indicated that Intralipid has a broadband emission between 360 and 650 nm with a primary peak at 500 nm and a secondary peak at 450 nm while polystyrene microspheres have a single peak at 500 nm. In addition, for similar scattering properties the fluorescence of Intralipid solutions is approximately three-fold stronger than that of the microsphere solutions. Furthermore, Intralipid phantoms with lipid concentrations ~2% (simulating the bottom layer of mucosa) produce up to seven times stronger fluorescent emission than phantoms with lipid concentration ~0.25% (simulating the top layer of mucosa). The fluoresence decays of Intralipid and microsphere solutions were also recorded for estimation of fluorescence lifetime.