Endoscopic fluorescence lifetime imaging for in vivo intraoperative diagnosis of oral carcinoma.

A clinically compatible fluorescence lifetime imaging microscopy (FLIM) system was developed. The system was applied to intraoperative in vivo imaging of head and neck squamous cell carcinoma (HNSCC). The endoscopic FLIM prototype integrates a gated (down to 0.2 ns) intensifier imaging system and a fiber-bundle endoscope (0.5-mm-diameter, 10,000 fibers with a gradient index lens objective 0.5 NA, 4-mm field of view), which provides intraoperative access to the surgical field. Tissue autofluorescence was induced by a pulsed laser (337 nm, 700 ps pulse width) and collected in the 460 ± 25 nm spectral band. FLIM experiments were conducted at 26 anatomic sites in ten patients during head and neck cancer surgery. HNSCC exhibited a weaker florescence intensity (~50% less) when compared with healthy tissue and a shorter average lifetime (τ(HNSCC) = 1.21 ± 0.04 ns) than the surrounding normal tissue (τN = 1.49 ± 0.06 ns). This work demonstrates the potential of FLIM for label-free head and neck tumor demarcation during intraoperative surgical procedures.

Fluorescence lifetime imaging for the characterization of the biochemical composition of atherosclerotic plaques.

This study investigates the ability of a flexible fiberoptic-based fluorescence lifetime imaging microscopy (FLIM) technique to resolve biochemical features in plaque fibrotic cap associated with plaque instability and based solely on fluorescence decay characteristics. Autofluorescence of atherosclerotic human aorta (11 autopsy samples) was measured at 48 locations through two filters, F377: 377∕50 and F460: 460∕60 nm (center wavelength∕bandwidth). The fluorescence decay dynamic was described by average lifetime (τ) and four Laguerre coefficients (LECs) retrieved through a Laguerre deconvolution technique. FLIM-derived parameters discriminated between four groups [elastin-rich (ER), elastin and macrophage-rich (E+M), collagen-rich (CR), and lipid-rich (LR)]. For example, τ(F377) discriminated ER from CR (R = 0.84); τ(F460) discriminated E+M from CR and ER (R = 0.60 and 0.54, respectively); LEC-1(F377) discriminated CR from LR and E+M (R = 0.69 and 0.77, respectively); P < 0.05 for all correlations. Linear discriminant analysis was used to classify this data set with specificity >87% (all cases) and sensitivity as high as 86%. Current results demonstrate for the first time that clinically relevant features (e.g., ratios of lipid versus collagen versus elastin) can be evaluated with a flexible-fiber based FLIM technique without the need for fluorescence intensity information or contrast agents.

A fluorescence lifetime spectroscopy study of matrix metalloproteinases-2 and -9 in human atherosclerotic plaque.

Matrix metalloproteinase (MMP)-2 and -9 play important roles in the progression of atherosclerosis. This study aims to determine whether MMP-2 and -9 content in the fibrotic caps of atherosclerotic plaque is correlated with plaque autofluorescence. A time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) system was used to measure the autofluorescence and assess the biochemical composition of human plaques obtained from carotid endarterectomy. Results presented here demonstrate for the first time the ability to characterize the biochemical composition as it relates to MMP-2 and -9 content in the atherosclerotic plaque cap using a label-free imaging technique implemented with a fiberoptic TR-LIFS system.

Fluorescence lifetime imaging microscopy for brain tumor image-guided surgery.

We demonstrate for the first time the application of an endoscopic fluorescence lifetime imaging microscopy (FLIM) system to the intraoperative diagnosis of glioblastoma multiforme (GBM). The clinically compatible FLIM prototype integrates a gated (down to 0.2 ns) intensifier imaging system with a fiber-bundle (fiber image guide of 0.5 mm diameter, 10,000 fibers with a gradient index lens objective 0.5 NA, and 4 mm field of view) to provide intraoperative access to the surgical field. Experiments conducted in three patients undergoing craniotomy for tumor resection demonstrate that FLIM-derived parameters allow for delineation of tumor from normal cortex. For example, at 460±25-nm wavelength band emission corresponding to NADH/NADPH fluorescence, GBM exhibited a weaker fluorescence intensity (35% less, p-value<0.05) and a longer lifetime τGBM-Amean=1.59±0.24 ns than normal cortex τNC-Amean=1.28±0.04 ns (p-value<0.005). Current results demonstrate the potential use of FLIM as a tool for image-guided surgery of brain tumors.

Time-resolved laser-induced fluorescence spectroscopy as a diagnostic instrument in head and neck carcinoma.

OBJECTIVE: The objectives of this study were to 1) determine differences in lifetime fluorescence between normal and malignant tissue of the upper aerodigestive tract, and 2) evaluate the potential of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) as a diagnostic instrument for head and neck squamous cell carcinoma (HNSCC). STUDY DESIGN: Cross-sectional study. SETTING: University-based medical center. SUBJECTS AND METHODS: Nine patients with suspected HNSCC were included. In the operating room, a nitrogen pulse laser (337 nm, 700-picosecond pulse width) was used to induce tissue autofluorescence of normal tissue and suspected malignant lesions. Spectral intensities and time-domain measurements were obtained and compared with the histopathology at each site. A total of 53 sites were measured. The fluorescence parameters that provided the most discrimination were determined. RESULTS: Differences in spectral intensities allowed for discrimination between malignant and normal tissue. The spectral intensity of malignant tissue was lower than that of normal tissue, and a shift of peak intensity to a longer wavelength was observed in the normalized spectrum of malignant tissue in the range of 360 to approximately 660 nm. Multiple time-resolved fluorescence parameters provided the best diagnostic discrimination between normal tissue and carcinoma, including average lifetimes (i.e., at 390 nm: 1.7 +/- 0.06 ns [not significant] for normal and 1.3 +/- 0.06 ns for tumor, P = 0.0025) and the second-order Laguerre expansion coefficient (LEC-2) (i.e., at 460 nm: 0.135 +/- 0.001 for normal and 0.155 +/- 0.007 for tumor, P < 0.05). CONCLUSION: These findings highlight some of the differences in lifetime fluorescence between normal and malignant tissue. TR-LIFS has potential as a noninvasive diagnostic technique for HNSCC.

Fluorescence lifetime imaging microscopy for the characterization of atherosclerotic plaques.

Atherosclerotic plaque composition has been associated with plaque instability and rupture. This study investigates the use of fluorescence lifetime imaging microscopy (FLIM) for mapping plaque composition and assessing features of vulnerability. Measurements were conducted in atherosclerotic human aortic samples using an endoscopic FLIM system (spatial resolution of 35 mum; temporal resolution 200 ps) developed in our lab which allows mapping in one measurement the composition within a volume of 4 mm diameter x 250 mum depth. Each pixel in the image represents a corresponding fluorescence lifetime value; images are formed through a flexible 0.6 mm side-viewing imaging bundle which allows for further intravascular applications. Based on previously recorded spectra of human atherosclerotic plaque, fluorescence emission was collected through two filters: f1: 377/50 and f2: 460/60 (center wavelength/bandwidth), which together provides the greatest discrimination between intrinsic fluorophores related to plaque vulnerability. We have imaged nine aortas and lifetime images were retrieved using a Laguerre expansion deconvolution technique and correlated with histopathology. Early results demonstrate discrimination using fluorescence lifetime between early, lipid-rich, and collagen-rich lesions which are consistent with previously reported time-resolved atherosclerotic plaque measurements.