Jennifer E. Phipps

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.

Multimodal characterization of compositional, structural and functional features of human atherosclerotic plaques

Detection of atherosclerotic plaque vulnerability has critical clinical implications for avoiding sudden death in patients with high risk of plaque rupture. We report on multimodality imaging of ex-vivo human carotid plaque samples using a system that integrates fluorescence lifetime imaging (FLIM), ultrasonic backscatter microscopy (UBM), and photoacoustic imaging (PAI). Biochemical composition is differentiated with a high temporal resolution and sensitivity at the surface of the plaque by the FLIM subsystem. 3D microanatomy of the whole plaque is reconstructed by the UBM. Functional imaging associated with optical absorption contrast is evaluated from the PAI component. Simultaneous recordings of the optical, ultrasonic, and photoacoustic data present a wealth of complementary information concerning the plaque composition, structure, and function that are related to plaque vulnerability. This approach is expected to improve our ability to study atherosclerotic plaques. The multimodal system presented here can be translated into a catheter based intraluminal system for future clinical studies.

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.

Dynamic tissue analysis using time- and wavelength-resolved fluorescence spectroscopy for atherosclerosis diagnosis

Simultaneous time- and wavelength-resolved fluorescence spectroscopy (STWRFS) was developed and tested for the dynamic characterization of atherosclerotic tissue ex vivo and arterial vessels in vivo. Autofluorescence, induced by a 337 nm, 700 ps pulsed laser, was split to three wavelength sub-bands using dichroic filters, with each sub-band coupled into a different length of optical fiber for temporal separation. STWRFS allows for fast recording/analysis (few microseconds) of time-resolved fluorescence emission in these sub-bands and rapid scanning. Distinct compositions of excised human atherosclerotic aorta were clearly discriminated over scanning lengths of several centimeters based on fluorescence lifetime and the intensity ratio between 390 and 452 nm. Operation of STWRFS blood flow was further validated in pig femoral arteries in vivo using a single-fiber probe integrated with an ultrasound imaging catheter. Current results demonstrate the potential of STWRFS as a tool for real-time optical characterization of arterial tissue composition and for atherosclerosis research and diagnosis.

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 imaging classification method to investigate the collagen to lipid ratio in fibrous caps of atherosclerotic plaque.

This study describes a novel fluorescence lifetime imaging (FLIM) classification method to determine the ratio of collagen to lipid content in the fibrous cap of atherosclerotic plaques. Additionally, an analytical process to assess risk of plaque rupture based on this ratio is proposed. Collagen to lipid ratio has been shown to be an important parameter to evaluate structural integrity of the fibrous cap. FLIM and other time‐resolved fluorescence techniques have recently been applied to the study of atherosclerosis based on the ability to assess biochemical composition.

Time-Resolved Fluorescence Spectroscopy as a Diagnostic Technique of Oral Carcinoma: Validation in the Hamster Buccal Pouch Model

OBJECTIVE To investigate the benefit of using time-resolved, laser-induced fluorescence spectroscopy for diagnosing malignant and premalignant lesions of the oral cavity. DESIGN The carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) was applied to 1 cheek pouch of 19 hamsters. The contralateral pouch and the cheek pouches of 3 hamsters without DMBA exposure served as controls. SETTING University of California, Davis. PARTICIPANTS Twenty-two golden/Syrian hamsters. INTERVENTION A nitrogen pulse laser was used to induce tissue autofluorescence between the wavelengths of 360 and 650 nm. MAIN OUTCOME MEASURES Spectral intensities and time-domain measurements were obtained and compared with the histopathologic findings at each corresponding site. RESULTS Spectral intensities and lifetime values at 3 spectral bands (SBs; SB1 = 380 +/- 10 nm; SB2 = 460 +/- 10 nm, and SB3 = 635 +/- 10 nm) allowed for discrimination among healthy epithelium, dysplasia, carcinoma in situ, and invasive carcinoma. The lifetime values at SB2 were the most important when distinguishing the lesions using only time-resolved parameters. An algorithm combining spectral fluorescence parameters derived from both spectral and time-domain parameters (peak intensities, average fluorescence lifetimes, and the Laguerre coefficient [zero-order]) for healthy epithelium, dysplasia, carcinoma in situ, and invasive carcinoma provided the best diagnostic discrimination, with 100%, 100%, 69.2%, and 76.5% sensitivity and 100%, 92.2%, 97.1%, and 96.2% specificity, respectively. CONCLUSIONS The addition of time-resolved fluorescence-derived parameters significantly improves the capability of fluorescence spectroscopy-based diagnostics in the hamster buccal pouch. This technique provides a potential noninvasive diagnostic instrument for head and neck cancer.

Diagnosis of Thin-Capped Fibroatheromas in Intravascular Optical Coherence Tomography Images

Background—Intravascular optical coherence tomography (IVOCT) images are recorded by detecting light backscattered within coronary arteries. We hypothesize that non–thin-capped fibroatheroma (TCFA) causes may scatter light to create the false appearance of IVOCT TCFA. Methods and Results—Ten human cadaver hearts were imaged with IVOCT (n=14 coronary arteries). IVOCT and histological TCFA images were coregistered and compared. Of 21 IVOCT TCFAs (fibrous cap <65 &mgr;m, lipid arc >1 quadrant), only 8 were true histological TCFA. Foam cell infiltration was responsible for 70% of false IVOCT TCFA and caused both thick-capped fibroatheromas to appear as TCFA, and the appearance of TCFAs when no lipid core was present. Other false IVOCT TCFA causes included smooth muscle cell–rich fibrous tissue (12%) and loose connective tissue (9%). If the lipid arc >1 quadrant (obtuse) criterion was disregarded, 45 IVOCT TCFAs were identified, and sensitivity of IVOCT TCFA detection increased from 63% to 87%, and specificity remained high at 92%. Conclusions—We demonstrate that IVOCT can exhibit 87% (95% CI, 75%–93%) sensitivity and 92% specificity (95% CI, 86%–96%) to detect all lipid arcs (both obtuse and acute, <1 quadrant) TCFA, and we also propose new mechanisms involving light scattering that explain why other plaque components can masquerade as TCFA and cause low positive predictive value of IVOCT for TCFA detection (47% for obtuse lipid arcs). Disregarding the lipid arc >1 quadrant requirement enhances the ability of IVOCT to detect TCFA.

Pulse Shape Discrimination and Classification Methods for Continuous Depth of Interaction Encoding PET Detectors

In previous work we demonstrated the potential of positron emission tomography (PET) detectors with depth-of-interaction (DOI) encoding capability based on phosphor-coated crystals. A DOI resolution of 8 mm full-width at half-maximum was obtained for 20 mm long scintillator crystals using a delayed charge integration linear regression method (DCI-LR). Phosphor-coated crystals modify the pulse shape to allow continuous DOI information determination, but the relationship between pulse shape and DOI is complex. We are therefore interested in developing a sensitive and robust method to estimate the DOI. Here, linear discriminant analysis (LDA) was implemented to classify the events based on information extracted from the pulse shape. Pulses were acquired with 2×2×20 mm(3) phosphor-coated crystals at five irradiation depths and characterized by their DCI values or Laguerre coefficients. These coefficients were obtained by expanding the pulses on a Laguerre basis set and constituted a unique signature for each pulse. The DOI of individual events was predicted using LDA based on Laguerre coefficients (Laguerre-LDA) or DCI values (DCI-LDA) as discriminant features. Predicted DOIs were compared to true irradiation depths. Laguerre-LDA showed higher sensitivity and accuracy than DCI-LDA and DCI-LR and was also more robust to predict the DOI of pulses with higher statistical noise due to low light levels (interaction depths further from the photodetector face). This indicates that Laguerre-LDA may be more suitable to DOI estimation in smaller crystals where lower collected light levels are expected. This novel approach is promising for calculating DOI using pulse shape discrimination in single-ended readout depth-encoding PET detectors.

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 µm; 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 µm 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.