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Fluorescent Lifetime Imaging and Spectroscopy

(Developmental Center-Driven Instrument Project)

UC Davis: Y Sun, J Park, J Phipps, Y Sun, R Liu, D Stephens, FYS Chuang, DG Farwell, L Marcu;

Imperial College of London: D Elson

Fluorescence lifetime imaging microscopy (FLIM) is a promising non-invasive imaging modality for the diagnosis of abnormal or malignant tissue. FLIM provides information about fluorescence intensity, lifetime, and specific spectrum, which can be used to characterize the chemical composition, metabolism, and environmental factors of tissues and biological samples

We have built a portable endoscopic FLIM system that can be used in the operating room to characterize tissue in vivo, without the need for performing excisional biopsy.  We are working with otolaryngologists at UC Davis Medical Center to validate the FLIM on the diagnosis of head-and-neck tumors. For in vivo and clinical applications, two key components − a compact gated intensifier imaging system (ICCD) and a fiber-based endoscope have been designed and integrated. The system performance was evaluated on standard fluorophores, biomolecules, and tissue in vitro and in vivo. The ability for FLIM to discriminate malignant and normal mucosa has been demonstrated.

The FLIM system includes a gated intensified CCD camera (ICCD), a pulsed nitrogen laser, an optical fiber imaging bundle with GRIN lens, a filter wheel, a microscope objective, and relay lenses. Here the nitrogen laser is used as excitation light source with the pulse width 0.7 ns and energy up to 70 μJ/pulse. For tissue imaging a 10−20 μJ /pulse energy was delivered to an area with the diameter of 5 mm. The ICCD has an adjustable gating time from 0.2 ns to DC with internal relay lens, an enclosed delay generator and a high voltage supply. The spatial resolution of the system was measured as 35 μm. The temporal resolution is approximately 0.5 ns.

A wide-field time-domain fluorescence lifetime endoscopy system is developed using a compact fast-gating intensifier and a imaging bundle cemented with a GRIN lens as objective. The imaging bundle consists of 10,000 optical fibers and has the length of 2 m and the outer diameter only 0.6 mm. The GRIN lens has the field of view of 4 mm diameter and provides either side view or front view. The compact apparatus is able to be installed on a mobile cart for clinical diagnosis and surgical service (see figure).

The endoscopic FLIM has been validated on standard fluorophores, biomolecules  collagen and elastin, and biological tissue  human head and neck tumor ex vivo and hamster buccal pouch in vivo with normal tissue and  carcinoma. In the hamster model, the auto-fluorescence of tumor, early-lesion, and normal tissue was analyzed by both FLIM and time-resolved laser-induced fluorescence spectroscopy. Diseased tissue showed difference in fluorescence intensity, spectrum, and lifetime compared with normal tissue. Tumor had weaker fluorescence and emission peak changed to 450 nm from 390 nm corresponding to NADH and collagen, respectively. The results from human tissue displayed similar variation. The FLIM system has been calibrated and assembled on a mobile cart. A semi-flexible endoscope was designed and fabricated with integrated imaging bungle, objective, and excitation fibers. Further applications on the human project of head & neck tumor are still undergoing. The FLIM system is expected to differentiate normal and malignant tissues establishing clear margins in cancer surgery, as well as to reduce negative biopsies.