Time Resolved Fluorescence Lifetime Imaging System For In Vivo Characterization of Tumors Amir H. Gandjbakhche 1 , Moinuddin Hassan 1 , Victor Chernomordik 1 , Sang Bong Lee 2 and Jacek Capala 2 1 National Institute of Child Health and Human Development, NIH, Bethesda, Maryland 2 National Cancer Institute, NIH, Bethesda, Maryland Abstract: We present a lifetime fluorescence imaging system for small animal imaging. The system uses a linear fiber array with given separations between a single source fiber and several detection fibers. The general goal is to detect and localize tumors, using specific fluorescent markers and investigate their progression. We investigated applications of the developed system to mouse imaging, using as contrast agent Alexa Fluor 750 conjugated to tumor specific antibodies (Herceptin). Realized 2D mapping of fluorescence lifetime indicate lower lifetime value in the tumor area. Introduction Fluorescence lifetime is known to be sensitive to an immediate environment of the fluorophore e.g., temperature, pH, oxygen content, nutrient supply and bioenergetic status (1). On the other hand, the fluorescence lifetime is practically independent of the concentration of the fluorescent agent and intensity of the excitation light. The lifetime tends to remain constant even when 5-fold fluctuations in intensity occur (2). The heterogeneity of tumor vasculature can be observed through changes in pH and temperature (3). By selecting fluorophores with known lifetime dependence on pH and / or temperature, this method can potentially be used for functional diagnosis of malignancies and drug targeting. In this paper, we present a small animal molecular imaging system and its application for in vivo mice study. The goal of our study is to develop efficient time-resolved system for in vivo imaging using suitable contrast agents in near infrared (NIR) spectral region, which provide spatial distributions of lifetime of fluorescent agent inside the turbid media. We present in vivo study on mice to validate the system for future applications to minimally invasive functional imaging. Materials and Methods: The schematic diagram of the NIR fluorescence small-animal imaging system is shown in Fig.1, the details of which have been described elsewhere (4). Briefly, the system was based on a time-domain technique, where an advance time-correlated single- photon counting device was used in conjunction with a high-speed repetition-rate tunable laser to detect individual photons. It used a photomultiplier tube as a detector, and a temperature-controlled scanning stage with an electrocardiogram and temperature monitoring device for small animals, and a scanning head. The scanning head consisted of multimode optical fibers that were used to deliver light from an excitation source and an emitted fluorescence signal to detectors. The imager had a laser source for fluorescence excitation (λ = 750 nm), an emission filter (λ = 790 nm) for fluorescence detection, and a computer for data analysis. The imager scanned in a raster pattern over skin or other tissue surfaces to produce a real-time two-dimensional image. A cooled, charge-coupled device (CCD) camera was also used to guide the scan to the region of interest (ROI) and to measure the fluorescence intensity distribution, which helped to locate the tumor inside the tissue. Animal study was performed with athymic female nude mice approximately six to eight weeks old. Breast cancer cells (SK-BR-3) were implanted to flank area of the mice. These cells express high levels of HER2 (HER2/neu, c-ErbB2) protein. In this study, Alexa Fluor 750 (Invitrogen, Inc) was conjugated with Herceptin (Genentach Inc. USA), a HER2-specific monoclonal antibody presently used in clinic for targeted therapy of HER2- overexpressing tumors. Fig.1. Schematic diagram of the imaging system. Ti: Sapphire Pulse width: f Repetition: 80 CF SYN PHOTON COUNTER Pr - Am PM Filter Optic Swit Fiber COMPUTE Len Len Fiber coupler with Scanner Bea splitt ND Filter Mirr Excitati fib Emissi fiber Obje Pin Phot diod Fluoroph (TCSPC C C Ti: Sapphire Pulse width: f Repetition: 80 CF SYN PHOTON COUNTER Pr - Am PM Filter Optic Swit Fiber COMPUTE Len Len Fiber coupler with Scanner Bea splitt ND Filter Mirr Excitati fib Emissi fiber Obje Pin Photo diod Fluoroph ND (TCSPC C C 7 MA3 2:30 PM – 2:45 PM U.S. Government work not protected by U.S. copyright