Spectrally Resolved Detector Arrays for Multiplexed Biomedical Fluorescence Imaging
The ability to resolve multiple fluorescent emissions from different biological targets in video rate applications, such as endoscopy and intraoperative imaging, has traditionally been limited by the use of filter-based imaging systems. Hyper and multispectral imaging facilitate the detection of both spatial and spectral information in a single data acquisition, however, instrumentation for spatiospectral data acquisition is typically complex, bulky and expensive. This thesis seeks to overcome these limitations by using recently commercialised compact and robust hyper/multispectral cameras based on spectrally resolved detector arrays. Following sensor calibrations, which devoted particular attention to the angular sensitivity of the sensors, we integrated spectrally resolved detector arrays into a wide-field and an endoscopic imaging platform. This allowed multiplexed reflectance and fluorescence imaging with spectrally resolved detector array technology in vitro, in tissue mimicking phantoms, in an ex vivo oesophageal model and in vivo in a mouse model. A hyperspectral linescan sensor was first integrated in a wide-field near-infrared reflectance based imaging set-up to assess the suitability of spectrally resolved detector arrays for in vivo imaging of exogenous fluorescent contrast agents. Using this fluorescence hyperspectral imaging system, we could accurately resolve the presence and concentration of seven fluorescent dyes in solution. We also demonstrated high spectral unmixing precision, signal linearity with dye concentration, at depth in tissue mimicking phantoms, and delineation of four fluorescent dyes in vivo. After the successful demonstration of multiplexed fluorescence imaging in a wide-field set-up, we proceeded to combine near-infrared multiplexed fluorescence imaging with visible light spectral reflectance imaging in an endoscopic set-up. A multispectral endoscopic imaging system, capable of simultaneous reflectance and fluorescence imaging, was developed around two snapshot spectrally resolved detector arrays. In the process of system integration and characterisation, methods to characterise and predict the imaging performance of spectral endoscopes were developed. With the endoscope we demonstrated simultaneous imaging and spectral unmixing of chemically oxy/deoxygenated blood and three fluorescent dyes in a tissue mimicking phantom, and of two fluorescent dyes in an ex vivo oesophageal porcine model. With further developments, this technology has the potential to become applicable in medical imaging for detection of diseases such as gastrointestinal cancers.
hyperspectral, biomedical, in vivo, multiplexed, fluorescence, SRDA, CMOS
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