Molecular imaging has become an emerging technology to assess tumor margins. As the imaging contrast agents are functionalized with multiple ligands that can bind to different biomarkers - multiplexed molecular imaging, this technique can achieve high sensitivity and specificity for tumor margin detection. Optical-based molecular imaging modalities provide non-hazardous optical radiation, multiplexing wavelengths, and higher spatial resolution than ionizing radiation tomography techniques. Two categories of optical contrast agents, fluorescent dyes and Surface-Enhanced Raman Spectroscopy (SERS) nanoparticles are introduced and mainly applied in this dissertation. However, because of the tissue-photon interactions, the imaging contrast and penetration depths are limited by the visible wavelengths. The light in the NIR regime (700~1700 nm) has shown a deeper imaging penetration and better contrast with lower autofluorescence background. Thus, in this dissertation, NIR fluorescent dyes and SERS NPs excited by 785 nm are used for ex vivo and in vivo imaging for biological studies. This work aims to develop a variety of optical instruments for NIR ex vivo and in vivo biomedical imaging applications with deeper penetration, better contrast, and higher sensitivity. The optical instruments include a spectrometric system for SERS Raman detection, a VO2 MEMS scanner for SERS imaging, portable confocal microscopes, and a PZT MEMS scanner-based macroscope for wide-field fluorescence imaging. Chapter 1 briefly introduced the research background, pros and cons of existing techniques, and motivations of this study. In Chapter 2, the spectrometric SERS Raman system and ratiometric analysis have been applied to the detection of Alzheimer's Disease biomarkers and breast cancer image-guided surgery, using different SERS NPs conjugated with ligands. The Raman results were confirmed with histological analysis. In Chapter 3, a VO2 MEMS scanner has been designed, fabricated, and characterized for the Lissajous scanning SERS imaging application. In Chapter 4, two variants of the portable confocal microscopes, the point-scan and line-scan systems were designed with reflective parabolic mirrors for broadband wavelengths from the visible to NIR ranges. Ex vivo and in vivo confocal imaging results have been demonstrated using tumor-bearing mouse tissues. In Chapter 5, a thin-film PZT MEMS scanner has been reported, characterized, and integrated into a wide-field macroscope for fluorescence imaging. In Chapter 6, a novel photodetector - SNSPD has been integrated into the point-scan portable confocal microscope and PZT MEMS scanner-based wide-field macroscope to increase the efficiency and contrast of fluorescent imaging in the NIR range. In the last chapter, the future applications of the advanced VO2 MEMS scanners and fluorescence lifetime imaging microscopy using SNSPD were discussed in detail.
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