Raman spectroscopy is a powerful technique for label-free molecular sensing and imaging in various fields. High molecular specificity, non-invasive sampling approach and the need for little or no sample preparation make Raman spectroscopy uniquely advantageous compared to other analytical techniques. However, Raman spectroscopy suffers from the intrinsic limitation of weak signal intensity. Therefore, time-sensitive studies such as diagnosis and clinical applications require improving the throughput of Raman instrumentation. Alternatively, surface-enhanced Raman scattering (SERS) improves the sensitivity by 10^6 to 10^14 times, making the weak Raman intensity no longer a limitation. Nevertheless, it is still a big challenge to engineer plasmonic substrates with high SERS enhancement, good uniformity and reproducibility. This thesis presents advances in: (1) Raman instrumentation towards high-throughput, environmental, biological and biomedical analysis; (2) SERS substrates with high enhancement factor (EF), uniformity and reproducibility; (3) biosensing applications including imaging of cell population and detection of biomolecules towards high time efficiency and sensitivity. In Raman instrumentation, we have built a high-throughput line-scan Raman microscope system and a novel parallel Raman microscope based on multiple-point active-illumination and wide-field hyperspectral data collection. Using the line-scan Raman microscope, we have performed chemical imaging of intact biological cells at the cell population level. We have also demonstrated more flexibility and throughput from the active-illumination Raman microscope in rapid chemical identification and screening of micro and nanoparticles and bacterial spores. Both Raman microscopes have been used to evaluate the large-area SERS uniformity of DC-sputtered gold nanoislands, a low-cost means to fabricate plasmonic substrates. In plasmonic engineering, we have introduced patterned nanoporous gold nanoparticles that feature 3-dimensional mesoporous network with pore size on the order of 10 nm throughput the sub-wavelength nanoparticles. We showed that the plasmonic resonance can be tuned by geometrical engineering of either the external nanoparticle size and shape or the nanoporous network. As an example, we have developed disk-shaped entities, also known as nanoporous gold disks (NPGD) with highly uniform and reproducible SERS EF exceeding 10^8. Label-free, multiplexed molecular sensing and imaging has been demonstrated on NPGD substrates. Using the line-scan Raman microscope and the NPGD substrates, we have successfully developed a label-free DNA hybridization sensor at the single-molecule level in microfluidics. We have observed discrete, individual DNA hybridization events by in situ monitoring the hybridization process using SERS. The advances and promising results presented in this thesis demonstrate potential impact in Raman/SERS imaging and sensing in environmental, biological and biomedical applications.Electrical and Computer Engineering, Department o
