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Coherent anti-Stokes Raman scattering (CARS) microscopy allows fast, label-free and chemically selective imaging of condensed-phase samples thanks to its high signal sensitivity. It also offers several other advantages such as intrinsic three-dimensional sectioning capability, longer penetration depth and high spatial resolution. In conventional CARS microscopy, two synchronized narrowband laser pulses are typically used to generate signals at a single vibrational resonance, from which vibrational images are constructed. Although this type of CARS methods has been proven to be an excellent visualizing tool for lipid in biological samples, it has two serious problems. First, the ubiquitous nonresonant background smears out vibrational signals, which makes quantitative image analysis very difficult. Second, the chemical information obtained in this method is seriously limited since only a single vibrational resonance is measured, which is far less information than full vibrational spectrum can offer. In the past few years, we have developed several novel CARS imaging techniques that can overcome these issues. All our methods require only a single broadband laser and produce background-free vibrational spectrum by combining the laser pulse shaping and various signal detection schemes. The first one obtains a vibrational spectrum over 800 ~ 1800 cm-1 in a single measurement by simultaneous excitation of multiple vibrational resonances and analysis of spectral interferences between the resonant and nonresonant signals. The second method adopts the spectral focusing mechanism, where stretched broadband pulses are used to excite a single vibrational resonance with great sensitivity. A novel frequency modulation (FM) scheme is invented to eliminate the non-resonant background. Complimentary spectral analysis algorithm is also developed to obtain quantitative CARS signals at the CH stretching region (2800 ~ 3100 cm-1). In this dissertation, the fundamental mechanisms, experimental implementations and various imaging applications of the above CARS methods are described in detail. / text

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A nanomanipulation system operating inside a scanning electron microscope (SEM) enables visual observation and physical interactions with objects at the nanometer scale. Compared to SEM that is a powerful imaging platform (‘eyes’), the development of nanomanipulation systems (‘hands) and techniques for transporting, modifying, and interacting with micro/nanoscaled objects is lagging behind. Two generations of nanomanipulation systems were developed with high SEM compatibility. The vacuum load-lock feature allows setup/sample/end-tools changes to be made within minutes instead of hours as with existing nanomanipulation systems. The integrated high resolution encoders and automation features significantly ease the skill dependency in nanomanipulation. Its small shape factor minimizes effects on SEM imaging performance, and does not restrict the use of the many detectors inside a SEM. The new nanomanipulation systems were applied to the manipulation of sub-cellular structures and the characterization of nano-structures. The first application involves the development of a technique to surgically extract sub-micrometer-sized subnuclear structures within a single cell’s nucleus, followed by biochemical analysis to amplify and sequence the genes contained within. Enabled by the technique, four novel genomic loci associations with promyelocytic leukemia nuclear bodies (PML NB) were discovered in Jurkat cells. The second application targets automated probing of nanostructures under poor imaging conditions. Through real-time image drift compensation and visual servoing of the nano probes, automated probing of nanostructures was achieved with a high success rate and a speed at least three times higher than skilled operator. To enhance the functions of the nanomanipulation system, new types of end-effectors were also developed. A MEMS tool with changeable tool tips was design and prototyped. In-situ (i.e., inside SEM) tool tip change was demonstrated for gripping objects that vary in size by two orders of magnitude (15 um to 100 nm) with a single microgripper body. Furthermore, a microfabrication process was developed to produce changeable nano-spatulas with tip size less than 10 nm, intended for use in the subnuclear structure extraction work. Finally, a local precursor sublimation technique compatible with the nanomanipulation system was developed for enhancing electron beam induced deposition (EBID) inside the SEM.

A nanomanipulation system operating inside a scanning electron microscope (SEM) enables visual observation and physical interactions with objects at the nanometer scale. Compared to SEM that is a powerful imaging platform (‘eyes’), the development of nanomanipulation systems (‘hands) and techniques for transporting, modifying, and interacting with micro/nanoscaled objects is lagging behind. Two generations of nanomanipulation systems were developed with high SEM compatibility. The vacuum load-lock feature allows setup/sample/end-tools changes to be made within minutes instead of hours as with existing nanomanipulation systems. The integrated high resolution encoders and automation features significantly ease the skill dependency in nanomanipulation. Its small shape factor minimizes effects on SEM imaging performance, and does not restrict the use of the many detectors inside a SEM. The new nanomanipulation systems were applied to the manipulation of sub-cellular structures and the characterization of nano-structures. The first application involves the development of a technique to surgically extract sub-micrometer-sized subnuclear structures within a single cell’s nucleus, followed by biochemical analysis to amplify and sequence the genes contained within. Enabled by the technique, four novel genomic loci associations with promyelocytic leukemia nuclear bodies (PML NB) were discovered in Jurkat cells. The second application targets automated probing of nanostructures under poor imaging conditions. Through real-time image drift compensation and visual servoing of the nano probes, automated probing of nanostructures was achieved with a high success rate and a speed at least three times higher than skilled operator. To enhance the functions of the nanomanipulation system, new types of end-effectors were also developed. A MEMS tool with changeable tool tips was design and prototyped. In-situ (i.e., inside SEM) tool tip change was demonstrated for gripping objects that vary in size by two orders of magnitude (15 um to 100 nm) with a single microgripper body. Furthermore, a microfabrication process was developed to produce changeable nano-spatulas with tip size less than 10 nm, intended for use in the subnuclear structure extraction work. Finally, a local precursor sublimation technique compatible with the nanomanipulation system was developed for enhancing electron beam induced deposition (EBID) inside the SEM.

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