The mid-infrared is a promising region for detection different materials. Many vibrational modes, including bending and stretching, are located in this regime. Photothermal spectroscopy and imaging in the mid-infrared region is an emerging new method for non-contact detection of molecular groups. Our approach to photothermal spectroscopy and imaging utilizes a near-infrared erbium doped fiber laser (EDFL) to detect the photothermal induced changes in the refractive index. These changes are excited by a mid-infrared quantum cascade laser (QCL) pump beam. The probe beam is detected by a commercially available near-infrared photodetector. This method has advantages of high sensitivity, label-free detection, high spatial resolution and high signal-to-noise ratio (SNR). Hydrogels such as pHEMA are polymers that are of interest for contact lens, drug delivery and soft tissue replacement. The pHEMA hydrogel can retain water content, causing the material to swell. Additionally, pHEMA has a critical temperature at which the hydrogel undergoes a glass transition. Photothermal spectroscopy of pHEMA is demonstrated in this thesis where the presence of this glass transition temperature can be revealed. Additionally, photothermal imaging of a PMMA USAF target sample is shown and ideal parameters for high-resolution photothermal imaging are determined. In this thesis, we report a spatial resolution much smaller than the diffraction limited spot size of the mid-infrared beam. / 2018-07-09T00:00:00Z

Atherosclerosis and specifically rupture of vulnerable plaques account for 23% of all deaths worldwide, far surpassing both infectious diseases and cancer. Plaque-based macrophages, often associated with lipid deposits, contribute to atherogenesis from initiation through progression, plaque rupture and ultimately, thrombosis. Therefore, the macrophage is an important early cellular marker related to vulnerability of atherosclerotic plaques. The objective of my research is to assess the ability of multiple optical imaging modalities to detect, and further characterize the distribution of macrophages (having taken up plasmonic gold nanoparticles as a contrast agent) and lipid deposits in atherosclerotic plaques. Tissue phantoms and macrophage cell cultures were used to investigate the capability of nanorose as an imaging contrast agent to target macrophages. Ex vivo aorta segments from a rabbit model of atherosclerosis after intravenous nanorose injection were imaged by optical coherence tomography (OCT), photothermal imaging (PTW) and two-photon luminescence microscopy (TPLM), respectively. OCT images depicted detailed surface structure of atherosclerotic plaques. PTW images identified nanorose-loaded macrophages (confirmed by co-registration of a TPLM image and corresponding RAM-11 stain on a histological section) associated with lipid deposits at multiple depths. TPLM images showed three-dimensional distribution of nanorose-loaded macrophages with a high spatial resolution. Imaging results suggest that superficial nanorose-loaded macrophages are distributed at shoulders on the upstream side of atherosclerotic plaques at the edges of lipid deposits. Combination of OCT with PTW or TPLM can simultaneously reveal plaque structure and composition, permitting assessment of plaque vulnerability during cardiovascular interventions. / text

Biological nanoparticles have enormous utility as well as potential adverse impacts in biotechnology, human health, and medicine. The physical and chemical properties of these nanoparticles have strong implications on their distribution, circulation, and clearance in vivo. Accurate morphological visualization and chemical characterization of nanoparticles by label-free (direct) optical microscopy would provide valuable insights into their natural and intrinsic properties. However, three major challenges related to label-free nanoparticle imaging must be overcome: (i) weak contrast due to exceptionally small size and low-refractive-index difference with the surrounding medium, (ii) inadequate spatial resolution to discern nanoscale features, and (iii) lack of chemical specificity. Advances in common-path interferometric microscopy have successfully overcome the weak contrast limitation and enabled direct detection of low-index biological nanoparticles down to single proteins. However, interferometric light microscopy does not overcome the diffraction limit, and studying the nanoparticle morphology at sub-wavelength spatial resolution remains a significant challenge. Moreover, chemical signature and composition are inaccessible in these interferometric optical measurements. This dissertation explores innovations in common-path interferometric microscopy to provide enhanced spatial resolution and chemical specificity in high-throughput imaging of individual nanoparticles. The dissertation research effort focuses on a particular modality of interferometric imaging, termed “single-particle interferometric reflectance (SPIR) microscopy”, that uses an oxide-coated silicon substrate for enhanced coherent detection of the weakly scattered light. We seek to advance three specific aspects of SPIR microscopy: sensitivity, spatial resolution, and chemical specificity. The first one is to enhance particle visibility via novel optical and computational methods that push optical detection sensitivity. The second one is to improve the lateral resolution beyond the system's classical limit by a new computational imaging method with an engineered illumination function that accesses high-resolution spatial information at the nanoscale. The last one is to extract a distinctive chemical signature by probing the mid-infrared absorption-induced photothermal effect. To realize these goals, we introduce new theoretical models and experimental concepts. This dissertation makes the following four major contributions in the wide-field common-path interferometric microscopy field: (1) formulating vectorial-optics based linear forward model that describes interferometric light scattering near planar interfaces in the quasi-static limit, (2) developing computationally efficient image reconstruction methods from defocus images to detect a single 25 nm dielectric nanoparticle, (3) developing asymmetric illumination based computational microscopy methods to achieve direct morphological visualization of nanoparticles at 150 nm, and (4) developing bond-selective interferometric microscopy to enable multispectral chemical imaging of sub-wavelength nanoparticles in the vibrational fingerprint region. Collectively, through these research projects, we demonstrate significant advancement in the wide-field common-path interferometric microscopy field to achieve high-resolution and accurate visualization and chemical characterization of a broad size range of individual biological nanoparticles with high sensitivity.

Mid-infrared spectroscopic imaging, which leverages the inherent vibrational contrast of chemical bonds, has been a powerful analytical tool for sample characterization. However, its use in studying living systems is limited by low spatial resolution and significant water absorption. Recently developed mid-infrared photothermal (MIP) microscopy addresses these limitations by probing the absorption-induced photothermal effect using visible light. MIP microscopy achieves sub-micrometer spatial resolution and reduces water background interference. Yet, the imaging speed of current MIP microscopy is constrained by the challenge of measuring a small modulation over the probe laser background. This low imaging throughput hinders the visualization of living dynamics, and the rich molecular information in the spectroscopic domain is obscured due to the slow acquisition process. This dissertation explores solutions for enhancing imaging speed and spectral throughput and extending MIP imaging into visualizing chemical dynamics in living systems. In the first part of the dissertation, the mid-infrared photothermal process is studied and modeled in the time, frequency, and spatial domains using heat transfer analysis. Photothermal dynamics imaging (PDI) is introduced with the ability to visualize nanosecond-scale thermodynamics in samples upon laser excitation. By capturing all higher-order harmonics, PDI achieves more than a four-fold improvement in signal-to-noise ratio compared to the lock-in method for detecting low-duty cycle photothermal signals. An imaging speed nearly two orders of magnitude faster than the lock-in counterpart has been reached. In addition, PDI captures the transient thermal field evolution, providing a tool to gauge the target’s physical properties and microenvironment. In the second part, a video-rate MIP microscope is introduced based on the PDI detection method. In the system, a synchronized IR and visible beam scanning scheme is developed, enabling photothermal detection with a single IR pulse at each pixel. Moreover, synchronized laser scanning allows uniform MIP imaging in a field of view over hundreds of micrometers while maintaining a high spatial resolution. This capability enabled the visualization of fast chemical dynamics inside living fungal cells, cancer cells, and living worms, providing an imaging platform for biology research. Having reached the speed limitation of single-pulse imaging, we further advanced the speed of spectroscopic imaging by moving beyond the conventional measurement of absorption contrast in the photothermal process. In the final part of this dissertation, we revisited the photothermal process from the perspective of energy deposition, discovering that the absorption coefficient is reflected in the slope of the heating process rather than its overall amplitude. We demonstrated mid-infrared energy deposition (MIRED) spectroscopy using a 32-channel quantum cascade laser array that emits a broadband pulse train in microseconds. With MIRED, we achieved hyperspectral mid-infrared imaging on a microsecond scale.