Quantitative Nonlinear Optical Microscopy with Applications in Nanostructure Imaging and Cancer Biology

Abstract

Quantitative Nonlinear Optical Microscopy with Applications in Nanostructure Imaging and Cancer Biology Abstract: Nonlinear optical microscopy is a powerful imaging technique that has been used for imaging for diverse scientific and biomedical applications for more than two decades. Much of the focus in this field has shifted more towards quantitative analysis of the obtained images to obtain quantifiable metrics from visual images. Quantitative analysis in nonlinear optical microscopy involves not just image processing to extraction numerical results from acquired images, it also broadly encompasses techniques to improve the imaging resolution using carefully designed phase masks, quantify the improvement in resolution, and also design of nonlinear optical media with tailored nonlinear optical properties for specific applications. This can play a crucial role in understanding new biological or nanoscience at smaller spatial scales with better precision. This work focuses on using quantitative nonlinear optical microscopy with application in cancer biology and nanostructure imaging. Engineering the excitation and detection volume is seen as a promising approach to improve the spatial resolution of these techniques. The initial part of the research focuses on super resolution techniques that enhance the resolution of nonlinear optical microscopy. We concentrate on developing beam shaping based super resolution technique to enhance the resolution of an infrared (IR) sensitive third-order SFG (TSFG) microscope. Simulation studies were conducted considering the effect of a suitable optical focusing element that suits well in both mid-IR and visible wavelength range. Parameters such as full width half maximum (FWHM) and energy in the sidelobes were obtained using full-vectorial focal field modelling, and used to optimize the phase mask profile for the TSFG process. Before implementing this technique, initial characterization was done by simulating the far-field emission of the non- linear TSFG signal from known samples such as gratings with varying pitch and isolated nanoparticles using Green’s function integral. We observed that along with resolution improvement, focal field engineering approach also offers resonant contrast enhancement when the modified focal field profile matches the periodicity of the features. Sub-diffraction resolution and contrast enhancement in focus engineered TSFG microscopy was experimentally demonstrated and applied to microbeads and multilayer two-dimensional material imaging. We also explored the detection volume engineering approach in focus engineered, confocal detection coherent anti-stokes roman scattering (CARS) microscopy. Simulation study modelling the field at the focus of non-degenerate pumped CARS microscopy using beam shaping approach was conducted. The imaging system for the confocal CARS was characterized by calculating the nonlinear polarization intensity signals in the sample plane for different sample offsets relative to the focal plane. The fields calculated at the far field were mapped onto the detector plane or image plane with a confocal pinhole using a lens function. Resolution improvement with the use of a phase mask and pinhole in the CARS microscope system was characterized based on the point spread function of the microscope system obtained through scans of an isolated particle. The resolution improvement and side-lobe suppression in confocal-focus engineered CARS microscope demonstrated in simulations were also compared with experimental studies. We next focused on the particle-swarm optimization-based design and fabrication of fixed phase masks that provide resolution improvement in second harmonic generation (SHG) microscope. Instead of using spatial light modulator (SLM) for phase mask generation as in the previous works, the proposed fixed phase masks offer the benefit of ease of integration with the microscope system and higher power handling capability. We designed phase mask of multi-step 0-π phase for better resolution improvement and sidelobe suppression using particle swarm optimization (PSO) algorithm. The SHG nonlinear polarization in the presence of phase mask was calculated by generating the three-dimensional vectorial fields at the focus of the objective lens and particle swarm optimization was used to optimize the mask design that achieved maximum resolution improvement with significant sidelobe suppression. The designed multi-step phase masks were fabricated and the resolution improvement with the use of optimized phase mask was validated by imaging isolated nanoparticle and collagen from mouse mesentery samples. We also reported the first experimental demonstration, to the best of our knowledge, of IR up-conversion imaging using a optimally designed multilayer two-dimensional (2D) nonlinear optical mirror. The proposed nonlinear optical mirror consists of a multilayer 2D material of optimum thickness on top of a gold reflector with silicon dioxide (SiO2) as the intermediate spacer layer. Particle swarm optimization (PSO) algorithm combined with nonlinear simulation modelled in COMSOL were used to optimize the thicknesses of the chosen Gallium Selenide (GaSe) 2D material and the intermediate silicon dioxide on gold film for efficient sum frequency generation at the far-field detector plane. The designed structure presented a promising alternative with enhanced power conversion efficiency atleast by two-order of magnitude over the conventional metasurfaces, which typically needs intricate fabrication processes. The potential application of these compact reflective up-conversion mirrors in imaging systems for up-conversion of infrared wavelengths to visible range was also illustrated in both real and Fourier plane imaging configurations. Basic image processing using Fourier transform based processing using these the 2D nonlinear optical mirror is also demonstrated. Next, in the context of application to biomedical imaging, we investigated in detail the role of Dermatan Sulfate (DS), a glycosaminoglycan in rearrangement of collagen fibrils associated with ovarian cancer development. Initial experiments were conducted on in-vitro samples to explore the role of varying concentrations of DS on structural changes in collagen architecture. Quantitative analysis using image processing approaches were done on the acquired images to extract parameters to differentiate samples of various compositions of DS. We observed that the extracted parameters like surface occupancy and means SHG signal were able to differentiate the samples of varying concentrations. In addition to this, we also study in detail the role of Decorin (a proteoglycans which is observed to be depleted not just in primary ovarian tumors but also at the site of metastasis, i.e., in the mesentery) in architectural property of collagen associated with metastasis of ovarian cancer. The wide field imaging performed using multimodal nonlinear microscopy imaging, combining SHG and two-photon excited fluorescence and coupled this with quantitative image analysis showed higher SHG signal from collagen fibers in omentum tissue (secondary site) with cancer deposits relative to their controls. Higher SHG signals signified greater collagen organization, which is known to promote cancer cell migration. Using in-vitro scaffold setups, greater spheroidal unpacking was observed on Collagen I relative to scaffolds where Collagen I has been infused with decorin. SHG signals for the latter are lower than only Collagen I control scaffolds, suggesting that the presence of extracellular decorin weakens the organization of Collagen potentially disallowing cancer cell migration. Hence its depletion or relative scarcity may signify sites for metastasis. This works opens up the possibility for SHG to be used as a predictive tool to identify prospective sites of cancer colonization. Thus, intra- and inter-patient heterogeneity in SHG signals could help define the kinetics of therapy in the near future.