Date of Award
Doctor of Philosophy
Carol J Hirschmugl
George Hanson, Chiu Law, Yongjin Sung, Achim Kohler
3D refractive index recovery, coherency, fringe correction, infrared hyperspectral imaging, scattering, spectroscopy
Fourier Transform Infrared (FTIR) microspectroscopy is a noninvasive technique for chemical imaging of micrometer size samples. Employing an infrared microscope, an infrared source and FTIR spectrometer coupled to a microscope with an array of detectors (128 x 128 detectors), enables collecting combined spectral and spatial information simultaneously. Wavelength dependent images are collected, that reveal biochemical signatures of disease pathology and cell cycle. Single cell biochemistry can be evaluated with this technique, since the wavelength of light is comparable to the size of the objects of interest, which leads to additional spectral and spatial effects disturb biological signatures and can confound the understanding and analysis. In the present research, the measured signatures are corrected by cleaning the spectra to improve the fundamental analysis of single cell samples.
In diffraction limit FTIR imaging, where the size of the sample is in the same range as the incident light, scattering phenomenon appear in spectra as a result of the interaction of light and matter. The observed scattering contribution depends on the physical and chemical structure of the sample as well as the focusing optics and the light source.
It is crucial to consider the light source to interpret any image, for example visible images taken by a flashlight or a laser provide distinct information about the sample. Synchrotron and thermal light sources are currently employed in FTIR imaging, and infrared laser sources are quickly being adopted for future application. Lasers are fully coherent light sources, while synchrotron and thermal sources (like flashlights) are partially coherent and non-coherent light sources, respectively. Coherency of the light can have a profound effect in the diffraction limited imaging and needs to be taken into account when analyzing hyperspectral images.
The objective of the thesis is to understand and remove scattering contributions in infrared hyperspectral images. The first Chapter of the thesis introduces the wide field FTIR imaging technique and describes the distinct observations for scattering objects using a large single detector vs a focal plane array, that is a two-dimensional array of small detectors. The former has been well established with many theories that accurately predict the detected signals, while the latter topic regarding what is detected in a single pixel detector is a central question that is addressed in this thesis.
In Chapter 2, an in-depth analysis of stressed, hydrated algal cells (Thalassiosira weissflogii) measured with FTIR spectromicroscopic imaging is presented. The spatially varying, pixelated, biochemical response to environmental stresses has been revealed. This prototypical experiment shows the potential of such imaging to monitor in-situ biochemical changes, for single cells that are maintained in a water environment with minimal scattering, but highlights the need to remove inherent spectral and spatial fringes from data that are inherent when maintaining cells in such an environment.
Many Hydrated single cells with distinct biochemistry have important chemical distributions (e.g. yeast cells) and are therefore interesting to measure without a hydrated environment. However, the infrared measurements of these cells are frequently dominated by scattering, since they are similar in size to the wavelength of the probing light. Due to the presence of scattering, the individual pixels in wide field FTIR imaging have spectral responses that represent both the chemistry and physical response of the sample. Pure absorbance spectra are desired to detect the subtle differences in biochemistry that are important. However, the pixelated data contain abrupt spectrally dependent inhomogeneities, since samples with geometrical shape, and refractive indices that are different from their environment strongly deviate direction of the incident light.
As a first step to understand the effects of light scattering in pixelated FTIR imaging, homogenous microspheres with size similar to the wavelength of light are studied, experimentally and theoretically. In Chapter 3 experimental results for polymer (PMMA) microspheres imaged with a synchrotron and a thermal source are provided. Interestingly, the pixelated spectra measured with synchrotron source are distinct from the ones measured with a thermal source. The distinction between the spectra measured with synchrotron and thermal sources is being related to the spatial coherency of the light source.
In Chapter 4, hyperspectral images of the microsphere are simulated using the resonant Mie scattering theory. The simulated images give an insight about the experimental results and help answer the question of what is detected in a single pixel. A full understanding of the impact of scattering effects on spatial and spectral responses will enable us to develop strategies for deconvolving the scattering contributions and recovering pure absorbance images.
With the insight gained from the experiments and simulations, we identified that removing scattering from pixelated spectra is feasible by an iterative inverse method. The outcome is presented in Chapter 5 as a new algorithm. It is shown that, the complex refractive index of the sample can be recovered by measuring amplitude and phase of the electric field. The algorithm is not sensitive to noise, and it can recover refractive indices of samples with high absorption.
By incorporating a finite element software and extending the algorithm, recovery of the complex refractive index of the sample with super resolution is expected.
Azarfar, Ghazal, "Light Scattering in Diffraction Limit Infrared Imaging" (2019). Theses and Dissertations. 2156.