Application of beam shaping in second-harmonic generation microscopy

Second-harmonic generation (SHG) microscopy, based on the second-order nonlinear susceptibility, has been established as a sub-micrometer resolution, label-free optical imaging method. Due to its selectivity for non-centrosymmetric structures and inherent confocality, SHG imaging has been used to perform three-dimensional sectioning of fibrillar biological structures such as collagen fibers. Quantitative analysis of SHG images has provided information about tissues, such as fibrillar organization, that can produce deeper insight into the relation between the tissue structure and its function. Further application of adaptive deconvolution algorithms for SHG images has allowed significant improvement in signal-to-noise ratio, revealing the underlying helical construct of collagen fibers. Despite the versatility of this imaging technique, the light-matter interaction has largely been limited to a Gaussian illumination intensity distribution. Restructuring the focal field intensity profile through beam shaping can be useful in reducing imaging artifacts and improving quantitative image analysis. In this dissertation, two different beam shapes are explored in the context of SHG imaging. The first approach incorporates a reflective microscope objective and a Bessel-Gauss beam illumination to reduce chromatic dispersion in SHG imaging. Using well-aligned, densely populated samples of collagen fibers, the performance of the reflective objective is systemically compared with a standard glass objective. For experiments traversing ~ 1 octave in the nearinfrared and visible wavelengths, a significant reduction in chromatic aberration is observed for the RO. This work highlights the potential applicability of ROs for multimodal multiphoton microscopy. The second research work focuses on adopting a square-shaped uniform illumination profile in a widefield imaging configuration to reduce intensity-based vignetting. The uniform illumination is obtained with a flat-top beam (FTB) that provides a comparatively higher two-photon excitation signal-to-noise ratio (SNR) distribution, as well as a reduction in the number of dark areas for a rectangular imaging region of interest. Utilizing well-aligned, densely populated tendon and randomly aligned, sparsely populated breast biopsy tissue, the effects of vignetting on quantitative SHG imaging are compared for a Gaussian beam and an FTB. This work emphasizes the utility of an FTB illumination for quantitative SHG imaging of fast, dynamic biological processes.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Electrical & Computer Engr

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2019

Author dc:creator

• Kabir, Mohammad Mahfuzul

Contributors dc:contributor

• Toussint, Kimani C
• Popescu, Gabriel
• Patel, Sanjay J
• Gao, Liang

• Biomedical Imaging, Optics, Microscopy

Statement dc:rights

• Copyright 2019 Mohammad Kabir

Language dc:language

en

Handle dc:identifier

http://hdl.handle.net/2142/105740

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/105740