Towards Multi-photon Light-sheet Microscopy through a Fiber

The gold standard for cancer diagnosis, including, but not limited to colorectal cancer (CRC), is the inherently invasive and time-consuming histopathological evaluation of resected tissue (biopsy). In contrast, the optical biopsy could assist the pathologist in early CRC diagnosis by exploiting the phenomena of light interaction with tissue with excellent optical sectioning capabilities, potentially preventing the need for tissue resection. Morphological and biochemical characteristics of the colorectal tissue could be obtained through multiple imaging modalities offering optical biopsy.
The focus of the thesis is to introduce a minimally invasive method that aids deeper and faster fluorescence imaging for real-time cancer diagnostics in vivo. Multi-photon light-sheet fluorescence microscopy (MP-LSFM) is a powerful imaging technology in the biomedical field that, when implemented through an optical fiber, will contribute a significant step towards the endoscopic adaptation of MP-LSFM, a requirement for clinical translation. This step allows for early implementations of in vivo testing and further expansions into a multimodal system. Moreover, the lensless endoscope adaptation of MP-LSFM could bypass the distal end optics and yet provide distal beam shaping and scanning through proximal wavefront control, thus allowing fast, non-phototoxic, 3D volumetric image acquisition at depth utilizing a miniaturized imaging probe.
The original contributions of this thesis are in the two-photon lensless adaptation of light-sheet fluorescence microscopy. Label-free two-photon light-sheet fluorescence microscopy (TP-LSFM) is implemented in free-space demonstrating fluorescence collection from colorectal mucosa in the absence of external contrast agents. Beam-shaping is applied to a MP-LSFM incorporating Airy beams for attenuation-compensation, thereby enhancing the field-of-view for better imaging depth through selective intensity delivery, using an inexpensive graded neutral density filter in a facile manner.
A numerical tool for simulating electric field propagation through optical fibers with arbitrary refractive index profiles using the computationally efficient Douglas-Gunn alternating direction implicit method is developed in MATLAB. The potency of the software is shown for choosing the optimal optical fiber for lensless light-sheet beam-shaping. Finally, steps are taken to implement the illumination arm of a two-photon lensless light-sheet endoscope using hexagonal multicore fiber, with particular attention to the challenges imposed by the heterogeneous intercore beam transmission.