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Development of Tools for Nonlinear Optical Imaging of Multiple Cells and Tissue Structures

Author
Bares, Amanda Josephine
Abstract
Studies of both normal and disease state physiology require the visualization of multiple
cells and cell types simultaneously. Multiphoton microscopy has enabled researchers to
visualize fluorescently-tagged cells with subcellular resolution in live animal models of
disease, overcoming the optical-scattering effects of tissue. However, most multiphoton
microscopes only provide two or four color channels, limiting the number of fluorescent
labels, and thus cell types, that can be simultaneously imaged.
This thesis describes the development and demonstration of a hyperspectral
multiphoton microscope that enables simultaneous visualization of multiple cell types.
Three laser excitation sources are multiplexed with multiple detection channels, each
providing improved spectral detection through the use of angle-tuned bandpass filters.
The detection system was designed to provide efficient detection of highly-scattered
light from tissue while minimizing the impact of scattered light on spectral resolution.
We demonstrated the ability of the instrument to image multiple, spectrally-similar
fluorescent labels, and developed methodology to post-process data to generate images
with each fluorescent label clearly separated and indicated with a unique color in a
composite image.
We demonstrated hyperspectral imaging and spectral separation of ten
overlapping colors of fluorescent beads, up to seven fluorescent labels in live cells, and
five labels in live mouse cortex. In addition, we demonstrated multicolor labeling
techniques enabling identification of morphologically-similar cells based on color
“hue”, and characterized color variability using the spectral capabilities of the
microscope.
In other work in this thesis, we explored third harmonic generation for label-free
visualization of myelinated nerves over large viewing areas for eventual surgery room
applications. Nerves are notoriously difficult to see in the surgical field, and nerve
injuries are a common cause of post-surgery morbidity. Third harmonic generation has
been established as an excellent tool for myelin visualization, but the requirements for
a high zoom microscope objective have limited the area of imaging to areas too small
for surgery room use. We demonstrated third harmonic generation imaging in both
mouse sciatic nerve and rat cavernous nerve on the prostate with low-zoom, low
numerical-aperture objectives, and found that myelin produces less signal than fat
deposits, potentially limiting the utility for nerve visualization in areas with high fat
content.
Date Issued
2017-08-30Subject
Biophysics; Biomedical engineering; Nonlinear Optics; Neurosciences; Hyperspectral; Imaging; Microscopy; Multiphoton; Multiplexing
Committee Chair
Schaffer, Chris
Committee Member
Fetcho, Joseph R.; Xu, Chunhui
Degree Discipline
Biomedical Engineering
Degree Name
Ph. D., Biomedical Engineering
Degree Level
Doctor of Philosophy
Rights
Attribution-NoDerivatives 2.0 Generic
Type
dissertation or thesis
Except where otherwise noted, this item's license is described as Attribution-NoDerivatives 2.0 Generic