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

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.