Development of a Multiphoton Photoacoustic Microscope

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Shelton, Ryan 1983-
ultrahigh resolution; hemoglobin; blood; transient absorption; microscopy; photoacoustic
thesis / dissertation description
Cellular/subcellular imaging of biological tissue is an important tool for understanding disease mechanisms. Many current techniques for subcellular absorption contrast imaging, such as two-photon excited fluorescence (TPEF), require exogenous contrast agents to gain access to many naturally occurring biomolecules. Non-fluorescent biomolecules must have a fluorescent marker (tag) chemically bound in order to be observed by TPEF. Contrast agents and markers, while effective, are not an optimal solution because they can change the local environment in the biological system and require FDA approval for human use. Photoacoustic microscopy (PAM) is an imaging modality with high endogenous absorption contrast and penetration depth due to its ability to detect acoustic waves, which are attenuated much less than light in tissue. However, this technique suffers from poor axial resolution, precluding it from consideration for subcellular imaging. This manuscript describes the author's efforts to improve the axial resolution of traditional PAM by merging it with pump-probe spectroscopy. Pump-probe spectroscopy is a non-linear optical technique that exploits a physical process called transient absorption, providing spatial resolution equivalent to two-photon microscopy and access to molecular-specific traits, such as the ground state recovery time and transient absorption spectrum. These traits provide molecular contrast to the imaging technique, which is highly desirable in a complex, multi-chromophore biological system. In this manuscript, a novel technique called transient absorption ultrasonic microscopy (TAUM) is designed and characterized in detail. A second-generation TAUM system is also described, which improves speed and sensitivity of TAUM by up to 1000-fold. This system is validated by collecting volumes of red blood cells in blood smears and tissue samples. These results constitute the first time single cells have been fully resolved using a photoacoustic microscope. Finally, the TAUM system is modified to measure chromophore ground state recovery times. This technique is validated by measuring the recovery time of Rhodamine 6G, which matches well with published values of the fluorescence lifetime. Recovery times of oxidized and reduced forms of hemoglobin are also measured and shown to statistically differ from one another, suggesting the possibility of subcellular measurements of oxygen saturation in future iterations of TAUM.