Ultra-High Frequency Photoacoustic Microscopy: From Organelles to Organisms

This dissertation describes novel signal analysis and imaging techniques for ultrahigh frequency (UHF, over 100 MHz) Photoacoustic Microscopy (PAM). New approaches for extracting information pertaining to object structure and scale are described, and novel sensing techniques and contrast mechanisms for imaging biological samples ranging from single cells to small organisms are presented. In the first section, I describe a methodology for assessing the structure of biological cells using UHF-PAM. The power spectra of ultrasound (US) pulses backscattered from MCF-7 cells, and photoacoustic (PA) waves emitted from their dyed nuclei were fit to analytical solutions to determine cell and nucleus diameter, respectively. The measured cell diameters (15.5±1.8 μm) and nucleus diameters (12±1.3 μm) were used to calculate the mean cell nucleus-to-cytoplasm ratio (1.9±1.0). Good agreement was observed between UHF-PAM measured values and literature. In the second section, I present a novel technique for PA image reconstruction that utilizes unique features in the PA power spectra as a source of contrast. The technique, termed F-Mode, provides a means for differentiating between objects of different scale that surpasses the capabilities of conventional reconstruction approaches. The ability of F-Mode to selectively accentuate absorbers of different size was demonstrated using experimental phantoms containing microspheres and cylindrical vessels, as well as in individual biological cells and live zebrafish larvae. Finally, I developed a new sensing technique, termed Photoacoustic Radiometry (PAR). Unlike PAM, which depicts optical absorption, PAR images depict the optical attenuation properties of the imaged object. It was demonstrated that PAR can be used to image transparent samples which generate no PA signals, and that simultaneous triplex PAR/PA/US imaging could be realized using our approach. Simultaneous PAR/PA imaging of biological cells, as well as zebrafish larvae in vivo, was also demonstrated. UHF-PAM provided excellent visualization of vascular organization in the larval trunk and head. The simultaneously acquired PAR images depicted anatomical structure (e.g. the notochord, muscle segments) not visible in PAM due to insufficient optical absorption. Potential areas of application for the new UHF-PAM techniques described in this dissertation include detection of cancer cells in blood samples, and investigation of tumour growth and metastasis. (6 zipped .mp4 files) https://digital.library.ryerson.ca/islandora/object/RULA:8618/datastream/Movies_Moore_mp4/view