Implementation of optical depth scanning and wide-field imaging in photoacoustic remote sensing (PARS) microscopy

Abstract

Optical resolution photoacoustic microscopy (OR-PAM) is a hybrid biomedical imaging technique that utilizes acoustic detection and optical absorption contrast. It is based on the photoacoustic effect, where the excitation light energy absorbed by biomolecules is converted into heat. The initial temperature rise causes thermo-elastic expansion in tissues, resulting in the generation of acoustic waves detected by an ultrasound transducer. OR-PAM takes advantage of tightly focused light as the excitation source to achieve micron to submicron optical lateral resolution at superficial depths (~1mm). Additionally, it provides high contrasts to endogenous chromophores allowing for label-free in-vivo imaging. As a result, OR-PAM is a powerful tool for morphological, functional, and molecular imaging of biological organisms and their vital processes. However, conventional OR-PAM architectures are usually limited by the need for an ultrasound transducer to be in direct contact with the sample through a coupling medium. This condition introduces complexity to optical design and implementation and might become a source of infection or contamination in applications where physical contact is undesirable or impossible. Photoacoustic remote sensing (PARS) microscopy is an all-optical, label-free technique first introduced in 2017. But unlike OR-PAM, PARS does not require physical contact with the sample. PARS microscopy employs an interrogation beam as an alternative to the conventional ultrasonic transducer. There are, however, two limitations to PARS that will be addressed in this thesis dissertation. Firstly, PARS lacks an inherent 3D imaging capability since photoacoustic pressures induced by pulsed lasers are detected at their origin. Instead, volumetric imaging is achieved by mechanical scanning, which presents some drawbacks, such as slow scanning rates and motion artifacts, as well as being bulky and expensive. Optical focus shifting may allow PARS to image larger volumes at higher speeds and with higher resolution. In this work, a novel continuous micro-electromechanical systems (MEMS) deformable mirror (DM) was integrated into a PARS microscope for imaging at varying depths. The first step was to create an optical model using the DM characteristics and use Zemax to predict the focal shift. Next, an experimental investigation of the focus shifting ability of the DM was conducted using a 532-nm scattering microscope, and a focal shift of 240 µm was achieved. Afterward, carbon fiber imaging was conducted to demonstrate the axial scanning capabilities of DM-based PARS microscopy. Lastly, the focal plane was optically shifted to perform in-vivo PARS imaging of blood vessels in chick embryo chorioallantoic membrane (CAM) models at different depths. The second limitation of PARS microscopy is its inability to provide fast wide field of view (WFOV) imaging. WFOV imaging is achieved by mechanically scanning small areas laterally at different positions and then stitching them together. Mechanical scanning, however, is slow, prone to motion artifacts, and might agitate sensitive samples. As part of this work, we demonstrated how an optical approach using a scan lens could be used to achieve 8.58.5 mm2 FOV imaging of carbon fibers in PARS. Moreover, the system was utilised in in-vivo studies by imaging CAM vasculature and reaching up to 3.343.34 mm2 FOV. Further system enhancements are needed to expand the FOV, increase imaging speeds, and improve resolution. This potentially can be realized by integrating adaptive optics elements to actively correct for system- and specimen-induced aberration and adjust focus over a large FOV.