In the current work by Song et al., this limitation of reflectance-based OR-PAM imaging systems has been overcome by configuring a miniature high-frequency ultrasonic transducer tightly under a water-immersion objective with an NA of one. Using a 532-nm laser for optical excitation, the lateral resolution of the system was measured to be ~320 nm with an axial resolution of ~29 nm. To date, OR-PAM has demonstrated the capability of providing optical-absorption-based anatomical, functional, and molecular information with broad applications in vascular biology, neurology, and ophthalmology. To that end, the investigators in this work demonstrate the resolution of their OR-PAM imaging system on red blood cells (RBCs), cancer cell lines, and mouse vasculature. Due to the strong optical absorption of hemoglobin in RBCs, the OR-PAM system offered high contrast for RBCs where the donut-like shape could be readily visualized. Compared with the RBC images from a conventional bright field optical microscope the OR-PAM system demonstrated ~25 dB improved contrast to noise ratio. OR-PAM images of B16 melanoma cancer cell line demonstrated high-resolution speckled images where the bright spots corresponded to melanin, which can produce high-amplitude photoacoustic signals due to the strong absorption of melanin. Example mouse vasculature in the ear of a murine model was imaged with the OR-PAM system. Excitingly, the submicron resolution of the novel OR-PAM system enabled visualization of individual RBCs in small capillaries.
In summary, the investigators have built and validated a subwavelength-resolution OR-PAM system with a compact optical-acoustic configuration, which allows laser focusing at high NA and photoacoustic signal detection in reflectance-based mode with improved resolution over other OR-PAM systems. In principle, the compact design of this OR-PAM system should enable integration with other widely used optical imaging techniques such as optical coherence tomography, confocal microscopy and multi-photon microscopy, facilitating visualization of complimentary optical contrast mechanisms.
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