Abstract
Photoacoustic microscopy (PAM) is an attractive imaging tool complementary to established optical microscopic modalities by providing additional molecular specificities through imaging optical absorption contrast. While the development of optical resolution photoacoustic microscopy (ORPAM) offers high lateral resolution, the acoustically-determined axial resolution is limited due to the constraint in ultrasonic detection bandwidth. ORPAM with isometric spatial resolution along both axial and lateral direction is yet to be developed. Although recently developed sophisticated optical illumination and reconstruction methods offer improved axial resolution in ORPAM, the image acquisition procedures are rather complicated, limiting their capabilities for high-speed imaging and being easily integrated with established optical microscopic modalities. Here we report an isometric ORPAM based on an optically transparent micro-ring resonator ultrasonic detector and a commercial inverted microscope platform. Owing to the superior spatial resolution and the ease of integrating our ORPAM with established microscopic modalities, single cell imaging with extrinsic fluorescence staining, intrinsic autofluorescence, and optical absorption can be achieved simultaneously. This technique holds promise to greatly improve the accessibility of PAM to the broader biomedical researchers.
Highlights
Light microscopy has become an indispensable tool for biological researchers
Using the current micro-ring resonator (MRR) detector, our Photoacoustic microscopy (PAM)’s axial resolution is 2.12 μm, which is solely determined by the ultrasonic detection bandwidth and it is better than the optical depth of field of the objective lens used in the experiment (~5.25 μm, see Supplementary Material for more details)
Employing high numerical apertures (NAs) objective lenses enables PAM a sub-micrometer lateral resolution to image samples at the cellular level. We demonstrated this capability by imaging healthy mouse red blood cells (RBCs)
Summary
Light microscopy has become an indispensable tool for biological researchers. It has evolved from the ancient “reading stone” to a powerful technology platform that hosts a broad range of imaging modalities for a comprehensive investigation of the physiological processes. In PAM, nanosecond laser pulses excite molecules in tissues through linear optical absorption, leading to a transient thermo-elastic expansion and subsequently generating ultrasonic waves with a wide-range of frequency components This effect is referred to as the photoacoustic (PA) effect. Only an objective lens with a large working distance, but low numerical apertures (NAs) can be used Such a limitation imposes constraints in the spatial resolution of PAM, but more importantly, it prevents a practical integration of PAM with established microscopic modalities that require high NAs, such as two-photon microscopy. We recently developed an opticallytransparent, micro-ring resonator (MRR)-based ultrasonic detector (diameter < 100 μm) and fabricated it on a microscope coverslip (thickness: 225 μm) [34] Such a miniaturized ultrasonic detector meets the strict dimension requirements for seamlessly integrating PAM with optical microscopic modalities using high NA objective lenses. We demonstrated a multimodal microscopic system developed around a commercial inverted microscopic platform to achieve ultrahigh-resolution, isometric PAM imaging of single red blood cells and the simultaneous autofluorescence, fluorescence labeling, and optical absorption imaging of retinal pigment epithelium
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