Optical-resolution photoacoustic microscopy (OR-PAM) has rapidly developed and is capable of characterizing optical absorption properties of biological tissue with high contrast and high resolution (micrometer-scale lateral resolution). However, the conventional excitation source of rapidly diverging Gaussian beam imposes limitations on the depth of focus (DOF) in OR-PAM, which in turn affects the depth-resolving ability and detection sensitivity. Here, we proposed a flexible DOF, depth-invariant resolution photoacoustic microscopy (FDIR-PAM) with nondiffraction of Airy beams. The spatial light modulator was incorporated into the optical pathway of the excitation source with matched switching phase patterns, achieving the flexibly adjustable modulation parameters of the Airy beam. We conducted experiments on phantoms and intravital tissue to validate the effectiveness of the proposed approach for high sensitivity and high-resolution characterization of variable topology of tissue, offering a promising DOF of 926 μm with an invariant lateral resolution of 3.2 μm, which is more than 17-fold larger compared to the Gaussian beam. In addition, FDIR-PAM successfully revealed clear individual zebrafish larvae and the pigment pattern of adult zebrafishes, as well as fine morphology of cerebral vasculature in a large depth range with high resolution, which has reached an evident resolving capability improvement of 62% mean value compared with the Gaussian beam.
KEYWORDS: Raman spectroscopy, Tomography, Optical tomography, Imaging systems, Signal detection, 3D image processing, 3D image reconstruction, Projection systems
Volumetric imaging enables rapid, quantitative and global measurements of cells, tissues or organisms to obtain their biomolecular information and has become a powerful tool for studying cellular metabolism, brain function and developmental biology. Optical projection tomography (OPT) plays an important role in whole-body imaging of cells, organs, embryos and organisms because it enables three-dimensional (3D) imaging with high spatial and temporal resolution of samples at the millimeter level. However, the OPT technique relies on fluorescent labels for chemical targeting, which can perturb the biological function of living system. As a label-free molecular imaging technique, widefield Raman imaging enables high-resolution analysis of large field-of-view samples. Its combination with projection tomographic strategy enables high-resolution 3D imaging of large-scale samples in a label-free manner. However, this technique was failure to determine the tissue microstructure and specific spatial distribution. Here, we proposed a concept of new label free volumetric imaging, dual-modality of optical-Raman projection tomography. In this concept, Raman projection tomography was assigned to achieve volumetric imaging of chemical composition and distribution in a 3D volume, and the OPT was used to obtain structural information of the 3D volume with micron-level spatial resolution. We further homebuilt a dual-modality imaging system for optical-Raman projection tomography and the feasibility of the system was validated by imaging polystyrene microspheres and dimethyl sulfoxide. Finally, we demonstrated the application potential by a series of bio-sample experiments.
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