Hyperspectral imaging has emerged as a valuable technique for analyzing biological tissue compositions by probing intrinsic or exogenous biomolecules. However, conventional hyperspectral imaging methods predominantly rely on fluorescent signatures, limiting their application to nonfluorescent samples. To overcome this limitation, a label-free reflection-mode hyperspectral photoacoustic microscopy (RHS-PAM) system has been developed. RHS-PAM enables the imaging of thick biological samples with a wide range of intrinsic contrasts using excitation wavelengths ranging from ultraviolet to near infrared. RHS-PAM eliminates the need for tissue staining, and has achieved cellular-level spatial resolution and automatic image coregistrations at all wavelengths. Proof-of-concept applications of RHS-PAM have been demonstrated on various model organisms, including Caenorhabditis elegans, frog tadpole, zebrafish, and mouse. The technique has successfully imaged a wealth of structural and molecular features in these organisms, utilizing the optical absorption contrast of nucleic acids, proteins, hemoglobin, melanin, and lipids. The results highlight the capability of RHS-PAM to provide rich optical contrast, high spatial resolution, and an extended spectral range for label-free imaging. We believe that RHS-PAM represents a highly promising tool for single-cell biochemical mapping of diverse biological tissues.
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