SIGNIFICANCE: Mid-infrared (IR) imaging based on the vibrational transition of biomolecules provides good chemical-specific contrast in label-free imaging of biology tissues, making it a popular tool in both biomedical studies and clinical applications. However, the current technology typically requires thin and dried or extremely flat samples, whose complicated processing limits this technology's broader translation. AIM: To address this issue, we report mid-IR photoacoustic microscopy (PAM), which can readily work with fresh and thick tissue samples, even when they have rough surfaces. APPROACH: We developed a transmission-mode mid-IR PAM system employing an optical parametric oscillation laser operating in the wavelength range from 2.5 to 12 μm. Due to its high sensitivity to optical absorption and the low ultrasonic attenuation of tissue, our PAM achieved greater probing depth than Fourier transform IR spectroscopy, thus enabling imaging fresh and thick tissue samples with rough surfaces. RESULTS: In our spectroscopy study, the CH2 symmetric stretching at 2850 cm - 1 (3508 nm) was found to be an excellent source of endogenous contrast for lipids. At this wavenumber, we demonstrated label-free imaging of the lipid composition in fresh, manually cut, and unprocessed tissue sections of up to 3-mm thickness. CONCLUSIONS: Our technology requires no time-consuming sample preparation procedure and has great potential in both fast clinical histological analysis and fundamental biological studies.
