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Description
Photoacoustic microscopy with optical resolution (OR-PAM) is a method that uses optical absorption contrast for generating microscopic images of mainly biological samples. The resolution is given by the focus dimensions of an optical objective, through which a pulsed laser illuminates a sample. Local heating in the focal volume gives rise to ultrasound transients, which are detected with a high-frequency, focused ultrasound receiver. With an excitation wavelength in the ultraviolet (UV) range, this method is able to visualize cell nuclei owing to the strong optical absorption of DNA molecules.In this study, we used excitation wavelengths between 258 and 264 nm to visualize UV-absorbing structures in biological samples. The photoacoustic images were compared to optical microscopy images that were obtained using standard hematoxylin-eosin (H&E) staining, showing the ability of OR-PAM to target cell nuclei. Special attention was drawn to the optimization of the scanning technique, using a fast galvo scanner to steer the excitation beam across the sample, thereby achieving a 512 µm wide scanning range with a static, focused ultrasound receiver. A special reconstruction method compensates variations in acoustic sensitivity and time-of-flight across the sensitive area of the detector. Due to the targeting of optically absorbing structures, the sample does not necessarily need to be optically transparent, such as in transmission optical microscopy. This is demonstrated by imaging not only standard histological sections with a thickness in the µm-range, but also up to 20 µm deep into about 2 mm thick slices of fresh biological tissue.The results show the ability of the method to obtain diagnostic information from fresh tissue without using the time-consuming standard staining techniques.