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Subwavelength-resolution photoacoustic microscopy for label-free detection of optical absorption in vivo

Mainstream optical microscopy technologies normally detect fluorescence or scattering, which may require undesirable labeling, but cannot directly sense optical absorption, which provides essential biological functional information. Here we reported in vivo and label-free subwavelength-resolution photoacoustic microscopy (SW-PAM) by using a waterimmersion optical objective with a 1.23 NA. Capable of detecting nonfluorescent endogenous pigments, SW-PAM provides exquisitely high optical-absorption contrast. And, as a result of background-free detection, the sensitivity of SW-PAM to optical absorption reaches 100%. SW-PAM was demonstrated with wide-field optical microscopy by imaging gold nanospheres, ex vivo cells, and in vivo vasculature and melanoma. It was shown that SW-PAM has approached the ultimate diffraction-limited optical resolution-220 nm resolution at 532 nm wavelength. Subcellular organelles, such as melanosomes, can be resolved by SW-PAM. Vasculature and early-stage melanoma were imaged with 21:1 and 34:1 contrasts, respectively, without labeling. For all these applications, SW-PAM has contrasts orders of magnitude higher than wide-field optical microscopy. Therefore, SW-PAM is expected to join the mainstream microscopy technologies.