Abstract
Microscopy based on non-fluorescent absorption dye staining is widely used in various fields of biomedicine for 400 years. Unlike its fluorescent counterpart, non-fluorescent absorption microscopy lacks proper methodologies to realize its in vivo applications with a sub-femtoliter 3D resolution. Regardless of the most advanced high-resolution photoacoustic microscopy, sub-femtoliter spatial resolution is still unattainable, and the imaging speed is relatively slow. In this paper, based on the two-photon photoacoustic mechanism, we demonstrated a in vivo label free laser-scanning photoacoustic imaging modality featuring high frame rates and sub-femtoliter 3D resolution simultaneously, which stands as a perfect solution to 3D high resolution non-fluorescent absorption microscopy. Furthermore, we first demonstrated in vivo label-free two-photon acoustic microscopy on the observation of non-fluorescent melanin distribution within mouse skin.
Highlights
Extreme case, the superiority of Photoacoustic microscopy (PAM) in penetration depth compared to other optical microscopy has already been sacrificed
To separate the two-photon acoustic signal from its single-photon background, we recently proposed the use of a loss modulation technique[13]
For in vivo imaging applications, we find this 2PAM to be ideal for high spatial resolution imaging of melanin distribution in animal skin
Summary
Extreme case, the superiority of PAM in penetration depth compared to other optical microscopy has already been sacrificed. Sub-femtoliter-resolution non-fluorescent absorption 3D microscopy remains a distant prospect. Another issue with in vivo high-resolution PAM is the imaging rate. We report our realization of in vivo 2PAM with a sub-femtoliter spatial resolution This was achieved with two-photon acoustic contrast excited by a Ti:sapphire femtosecond laser, combining a loss modulation system to remove the linear photoacoustic background. We optimized the efficiency of two-photon acoustic excitation by ameliorating a material dispersion effect, carefully characterized the spatial resolution of 2PAM in both lateral and axial directions, and solved the imaging speed problem by integration with a galvo-scanner. We demonstrated the first in vivo laser-scanning high-frame-rate 2PAM based on non-fluorescent absorption contrasts with a sub-femtoliter 3D spatial resolution. For in vivo imaging applications, we find this 2PAM to be ideal for high spatial resolution imaging of melanin distribution in animal skin
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