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Two-photon line-scanning structured illumination microscopy combined with lightsheet scanning mode for super-resolution imaging in deep tissue

preprint
posted on 2024-06-06, 03:39 authored by Patrick Byers, Thomas Kellerer, Miaomiao Li, Zhifen Chen, Thomas Huser, Thomas Hellerer
Imaging structures deep within biological tissue and organisms with a spatial resolution well below the optical diffraction limit is still underrepresented. This is mostly due to the complexity and high cost of microscopes that can facilitate such challenges. The majority of deep tissue imaging is still accomplished by exciting fluorescence and other nonlinear light-sample interactions with ultrashort pulsed lasers. Here, we demonstrate a cost efficient and easy to implement method to turn most two-photon laser-scanning microscopes into a super-resolution microscope for deep tissue imaging. We realize this by adding a compact and cost-efficient line-scanning module, a field rotator, and a sCMOS camera to these systems. By combining two-photon excitation with patterned line-scanning and subsequent image reconstruction, we achieve imaging with a spatial resolution well below the diffraction limit. Furthermore, by synchronizing the two-photon line illumination with camera-based line confocalization, a significant contrast enhancement deep within biological tissue can be achieved. We demonstrate this by imaging subwavelength structures in extended Pinus radiata, mouse heart muscle and zebrafish samples.

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Preprint ID

113933

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