High Refractive\nIndex Imaging Buffer for Dual-Color\n3D SMLM Imaging of Thick Samples

The current limitations of single-molecule localization\nmicroscopy\n(SMLM) in deep tissue imaging, primarily due to depth-dependent aberrations\ncaused by refractive index (RI) mismatch, present a significant challenge\nin achieving high-resolution images at greater depths. To extend the\nimaging depth, we optimized the imaging buffer of SMLM with the RI\nmatched to that of the objective immersion medium and systematically\nevaluated five different RI-matched buffers, focusing on their impact\non the blinking behavior of red-absorbing dyes and the quality of\nreconstructed super-resolution images. Particularly, we found that\nclear unobstructed brain imaging cocktails-based imaging buffer could\nmatch the RI of oil and was able to clear the tissue samples. With\nthe help of the RI-matched imaging buffers, we showed high-quality\ndual-color 3D SMLM images with imaging depths ranging from a few micrometers\nto tens of micrometers in both cultured cells and sectioned tissue\nsamples. This advancement offers a practical and accessible method\nfor high-resolution imaging at greater depths without the need for\nspecialized optical equipment or expertise.