High Refractive Index Imaging Buffer for Dual Color 3D SMLM Imaging of Thick Samples

Abstract

AbstractThe current limitations of single-molecule localization microscopy (SMLM) in deep tissue imaging, primarily due to depth-dependent aberrations caused by refractive index (RI) mismatch, present a significant challenge in achieving high-resolution images at greater depths. To extend the imaging depth, we optimized the imaging buffer of SMLM with RI matched to the objective immersion medium, and systematically evaluated five different RI matched buffers, focusing on their impact on the blinking behavior of red-absorbing dyes and the quality of reconstructed super-resolution images. Particularly, we found clear unobstructed brain imaging cocktails (CUBIC) based imaging buffer could match the RI of oil and was able to clear the tissue samples. With the help of RI matched imaging buffer, we showed high quality dual color 3D SMLM images with imaging depth ranging from few microns to tens of microns in both cultured cells and sectioned tissue samples. This advancement offers a practical and accessible method for high-resolution imaging at greater depths without specialized optical equipment or expertise.