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Nanoscale localization of point emitters is critical to several methods in optical fluorescence microscopy, including single-molecule super-resolution imaging and tracking. While the precision of the localization procedure has been the topic of extensive study, localization accuracy has been less emphasized, in part due to the challenge of producing an experimental sample containing unperturbed point emitters at known three-dimensional positions in a relevant geometry. We report a new experimental system which reproduces a widely-adopted geometry in high-numerical aperture localization microscopy, in which molecules are situated in an aqueous medium above a glass coverslip imaged with an oil-immersion objective. We demonstrate a calibration procedure that enables measurement of the depth-dependent point spread function (PSF) for open aperture imaging as well as imaging with engineered PSFs with index mismatch. We reveal the complicated, depth-varying behavior of the focal plane position in this system and discuss the axial localization biases incurred by common approximations of this behavior. We compare our results to theoretical calculations.

Glyco-immune checkpoint receptors, molecules that inhibit immune cell activity following binding to glycosylated cell-surface antigens, are emerging as attractive targets for cancer immunotherapy. Defining biologically relevant ligands that bind and activate such receptors, however, has historically been a significant challenge. Here, we present a CRISPRi genomic screening strategy that allowed unbiased identification of the key genes required for cell-surface presentation of glycan ligands on leukemia cells that bind the glyco-immune checkpoint receptors Siglec-7 and Siglec-9. This approach revealed a selective interaction between Siglec-7 and the mucin-type glycoprotein CD43. Further work identified a specific N-terminal glycopeptide region of CD43 containing clusters of disialylated O-glycan tetrasaccharides that form specific Siglec-7 binding motifs. Knockout or blockade of CD43 in leukemia cells relieves Siglec-7-mediated inhibition of immune killing activity. This work identifies a potential target for immune checkpoint blockade therapy and represents a generalizable approach to dissection of glycan–receptor interactions in living cells.