Optimization of Spc42-MTH staining during freeze substitution. (A) Au(III)Cl3 was added to acetone during freeze substitution, and it provided staining with good SNR (Table S2, condition #3, Lowicryl HM20 resin). This staining could be enhanced by the addition of more heavy metals, as described in He et al., 2008, but a few additional particles of heavy metal are now seen not associated with the SPB (B, arrows; Table S2, condition #12, Lowicryl HM20 resin). Samples that had been treated with diglyme followed by silver enhancement (as described in Morphew et al. 2008) gave the strongest label (C) and distinct particles could now be detected (C, inset; Table S2, condition #8, Durcupan resin). Control cells freeze treated with diglyme followed by silver enhancement showed no SPB staining (D; Table S2, condition #8, Durcupan resin). Bars = 100 nm. Movies of tomographic volumes corresponding to (A)—(D) can be found in the supplementary materials.