A 50-50 mixture of Me and Et was purchased from Scott Specialty Gasses. This mixture is likely to be close to the eutectic, and if the mixture was not itself liquid at 77 K, the component in excess should freeze out, leaving a mixture whose freezing point was 77 K. Experiments were performed with a gravity-operated plunge freezer and with a Vitrobot. In each case, a few µL of a specimen of Caulobacter crescentus in culture medium were placed on a Quantifoil grid, the excess liquid was blotted off, leaving a layer of liquid that was a few hundred nanometers thick, and this was plunged into the cryogen. The frozen specimens were placed in LN, loaded into a Gatan 626 cryoholder, and examined on a FEI T12 electron microscope.
Pr was obtained from a local hardware store, and Et was purchased from Gilmore Liquid Air. The appropriate mixture of Pr and Et was prepared in either of two ways. The cryogen cup of the gravity-operated plunge freezer was filled to about 60% of capacity with Pr, which was allowed to solidify, then Et was added both to melt the Pr and fill the cup. Alternatively, the cryogen cup of the Vitrobot was filled to about 40% of capacity with Et, which was allowed to solidify, then Pr was added to melt the Et and fill the cup. The specimens and freezing steps were the same as for the Me-Et experiments.
Because both Me-Et and Pr-Et mixtures exist that are liquid at 77 K, a redesigned cryogen cup for the Vitrobot was constructed (see Fig. 1C,D). The main differences between this and previous designs are, first, the new design has much greater thermal contact between the LN and the cryogen, and, second, it incorporates the grid box storage and barrier features of newer Vitrobot cryogen cups with the outer insulating construction of the older Vitrobot cups. The redesigned cryogen cup was fabricated simply by milling the central hole and the outer annulus from an aluminum cylinder.