Graphene sandwich–based biological specimen preparation for cryo-EM analysis

Significance Although cryo-EM has become a widely used technique for analyzing high-resolution structures of macromolecules, it still faces several challenges, including significant background noise, beam-induced particle motion, charging effect, and the air–water interface. In this study, we have introduced a graphene sandwich technique that utilizes graphene membranes as both the coverslip and slide to enclose macromolecules for cryo-EM specimen preparation. Due to the superior properties of graphene, such as low background noise and strong mechanical strength, this graphene sandwich-based cryo-EM specimen preparation method has demonstrated an improvement in the quality of collected cryo-EM datasets when compared to the conventional approach of using graphene support on just one side.

the free-standing graphene on the solution surface was obtained by etching off the copper foil.In the second step, sample was deposited onto a graphene grid.In the third step, we employed two alternative methods to transfer the free-standing graphene onto the sample-loaded graphene grid, using a loop-assisted transfer method (upper) or using the graphene grid to directly scoop the free-standing graphene (lower).In the last step, the graphene sandwich grid was placed onto a piece of filter paper to enable the paper to absorb any excess solution from the sandwich area.

Figure S1 .
Figure S1.Production of free-standing graphene film.a, The graphene on copper foil was firstly coated by stearic acid molecules and floated on the etchant (ammonium persulfate) surface.b-c, The copper foil was being etched while the graphene was kept stretched (b) and finally free-standing on the etchant surface (c).The four corners of graphene are labeled by red dotted lines for clarity in (c).

Figure S2 .
Figure S2.Diagram showing the fabrication procedure of the graphene sandwich.In the first step,

Figure S3 .
Figure S3.Room-temperature TEM characterization of graphene sandwich.a, A representative micrograph of the graphene sandwich prior to high-dose electron radiation damage.b, A representative micrograph of the graphene sandwich after electron radiation damage.Numerous bubbles have emerged in the irradiation area.

Figure S4 .
Figure S4.Micrographs of graphene with stearic acid coating.a, The image before high-dose electron radiation.b, The image after high-dose electron radiation.

Figure S5 .
Figure S5.Room-temperature TEM characterization of graphene sandwich.a, A representative micrograph of the graphene sandwich after irradiation of 114 e -/Å 2 electron dose.b, A representative micrograph of the graphene sandwich after 342 e -/Å 2 electron dose irradiation.After longer irradiation, the majority of the bubbles increased in size.Bubble fusion events were labeled by the dotted boxes.

Figure S6 .
Figure S6.Imaging two stacked graphene layers at room temperature under various electron doses reveals no visible bubbles.The particles showing significant contrast in these micrographs are likely contaminants introduced during the graphene transfer process.

Figure S7 .Figure S8 .
Figure S7.Representative grid atlases of graphene-sandwiched samples.a-c, Grid atlas of good graphene-sandwiched sample, with typical areas for data collection marked by yellow boxes.d, Grid atlas of not-so-good graphene-sandwiched sample, where the ice thickness was too thin.

Figure S12 .
Figure S12.Diagrams showing the buckling of the graphene-sandwiched ice (a) and the graphenesupported ice (b).N is the compressive stress and h is the ice thickness in (a).

Figure S13 .
Figure S13.Particle motion with electron dose.The average particle displacement was measured for both the graphene-sandwiched sample (orange) and the graphene-supported sample (black) as a function of accumulated dose.The particle displacement was determined by measuring the distance between particles in the current frame and the previous frame.In the first 8~9 e − /Å 2 , the particle displacement on the graphene support was notably larger, approximately 1.5-2.0times greater, compared to that observed in the graphene sandwich.Supplementary Video 1.The process of transferring graphene occurred after the copper substrate had been etched away.