CryoCycle your grids: Plunge vitrifying and reusing clipped grids to advance cryoEM democratization

CryoEM democratization is hampered by access to costly plunge-freezing supplies. We introduce methods, called CryoCycle, for reliably blotting, vitrifying, and reusing clipped cryoEM grids. We demonstrate that vitreous ice may be produced by plunging clipped grids with purified proteins into liquid ethane and that clipped grids may be reused several times for different protein samples. Furthermore, we demonstrate the vitrification of thin areas of cells prepared on gold-coated, pre-clipped grids.

Furthermore, we show that the blotting method is sufficient for vitrifying thin areas of cells grown on grids and provide a protocol for preparing cell-compatible, pre-clipped grids (Supplementary Protocol 3). Figure 1c-h shows examples of vitrified samples of single particles (globular proteins and a long complex) and cells (human cells grown on grids; Supplementary Fig. 4) prepared on multiple different grid types using blotting pipette tips.We compared orientations of the p97/selenos complex prepared with a blotting pipette tip (Fig. 1f) versus conventionally (Supplementary Fig. 5a) and found them to be comparable (Supplementary Fig. 5b,c).These methods, called CryoCycle, significantly reduce costs and demand for consumables, thus democratizing cryoEM by enabling more widespread and uninterrupted adoption.
The key components for blotting clipped grids for vitrification are rigid, flat-tipped tweezers and a blotting pipette tip that uniformly contacts the grid.For the former, we found that conventional fine-tipped Vitrobot tweezers insecurely hold clipped grids due to their flexible tips, causing rotation into the tweezers when handled (Supplementary Fig. 6a,b).Trimming the tweezer tips increases rigidity sufficiently to handle the autogrid without rotating into the grid, which is essential for blotting (Supplementary Fig. 6a,c & 7; Supplementary Video 1; Supplementary Protocol 1).For the latter, a blotting pipette tip is created by cutting the narrow end of a pipette tip to a 3 mm inner diameter then inserting a ~1 cm piece of filter paper until 1 mm protrudes out of the small end (Fig. 1a,b; Supplementary Protocol 1).Rounding the protruding filter paper's edges facilitates blotting within the clip ring, while flattening the end helps ensure uniform grid contact (Supplementary Fig. 8).200 µL and 1,000 µL blotting pipette tips accommodate enough blotting paper to absorb 3+ µL of sample (Fig. 1a,b).Supplementary Video 2 shows the CryoCycle blotting pipette tip assembly process.Supplementary Figure 3 depicts sample application, blotting direction, and orientation of the grid and autogrid for single particle and cell samples.
We developed a washing protocol for reusing single particle clipped grids, taking into account the sensitivity of the grid film and the autogrid assembly.Clipped EM grids are typically composed of copper, carbon, gold, and palladium.Washing them first with water removes bulk contamination, then subsequent isopropanol washes displace and rinse away remaining samples and contaminants.Isopropanol was selected for its minimal reactivity with these metals at room temperature and high purity (99+%) to prevent altering their chemical composition.We determined that one 5-minute wash with water followed by two 5-minute isopropanol washes sufficiently cleans clipped carbon and gold grids (Fig. 2; Supplementary Fig. 9; Supplementary Protocol 2). Figure 2e shows a micrograph from a clipped carbon grid that was reused twice (i.e. 3 samples, 2 washings) and Supplementary Figure 10 shows their grid atlases with the vast majority of squares intact, exemplifying the protocol's robustness.The maximum number of times clipped grids may be reused has not yet been determined.
The CryoCycle clipped grid preparation methods offer significant advantages over conventional unclipped grid preparation in four key areas.1) Room temperature grid clipping is simpler and safer, as it avoids mechanical and visual disruptions caused by LN2, enables easy visual verification of clipped ring placement by eye, and eliminates risks of grids thawing, fingers freezing, and ice contamination.Additionally, a clean, flat surface can be used to clip instead of a clipping station, saving a one-time cost of thousands of dollars.2) Handling pre-clipped grids mitigates mechanical damage and user stress (Supplementary Fig. 11) compared to handling unclipped grids (Supplementary Fig. 7); ideally, a grid is handled once for clipping, then only the autogrid is touched thereafter.Handling clipped grids substantially reduces bent grids, which expedites screening due to minimal defocus gradients.Moreover, clipped grid handling eliminates the need to purchase unclipped grid boxes.3) CryoCycle blotting and freezing only requires a humidity chamber; popular commercial semi-automated plunge freezing devices are not needed, which may reduce one-time costs by tens of thousands of dollars.While we illustrate the use of CryoCycle in a Vitrobot, we emphasize that only the humidity chamber and plunger are used (Supplementary Video 1); CryoCycle grid preparation using a gravity plunger (Comolli et al., 2012;Depelteau et al., 2020) or manually plunging by hand is possible, although the latter has been minimally tested (Supplementary Fig. 12).4) Reusing clipped grids reduces initial and recurring costs.Each pre-clipped grid assembly costs about $45 in 2024, so for a typical cryoEM sample where 24 grids are required to be screened before conditions suitable for data collection are found, reusing 8 grids three times each would reduce recurring costs by $720.Most cryoEM projects require optimization of several samples, thus using CryoCycle methods reduces costs of cryoEM projects by thousands of dollars per project and reduces costs for cryoEM labs by tens of thousands of dollars per year.Table 1 lists where CryoCycle methods can reduce both initial and yearly costs by tens of thousands of dollars each for typical cryoEM labs.Additionally, CryoCycle methods enable vitrification of clipped grids by plunge freezing, circumventing the previous requirement of using ethane jet freezers that cost hundreds of thousands of dollars.The advantages listed here particularly benefit new cryoEM users, making cryoEM more accessible.

Protein mixture:
Purified apoferritin and tobacco mosaic virus (TMV) were mixed in phosphate buffered saline at concentrations of 2.9 mg/mL and 13.6 mg/mL, respectively.

p97/selenos complex:
The AAA ATPase p97 was combined with an excess molar quantity of full-length selenoprotein S U188C (selenos), resulting in a final concentration of the p97/selenos complex at 11 mg/mL.This mixture was incubated on ice for 30 minutes, including 5 mM ATPS and 1.2 mM DDM (1 CMC), prior to grid application.The preparation of selenos followed the method outlined in (Ghelichkhani et al., 2023).

Grid Preparation and Vitrification
For samples prepared with the CryoCycle blotting method inside a TFS Vitrobot Mark IV chamber (Thermo Fisher Scientific) (Fig. 1c-h, Fig. 2d,e, Supplementary Fig. 4), all time and force parameters were set to zero (Blot Time, Blot Force, Wait Time, and Drain Time) and humidity was set to 80%.The instrument was operated at room temperature.Quantifoil (Quantifoil Micro Tools GmbH) holey carbon, gold, and UltrAuFoil grids were used.Single particle samples were blotted on the same side as sample application following the Procedure in Supplementary Protocol 1 and cell samples were blotted on the opposite side as cell growth (see Supplementary Fig. 3).
For samples prepared conventionally using multiple blotting materials in the TFS Vitrobot Mark IV and Leica EM GP2 chambers (Supplementary Fig. 1), several parameters were varied across different test samples (primarily apoferritin, VLPs, and the protein mixture) including: Quantifoil and C-Flat (Protochips Inc.) carbon and gold grids, blot times from 1 to 4 seconds, and 70-85% humidity.
The p97/selenos complex prepared conventionally with a TFS Vitrobot Mark IV (one Quantifoil carbon grid) and screened on a TFS Glacios cryoTEM resulted in 2,392 micrographs (counting mode).Micrographs were processed in Cryosparc v4.4.1 (Punjani et al., 2017) before selecting a random subset of 24,584 particles for a final 2D classification (Supplementary Fig. 5b) to match the number of particles from the CryoCycle method dataset.

CryoFLM imaging
CryoFLM images (Supplementary Fig. 4) were collected on a Zeiss LSM 900 with Airyscan 2 (Carl Zeiss Microscopy GmbH) configured to excite the sfGFP bound to the CENP-A nucleosomes.

Verification of Consent
Photo (Supplementary Fig. 12a) and Supplementary Videos 1 & 2 of Viacheslav Serbynovskyi were taken with approval and are shown here with consent.3a.The inset shows the contact between the blotting paper and the grid inside the autogrid ring.Note: Blotting for cell samples is from the opposite side of the grid, as shown in Supplementary Figure 3b. (c-h) A selection of micrographs of vitreous single particle and cell samples on multiple grid types (gold and carbon) prepared with the CryoCycle method; (c) Virus-like Particles (VLPs), (d) Apoferritin, (e) A protein mixture of apoferritin and tobacco mosaic virus (TMV), (f) A p97/selenos complex (conventional preparation comparison in Supplementary Fig. 5), (g-h) Thin edges of human retinal pigment epithelial-1 (RPE-1) cells, showing intact membrane bilayers (cryoFLM images shown in Supplementary Fig. 4).Scale bars: 100 nm for (c-h).d-e).Supplementary Figure 9 shows CryoCycle reused grid washing for a gold grid.Supplementary Figure 10 shows grid atlases of (d) & (e).

Figure 1 |
Figure 1 | CryoCycle clipped grid blotting with various designs, grids, and samples.(a) Two blotting pipette tip sizes: Trimmed 200 μL (left) and 1,000 μL (right) tips, each with a piece of filter paper inserted for blotting 3+ μL of sample.(b) A 200 μL blotting pipette tip applied to the same side of the grid as the single particle sample; see orientations in Supplementary Figure3a.The inset shows the contact between the blotting paper and the grid inside the autogrid ring.Note: Blotting for cell samples is from the opposite side of the grid, as shown in Supplementary Figure3b.(c-h) A selection of micrographs of vitreous single particle and cell samples on multiple grid types (gold and carbon) prepared with the CryoCycle method; (c) Virus-like Particles (VLPs), (d) Apoferritin, (e) A protein mixture of apoferritin and tobacco mosaic virus (TMV), (f) A p97/selenos complex (conventional preparation comparison in Supplementary Fig.5), (g-h) Thin edges of human retinal pigment epithelial-1 (RPE-1) cells, showing intact membrane bilayers (cryoFLM images shown in Supplementary Fig.4).Scale bars: 100 nm for (c-h).

Table 1 |
CryoCycle savings versus conventional preparation costs.CryoCycle methods save tens to hundreds of thousands of dollars initially and tens of thousands of dollars yearly for a typical cryoEM lab (bold).Added costs are ~$5,000 initially (bold), $0 recurring.Grid savings assume reusing clipped grids 3 times each.Conventional preparation may incur additional losses of unclipped grids, varying with user expertise.Staffing costs or savings may vary depending on how the protocols are implemented.