This manuscript outlines the blot-and-plunge method to manually freeze biological specimens for single-particle cryogenic electron microscopy.
Imaging biological specimens with electrons for high-resolution structure determination by single-particle cryogenic electron microscopy (cryoEM) requires a thin layer of vitreous ice containing the biomolecules of interest. Despite numerous technological advances in recent years that have propelled single-particle cryoEM to the forefront of structural biology, the methods by which specimens are vitrified for high-resolution imaging often remain the rate-limiting step. Although numerous recent efforts have provided means to overcome hurdles frequently encountered during specimen vitrification, including the development of novel sample supports and innovative vitrification instrumentation, the traditional manually operated plunger remains a staple in the cryoEM community due to the low cost to purchase and ease of operation. Here, we provide detailed methods for using a standard, guillotine-style manually operated blot-and-plunge device for the vitrification of biological specimens for high-resolution imaging by single-particle cryoEM. Additionally, commonly encountered issues and troubleshooting recommendations for when a standard preparation fails to yield a suitable specimen are also described.
Single-particle cryogenic electron microscopy (cryoEM) is a powerful structural technique that can be used to solve structures of dynamic biological specimens to near-atomic resolution1,2,3,4. Indeed, recent advances in direct electron detector technologies4,5,6,7,8,9,10, improvements in electron sources4,11,12,13,14, and electromagnetic lens stability15, coupled with the continued development of data acquisition16,17 and analysis software packages18,19, have enabled researchers to now routinely determine structures of well-behaved specimens to 3 Å resolution or better4,11,13,14,20,21,22,23. Despite these improved imaging and data processing capabilities, cryoEM grid preparation remains the largest impediment for successful high-resolution structure determination and often serves as a considerable bottleneck in the EM workflow24,25,26,27.
CryoEM relies on the imaging of biological samples in aqueous solutions that are frozen to form a thin film of "glass-like" ice – a process known as vitrification – that preserves the native biochemical state. Vitrification of biological samples for cryoEM dates back over 40 years28,29,30 and many techniques and equipment that have been developed for this process rely on the originally detailed blot-and-plunge method31,32,33,34,35, whereby a small volume of sample (e.g., 1-5 µL) is applied to a specialized EM grid before the excess solution is removed using physical interaction of the grid with blotting paper. The timing of this process is usually empirically determined for each specimen as a critical component of freezing samples is the thickness of the vitreous ice film – if the ice is too thick then imaging quality deteriorates dramatically due to increased scattering of the electron beam while ice that is too thin can restrict protein orientations and/or exclude particles from the center of the grid foil holes36. This reliance on the perfect ice thickness for single-particle cryoEM has led to a wide array of techniques and equipment that can freeze samples, including robotics37,38, microfluidics42, and ultrasonic or spraying devices27,39,40,41,42,43,44. In recent years, some of the most popular sample preparation devices rely on the use of robotics for automated freezing of samples using the blot-and-plunge technique45. While these devices are designed to reproducibly create proper ice thickness for imaging, they often remain too expensive for individual labs to purchase and operate and are generally found within cryoEM facilities at hourly rates for usage. In recent years, the original manual blot-and-plunge technique has come back into increased use3,47,48,49,50,51,52. Indeed, a manually operated blot-and-plunge device can achieve high-quality cryoEM grids at a fraction of the cost of robotic counterparts. Furthermore, manual blotting also offers more users control over blotting as researchers can adjust the type of blotting (i.e., back-blotting of the grid, front-blotting of the grid, etc.), and blotting time based on each individual sample and research questions.
In this article, we provide details on how to effectively freeze biological samples using a traditional manual blot-and-plunge vitrification device coupled with a custom-designed dewar platform53. Best practices, including preparation of the cryogen, grid handling, sample application, and blotting, as well as common pitfalls and recommendations on how to overcome these hurdles are provided. Advice on how to increase ice thickness reproducibility between grid preparations and how to modify sample blotting based on biological specimen type are discussed. Given the low cost associated with the purchase and operation of the manual plunger described in this manuscript, labs across the globe can prepare biological specimens for cryoEM in a cost-effective and reproducible manner.
1. Prepare the manual plunging environment
NOTE: Estimated operating time: 5-30 minutes
2. Prepare plunging materials and accessories
NOTE: Estimated operating time: 1-5 minutes
3. Prepare the cryogen dewar and manual plunger
NOTE: Estimated operating time: 5-15 minutes
4. Prepare the cryogen
NOTE: Estimated operating time: 10-30 minutes
5. Prepare EM grids
NOTE: Estimated operating time: 1-5 minutes
6. Prepare cryoEM specimen by plunge freezing
NOTE: Estimated operating time: >10 minutes (~1-3 min per grid)
Successful execution of the blot-and-plunge protocol described here will result in a thin, uniform layer of vitreous ice that is free of any hexagonal ice, contaminants, and large gradients of unusable ice which can be observed under the electron microscope (Figure 3). Inconsistent contact of the blotting paper with the grid surface, prematurely removing the blotting paper, or moving the blotting paper during grid contact can decrease the quality of the vitreous ice and lead to inconsistent ice thickness across the EM grid (Figure 4)
Figure 1: Specimen plunging room and required equipment. A) Staged cold room for the manual freezing of biological specimens using a traditional blot-and-plunge device outlined in this article. Necessary equipment is shown and labeled accordingly. B) To adjust the working height of the manual plunger, adjust the bump stop by sliding it up and down the manual plunging arm and securing it by tightening the screw. C) Zoomed-in view of the ethane vessel and spinning grid storage platform to indicate proper height and location of the clamping tweezers and grid inside the empty brass ethane vessel. The tweezers and grid should not contact the sides or bottom of the brass ethane to avoid damage. D) Proper height and location of the clamping tweezers and grid in liquid ethane. The tweezers and grid should enter the liquid ethane in the center, avoiding contact with the solid ethane at the perimeter. Please click here to view a larger version of this figure.
Figure 2: Prepared liquid ethane. Zoomed-in view of the plunging dewar showing the state of liquid ethane in the brass ethane vessel prior to specimen freezing. The 2-3 mm ring of solid ethane within the brass ethane vessel is clearly visible. Please click here to view a larger version of this figure.
Figure 3: Representative apoferritin images obtained using the manual blot-and-plunge technique. (A) Representative atlas of a cryoEM grid showing the ice thickness and quality of grid squares that can be obtained using the manual blot-and-plunge technique. (B) Motion-corrected micrograph of vitrified mouse apoferritin acquired using a 200 kV transmission electron microscope equipped with a direct electron detector at the University of California, San Diego's CryoEM Facility. Please click here to view a larger version of this figure.
Figure 4: Representative atlas of a sub-optimal cryoEM grid showing inconsistent ice thickness across the grid, numerous broken squares, and areas in which the ice is too thick to image the specimen. Please click here to view a larger version of this figure.
The vitrification of biological specimens for imaging by single-particle cryogenic electron microscopy (cryoEM) remains a critically important step for successful structure determination. The manual blot-and-plunge method described in this protocol represents a cost-effective, reliable, and robust method for quickly freezing biological samples in thin films of vitreous ice for cryoEM imaging. Using the methods outlined in the manuscript, researchers will be able to assemble and operate the manual plunger, prepare cryogen suitable for flash-freezing biological samples, and manually blot-and-plunge EM grids containing biological specimens. While this method is quite robust, care should be taken during critical steps in this procedure to obtain optimal ice thickness and quality for high-resolution imaging. We have outlined several of these critical steps below and provide recommendations on how to troubleshoot these steps.
It is imperative to properly position the manual plunging arm to ensure that the grid locates at the center of the liquid ethane within the brass vessel after plunging. Improper height or position of the plunging arm and/or not securing the tweezers properly will lead to damage to the clamping tweezers, the EM grid, and possibly the manual plunger. As discussed above, we always perform at least one trial run prior to preparing biological specimens to verify that the EM grid will locate to the center of the brass ethane vessel after successful plunging (Figure 1C). In addition, we also make minor adjustments to the location of the plunging dewar after each grid freezing to fine-tune grid placement within the ethane vessel (Figure 1D).
The proper preparation of the ethane cryogen is critical for obtaining thin films of biological specimens in vitreous ice. We have observed that the presence of a 2-3 mm ring of solid ethane around the inner edge of the brass ethane vessel ensures that the temperature of the liquid ethane is optimal for sample vitrification (Figure 2). Indeed, after each grid has been frozen, we monitor the quality of the ethane and make minor adjustments – slightly warming the vessel if too much ethane has solidified or cooling the ethane if the system has warmed up – as needed. We have found that the edge of a room temperature tweezer is sufficient to liquefy solid ethane while covering the dewar with the foam lid for 1-5 min is enough time to allow the ethane to cool. Importantly, we make these adjustments prior to preparing the grid surface (i.e., plasma cleaning) and applying the sample to the grid as this can introduce another variable to grid preparation that is not reproducible.
Finally, we recommend developing a standardized blot-and-plunge routine – sample application, sample blotting, and blotting time – to increase grid-to-grid reproducibility. Bending the blotting paper towards the EM grid allows for uniform contact of the paper with the grid and produces more consistent ice thickness across the entire grid, resulting in even particle distribution within the grid holes (Figure 3A and Figure B, respectively). This method of blotting is in contrast to robotic blotting devices that interact with the specimen at an angle that may result in a gradient of ice thickness across the grid. In addition, this bending of the blotting paper also decreases the chance of damaging the EM grid upon contact with the blotting paper by buffering the force being applied by the user. After the desired blotting time, quickly straighten the blotting paper by performing a snapping motion to rapidly move the blotting paper away from the grid surface before plunging to prevent damage to the grid upon release of the manual plunging arm. We have found this blotting method and the snapping motion of the blotting paper, when timed with the simultaneous release of the manual plunging arm via the foot pedal, limits evaporation of the thin film before vitrification and increases grid-to-grid reproducibility.
The manual blot-and-plunge method described here is a robust and reliable method that helps lessen some of the financial burden cryoEM can place on emerging labs. While this method is reproducible, creating high-quality vitreous ice that is suitable for cryoEM relies on the experience and skill of the individual researcher. Although robotic plungers and other emerging technologies automate several aspects of the freezing process, they are generally limited by how much control they offer to researchers and often incur a high price to purchase and operate. With the method outlined in this protocol, researchers will be able to utilize an affordable and versatile EM grid preparation platform that offers flexibility to optimize the plunging conditions (i.e., blotting paper types, blotting angle, blotting durations, blotting directions, etc.) based on sample types and characteristics.
The authors have nothing to disclose.
We thank the Herzik lab members for critically thinking and providing feedback on this manuscript and the video content. M.A.H.Jr. is supported by NIH R35 GM138206 and as a Searle Scholar. H.P.M.N is supported by the Molecular Biophysics Training Grant (NIH T32 GM008326). We would also like to thank Bill Anderson, Charles Bowman, and Dr. Gabriel Lander at the Scripps Research Institute for help designing, assembling, and testing the manual plunger shown in the video.
4 slot grid storage box | Ted Pella | 160-40 | |
14 gauge flat metal dispensing tip | Amazon | B07M7YWWLT | |
22×22 mm square glass coverslip | Sigma | C9802-1PAK | |
60 mm glass Petri dish to store grids | Fisher | 08-747A | |
100 mm glass Petri dish to store Whatman paper | Fisher | 08-747D | |
150 mm glass Petri dish to store Whatman paper | Fisher | 08-747F | |
250 mL beaker | Fisher | 02-555-25B | |
Blue styrofoam dewar | Spear Lab | FD-500 | |
Brass ethane vessel | Lasco | 17-4075 | |
Clamping tweezers | Ted Pella | 38825 | |
Delicate task wipes | Fisher | 06-666 | |
Dual-stage regulator with control valve | Airgas | Y12N245D580-AG | |
Dewer grid base | UCSD | ||
Ethane platform | UCSD | ||
Ethane propane tank | Praxair | ET PR50ZU-G | ethane (50%) : propane (50%) in a high-pressure tank |
Ethane tank | Praxair | UN1035 | ethane (100%) |
Flexible arm task light | Amscope | LED-11CR | |
Grids (UltrAufoil R 1.2/1.3 300 mesh) | Electron Microscopy Sciences | Q325AR1.3 | |
Humidifier | Target | 719438 | |
Hygrometer | ThermoPro | B01H1R0K68 | |
Lab coat | UCSD | ||
Liquid Nitrogen dewar | Worthington | LD4 | |
Liquid Nitrogen gloves | Fisher | 19-059-925 | |
Manual plunger stand (black stand + foot pedal) | UCSD | ||
Mark 5 (plunging platform) | UCSD | ||
Nitrile gloves | VWR | 82026-424 | |
P20 pipette | Eppendorf | 13-690-029 | |
PCR tubes | Eppendorf | E0030124286 | |
Pipette tips | ibis scientific | 63300005 | |
Ring lamp | Amazon | B07HMR4H8G | |
Safety glasses | UCSD | ||
Scissors | Amazon | Fiskars 01-004761J | |
Screw driver | Ironside | 354711 | |
Tape | Fisher | 15-901-10R | |
Tweezer to transfer grid box | Amazon | LTS-3 | |
Tygon tubing | Fisher | 14-171-130 | |
Whatman blotting paper | Fisher | 1001-090 |