Manual Blot-and-Plunge Freezing of Biological Specimens for Single-Particle Cryogenic Electron Microscopy

Hoang P. M. Nguyen; Kelly L. McGuire; Brian D. Cook; Mark A. Herzik, Jr.

doi:10.3791/62765

Biochemistry

Manual Blot-and-Plunge Freezing of Biological Specimens for Single-Particle Cryogenic Electron Microscopy

Published: February 7, 2022 doi: 10.3791/62765

Hoang P. M. Nguyen*¹, Kelly L. McGuire*¹, Brian D. Cook*¹, Mark A. Herzik, Jr.¹

¹Department of Chemistry and Biochemistry, University of California, San Diego

* These authors contributed equally

Summary

This manuscript outlines the blot-and-plunge method to manually freeze biological specimens for single-particle cryogenic electron microscopy.

Abstract

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.

Introduction

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.

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Protocol

1. Prepare the manual plunging environment

NOTE: Estimated operating time: 5-30 minutes

Locate the manual plunger in a 4 °C cold room where a humidifier can be co-located to maintain the room close to 100% relative humidity (RH) (Figure 1A).
CAUTION: Please consult with the institution's Environmental Health and Safety guidelines for the safe location of the manual plunger and recommended operations.
Prior to grid preparation, turn on the humidifier in the cold room to ensure the RH of the cold room is ≥ 95 %.
NOTE: Grid preparation in low humidity can result in dehydration of thin films, alteration of buffer components due to evaporation, and a decrease in grid-to-grid reproducibility⁴⁶. It is not recommended to freeze grids at <80 % RH.
Ensure the temperature of the cold room is at 4 °C.
Locate the manual plunger away from strong air currents (i.e., away from air conditioning unit vents) as they can lead to turbulence near grids and/or prominent ice accumulation on cold surfaces.

2. Prepare plunging materials and accessories

NOTE: Estimated operating time: 1-5 minutes

Use clean scissors to cut blotting paper circles into 1-1.5 cm wide and ~9 cm long strips. Avoid touching the center of the blotting paper and discard the smaller end pieces. It is important to ensure that the blotting paper strips are dry, clean, and free of contaminants. Separate the strips and place them in a 100 mm Petri dish.
Place a 22x22 mm square glass coverslip into a separate 60 mm glass Petri dish. This coverslip-containing glass Petri dish will be used to store, transfer, and glow-discharge grids.
NOTE: It is recommended to use an air-duster can to remove any visible debris from the slide prior to adding grids. An anti-static gun can also be used to remove any static electricity that accumulates.
Assemble and label grid storage boxes.
Acquire 4 to 6 clean and dry clamping tweezers and locate them to the manual plunger. Visually inspect each tweezer prior to plunging to ensure they are not damaged and are free of contaminants.

3. Prepare the cryogen dewar and manual plunger

NOTE: Estimated operating time: 5-15 minutes

Install the platform base at the bottom of the plunging dewar. Place the ethane vessel dewar on top of the platform base, add the brass ethane vessel, and then install the spinning grid storage platform.
1. Once liquid nitrogen (LN₂) is added to the plunging dewar, the height of the platform base can no longer be adjusted. Ensure that the grid base is installed properly and is level to limit grid and/or tweezer damage due to improper plunger height.
Prior to freezing, check the manual plunger and all ancillary equipment to ensure they are functioning properly to limit sample and/or grid loss.
Prior to each freezing session, replace the tape on the manual plunger arm used to hold the tweezers. The high humidity of the room can deteriorate the tape adhesive and decrease the ability of the tape to hold the tweezers, increasing the likelihood of tweezer damage and/or grid loss.
Adjust the lamp(s) near the manual plunger to ensure there is sufficient light to monitor sample wicking and ensure grid transfer is easily visualized to prevent grid damage and/or loss (see step 3.7). Use a ring lamp directly behind the plunger to visualize liquid movement and a flexible arm task light near the dewar to illuminate the frozen grids.
Adjust the foot pedal tension to ensure the dewar plunging arm is securely retained in place while in the raised position and is fully released when the pedal is depressed. Perform several "dry" runs prior to sample application to ensure the plunger is working properly.
NOTE: Improper adjustment of the foot pedal tension will result in premature release of the plunging arm (i.e., tension is set too low) and grid loss or incomplete plunging of the grid into the ethane vessel (i.e., the tension is set too high).
Place the plunging dewar at the base of the manual plunger directly below the plunging arm and secure it in place. Attach a pair of tweezers to the plunging arm using the attached tape. While holding the manual plunging arm, depress the foot pedal to carefully lower the plunging arm and adjust the travel of the plunging arm to ensure the grid will locate within the middle of the ethane vessel.
Use the bump stop at the top of the plunging arm to determine the final position of the plunging arm when the foot pedal is fully depressed (Figure 1B). Adjust the bump stop height on the plunging arm to adjust the location of the grid in the ethane vessel (Figure 1C).
NOTE: Improperly setting the plunging arm height can lead to the grid and/or tweezer damage (e.g., plunging arm height is set too low) or inadequate vitrification (e.g., plunging arm height is set too high).
Locate the dewar outside the cold room and proceed to preparing liquid ethane (step 4).

4. Prepare the cryogen

NOTE: Estimated operating time: 10-30 minutes

Assess the ethane tank, regulator, tubing, and ethane dispensing tip for any signs of damage. Immediately report and rectify any signs of damage before proceeding.
CAUTION: Compressed ethane and ethane:propane gas mixes are flammable and can pose a serious threat to life and/or result in injury if improperly handled. Please consult an expert if unsure how to operate or handle compressed gas tanks. Please refer to the institution's Environmental Health and Safety guidelines when handling flammable compressed gases. In addition, liquified ethane is a powerful cryogen that can pose a serious threat to life and/or injury if not handled properly. Please refer to the institution's Environmental Health and Safety guidelines when handling cryogens.
Acquire sufficient LN₂ in an appropriate LN₂ handling dewar (i.e., 3-4 L is typical for grid preparation and storage).
CAUTION: LN₂ is a cryogen that can pose a serious threat to life and/or injury if not handled properly.
1. Ensure all personal protective equipment are utilized to minimize the risk of injury. The vapor of LN₂ is an asphyxiant and should be handled in well-ventilated areas. Please consult an expert if unsure how to operate or handle cryogenic vessels and cryogens. Please refer to the institution's Environmental Health and Safety guidelines when handling cryogens. For situations in which liquid nitrogen cannot be used in a cold room, we recommend plunge freezing in a cool and well-ventilated space.
Outside of the cold room, cool down the plunging dewar by pouring LN₂ directly into the brass ethane vessel until the liquid nitrogen level reaches the top of the ethane vessel (i.e., just above the platform). Top off the LN₂ outside the ethane vessel as needed. Proceed to the next step when the LN₂ stops bubbling violently (approximately 5 minutes).
1. Add LN₂ directly to the brass ethane vessel to sufficiently cool the vessel prior to condensing ethane gas. Failure to properly cool the brass ethane vessel will dramatically increase the time it takes to condense ethane.
2. Once the dewar has reached LN₂ temperature, avoid overfilling the dewar such that LN₂ spills into the ethane vessel.
Ethane comes as a compressed gas and needs to be liquefied for use. The ethane tank utilizes a high-purity dual-stage regulator to control the gas flow. Connect tubing to the regulator outlet valve and use a 14-gauge flat metal dispensing tip connected at the end for dispensing ethane.
Prior to opening the ethane main tank valve, make sure the pressure adjusting knob and outlet valve are closed all the way. Fully open the main tank valve and then open the outlet valve to ~50%. Slowly open the pressure adjusting knob until a slow gas flow is observed. Use the outlet valve to fine-tune the gas flow.
CAUTION: Always point the metal dispensing tip away from self when opening valves or making gas flow adjustments.
Slowly start the gas flow and assess the flow rate by inserting the tip of the ethane gas line into a small beaker of deionized water. Adjust the gas flow until the flow rate moderately disturbs the water.
1. Adjust the gas flow rate to ensure proper ethane condensing occurs - too slow of a flow rate will cause the ethane gas to solidify in the dispensing tip and too fast of a flow rate will result in intense bubbling and prevent freezing.
Prior to inserting the tip of the ethane gas line into the brass ethane vessel, clean and wipe the dispensing tip with delicate task wipes to remove any water.
In a smooth and quick motion, locate the gas dispensing tip at the bottom of the brass ethane vessel and begin moving the dispensing tip in a slow circle around the bottom of the ethane vessel. Solid ethane will form immediately but will quickly liquefy as more ethane gas is added/condensed.
1. Continue to move the metal ethane dispensing tip around at the bottom of the ethane vessel to liquefy the solid ethane. Fill up the ethane vessel to ¾ full of liquid ethane (2-3 threads from the top). Stop ethane gas flow by carefully removing the metal ethane dispensing tip from the brass ethane vessel and closing the outlet valve.
Top off the plunging dewar with LN₂pouring gently on the side of the dewar to avoid LN₂ addition to the brass ethane vessel, until the liquid level just touches the brass ethane vessel. Place the foam lid on the plunging dewar to facilitate ethane solidification.
1. LN₂ must directly contact the brass ethane vessel to aid in the solidification of ethane. After approximately 5 minutes, the ethane within the brass vessel will become completely frozen solid. Add more LN₂ until it just touches the ethane vessel and proceed to the next step.
If the ethane has not frozen solid, then the brass ethane vessel is not cold enough for the remaining steps. Add more LN₂ until it just touches the ethane vessel and wait an additional 5 minutes. Ensure that the ethane within the brass vessel is completely frozen solid.
Open the gas outlet valve to produce an ethane gas flow at a similar rate as determined in step 4.6. Vertically place the metal ethane dispensing tip into the solid ethane and continue to move the ethane dispensing tip in a circular motion to melt the solid ethane.
1. Continue to add ethane until it is level with the top of the brass ethane vessel. Slowly remove the tip from the ethane vessel and close the ethane tank outlet valve. Cover the dewar with the lid for ~1-4 minute(s) to let the ethane solidify around the edges of the brass ethane vessel.
  NOTE: An ideal ethane vessel will have a 2-3 mm symmetric ring of solid ethane at the perimeter of the brass ethane vessel with liquid ethane in the center (Figure 1D and Figure 2).
2. Ensure that the ethane be as cold as possible without solidifying to ensure proper vitrification of the biological specimen. Failure to properly prepare the liquid ethane can result in inadequate vitrification of the specimen, ice accumulation, and/or loss of specimen, each contributing to the deterioration of specimen quality for imaging.
3. If a solid ring of ethane does not form after 2-3 minutes, add more LN₂ to the dewar and cover for 2-5 more minutes.
4. If the ethane is solidifying too quickly, then use a large pair of clean tweezers to gently warm the ethane vessel and/or solid ethane to prevent complete ethane solidification. Once the ethane and LN₂ are stable, close all the ethane tank valves and locate the plunging dewar to the manual plunger. Secure the plunging dewar to the manual plunger.
  CAUTION: Be extra careful when transferring the dewar as LN₂ can spill into the ethane vessel and solidify the liquid ethane. If need be, a clean set of tweezers can be used to melt any solid ethane in the middle of the vessel.
Test the location of the ethane dewar with a pair of empty tweezers to ensure the tweezer tip locates at the center of the ethane vessel and there is sufficient space for the grid and tweezer tip inside the liquid ethane (Figure 1D).
1. If the solid ethane ring is too thick for easy grid handling, then use a pair of room temperature, clean tweezers to melt the solid ethane and create more freezing area at the center of the ethane vessel.

5. Prepare EM grids

NOTE: Estimated operating time: 1-5 minutes

Add the grid storage box(es) to the dewar, unscrew the grid storage box lid(s), and make sure each lid can freely rotate to a new grid slot.
Carefully transfer grids from the grid storage box to the edge of the square glass coverslip with ~30-40% of the grid off the slide edge. Ensure that the grid foil is facing up. Ensure grids are covered when not in use. Placing the grids over the edge of the square glass coverslip provides ease of grid handling and decreases the chance of bending or damaging the grid during transfer.
NOTE: 4-6 grids are typically prepared at a time.
Render grids hydrophilic using a glow discharger or plasma cleaner.
NOTE: Please refer to the recommended guidelines for grid cleaning provided by the glow discharger/plasma cleaner manufacturer.
1. Use the grids within 10 minutes of plasma cleaning as the grids lose hydrophilicity and grid-to-grid reproducibility decreases after this time.

6. Prepare cryoEM specimen by plunge freezing

NOTE: Estimated operating time: >10 minutes (~1-3 min per grid)

Use a clean and dry clamping tweezer to pick up a cleaned grid, slide the plastic clamp down to fix the grid in place, and gently tap the tweezers to ensure the grid is properly secured.
1. Handle grids by the outer ring to prevent damage to the grid foil.
Apply 1 to 5 µL of sample to the prepared side (e.g., front or foil side) of the grid.
NOTE: The optimal volume and blotting time depends on the sample and needs to be optimized for each sample; larger volumes and more viscous samples require longer blotting times.
Secure the tweezer-grid-sample assembly to the manual plunging arm by wrapping tape around the tweezer handle.
1. Face the sample towards the user for traditional front blotting. If the sample requires back blotting, then locate the sample away from the user.
Hold a clean, dry, cut piece of blotting paper between the thumb and index finger of each hand.
1. Only handle the blotting papers from the edges and never touch the center as oils and other contaminants from hands/gloves can alter grid quality.
Rest hands on the edge of the plunging dewar to establish a stable position. Locate the blotting paper parallel to the grid surface approximately 1 cm away from the grid surface.
1. Use the middle section of the blotting paper to allow for complete fluid mobility and even wicking across the grid surface.
Gently slide and rotate the thumb and ring fingers towards each other to bend blotting paper toward the grid to initiate blotting. Maintain contact between the blotting paper and the grid surface during the entire blotting process.
1. Directly contact the blotting paper to the grid surface and maintain consistent contact across the grid surface.
2. Gently bending the blotting paper decreases bending of the grid and/or damage to the grid surface, and results in more consistent ice across the grid (Figure 3A).
Observe the mobile liquid front and once it stops progressing into the blotting paper begin counting for 4 to 6 seconds.
NOTE: Counting can occur once the blotting paper contacts the grid surface but lower grid-to-grid reproducibility can occur. The total blot time will depend on the grid type, foil type, sample concentration, and sample type (e.g., soluble versus membrane versus filamentous proteins). For more viscous samples longer blot times (e.g.., 5 to 7 seconds) will be required.
1. Important: Develop a reliable and consistent counting scheme to greatly enhance reproducibility during the freezing process.
Move the left, right thumb and index fingers in opposite directions to remove the blotting paper in a "snapping motion" away from the grid surface. Immediately depress the foot pedal to release the plunging arm and plunge the grid into liquid ethane.
1. Simultaneously remove the blotting paper and press the foot pedal to plunge the grid into the ethane as soon as possible for best freezing results. The longer the time between blotting paper removal and plunging, the more evaporation of thin films will occur and decrease grid-to-grid reproducibility.
Use one hand to stabilize the clamping tweezers, unwind tape carefully from around the tweezers and manual plunging arm.
1. Always maintain contact with the tweezer to prevent movement of the tweezer-grid and limit grid damage that occur from knocking the grid against the solid ethane.
Once clamping tweezers are free from the manual plunging arm, maintain the tweezer in one hand resting on top of the plunging dewar, ensure the grid remains in the liquid ethane. Carefully slide the plastic clamp away from the grid so the grid can be transferred. Maintain pressure on the tweezers to retain the grid.
1. With one swift motion, quickly transfer the grid from the ethane vessel into the LN₂ reservoir. Carefully place the grid in the grid storage box.
  NOTE: Some ethane may solidify on the grid surface. Opening the tweezer slightly will break the ethane and allow for the grid to be dropped into the grid box.
Wrap the tip of the tweezers in a delicate task wipe to prevent frost accumulation. Set aside until the tweezers have returned to room temperature.
1. Have 4 to 6 tweezers on hand for ease of use. Each tweezer will be used for sample freezing and warmed before subsequent use.
Repeat steps 6.1-6.11 for each grid.
Once freezing has culminated, securely close the grid box and transfer to a proper storage location.
Carefully dispose of the liquid ethane and LN₂ and store all materials in a dry location.

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Representative Results

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.

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Discussion

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.

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Disclosures

We have nothing to disclose.

Acknowledgments

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.

Materials

Name	Company	Catalog Number	Comments
4 slot grid storage box	Ted Pella	160-40
14 gauge flat metal dispensing tip	Amazon	B07M7YWWLT
22x22 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

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