Name: manual blot-and-plunge freezing of biological specimens for single-particle cryogenic electron microscopy
Uploaded: 2022-02-07T12:30:00
Description: Watch this Scientific Journal Video about Manual Blot-and-Plunge Freezing of Biological Specimens for Single-Particle Cryogenic Electron Microscopy at JoVE.com

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.

1. Prepare the manual plunging environment
NOTE: Estimated operating time: 5-30 minutes
<ol>
	<li>Locate the manual plunger in a 4 &#176;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&#39;s Environmental Health and Safety guidelines for the safe location of the manual plunger and recommended operations.</li>
	<li>Prior to grid preparation,...

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...

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 &#197; 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 &#34;glass-like&#34; 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 &#181;L) 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.&#160;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4 slot grid storage box</td>
			<td>Ted Pella</td>
			<td>160-40</td>
			<td></td>
		</tr>
		<tr>
			<td>14 gauge flat metal dispensing tip</td>
			<td>Amazon</td>
			<td>B07M7YWWLT</td>
			<td></td>
		</tr>
		<tr>
			<td>22x22 mm square glass coverslip</td>
			<td>Sigma</td>
			<td>C9802-1PAK</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm glass Petri dish to store grids</td>
			<td>Fisher</td>
			<td>08-747A</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm glass Petri dish to store Whatman paper</td>
			<td>Fisher</td>
			<td>08-747D</td>
			<td></td>
		</tr>
		<tr>
			<td>150 mm glass Petri dish to store Whatman paper</td>
			<td>Fisher</td>
			<td>08-747F</td>
			<td></td>
		</tr>
		<tr>
			<td>250 mL beaker</td>
			<td>Fisher</td>
			<td>02-555-25B</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue styrofoam dewar</td>
			<td>Spear Lab</td>
			<td>FD-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Brass ethane vessel</td>
			<td>Lasco</td>
			<td>17-4075</td>
			<td></td>
		</tr>
		<tr>
			<td>Clamping tweezers</td>
			<td>Ted Pella</td>
			<td>38825</td>
			<td></td>
		</tr>
		<tr>
			<td>Delicate task wipes</td>
			<td>Fisher</td>
			<td>06-666</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual-stage regulator with control valve</td>
			<td>Airgas</td>
			<td>Y12N245D580-AG</td>
			<td></td>
		</tr>
		<tr>
			<td>Dewer grid base</td>
			<td>UCSD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethane platform</td>
			<td>UCSD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethane propane tank</td>
			<td>Praxair</td>
			<td>ET PR50ZU-G</td>
			<td>ethane (50%) : propane (50%) in a high-pressure tank</td>
		</tr>
		<tr>
			<td>Ethane tank</td>
			<td>Praxair</td>
			<td>UN1035</td>
			<td>ethane (100%)</td>
		</tr>
		<tr>
			<td>Flexible arm task light</td>
			<td>Amscope</td>
			<td>LED-11CR</td>
			<td></td>
		</tr>
		<tr>
			<td>Grids (UltrAufoil R 1.2/1.3 300 mesh)</td>
			<td>Electron Microscopy Sciences</td>
			<td>Q325AR1.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Humidifier</td>
			<td>Target</td>
			<td>719438</td>
			<td></td>
		</tr>
		<tr>
			<td>Hygrometer</td>
			<td>ThermoPro</td>
			<td>B01H1R0K68</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab coat</td>
			<td>UCSD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid Nitrogen dewar</td>
			<td>Worthington</td>
			<td>LD4</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid Nitrogen gloves</td>
			<td>Fisher</td>
			<td>19-059-925</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual plunger stand (black stand + foot pedal)</td>
			<td>UCSD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mark 5 (plunging platform)</td>
			<td>UCSD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrile gloves</td>
			<td>VWR</td>
			<td>82026-424</td>
			<td></td>
		</tr>
		<tr>
			<td>P20 pipette</td>
			<td>Eppendorf</td>
			<td>13-690-029</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR tubes</td>
			<td>Eppendorf</td>
			<td>E0030124286</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips</td>
			<td>ibis scientific</td>
			<td>63300005</td>
			<td></td>
		</tr>
		<tr>
			<td>Ring lamp</td>
			<td>Amazon</td>
			<td>B07HMR4H8G</td>
			<td></td>
		</tr>
		<tr>
			<td>Safety glasses</td>
			<td>UCSD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Amazon</td>
			<td>Fiskars 01-004761J</td>
			<td></td>
		</tr>
		<tr>
			<td>Screw driver</td>
			<td>Ironside</td>
			<td>354711</td>
			<td></td>
		</tr>
		<tr>
			<td>Tape</td>
			<td>Fisher</td>
			<td>15-901-10R</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezer to transfer grid box</td>
			<td>Amazon</td>
			<td>LTS-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Tygon tubing</td>
			<td>Fisher</td>
			<td>14-171-130</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman blotting paper</td>
			<td>Fisher</td>
			<td>1001-090</td>
			<td></td>
		</tr>
	</tbody>
</table>

manual blot-and-plunge freezing of 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.

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 ...

Watch this Scientific Journal Video about Manual Blot-and-Plunge Freezing of Biological Specimens for Single-Particle Cryogenic Electron Microscopy at JoVE.com

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

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 ...

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