The authors would like to thank the workshop of the Biozentrum of the University Basel for their support, S. A. M&#252;ller for critical discussions and for carefully reading the manuscript, A. Fecteau-LeFebvre for technical assistance with EM, Ricardo Adaixo, Frank Lehmann for membrane protein test samples (all from the C-CINA, Biozentrum, University of Basel) and A. Engel, emeritus University Basel for his inspiring conversations. Test samples were kindly provided by P. Ringler, M.-A. Mahi, and T. Schwede (Biozentrum, University of Basel), P. Leiman (Laboratory of Structural Biology and Biophysics, EPFL) and R. Diaz- Avalos (New York Structural Biology Center, USA). The project was supported by the Swiss Nanoscience Institute (SNI, project P1401, ARGOVIA project MiPIS) and the Swiss National Science Foundation (SNF, project 200021_162521).

<ol>
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A., Ringler, P., Goldie, K. N., Goedecke, N., Hierlemann, A., Stahlberg, H., Engel, A., Braun, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connecting+&#181;-fluidics+to+electron+microscopy.">Connecting &#181;-fluidics to electron microscopy.</a> J Struct Biol. 177 (1), 128-134 (2012).</li><li>Arnold, S. A., Albiez, S., Opara, N., Chami, M., Schmidli, C., Bieri, A., Padeste, C., Stahlberg, H., Braun, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+sample+conditioning+and+preparation+of+nanoliter+volumes+for+electron+microscopy.">Total sample conditioning and preparation of nanoliter volumes for electron microscopy.</a> ACS nano. 10 (5), 4981-4988 (2016).</li><li>Kemmerling, S., Arnold, S. A., Bircher, B. 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The authors Stefan A. Arnold, Henning Stahlberg and Thomas Braun declare the following competing financial interest: The cryoWriter concept is part of patent application PCT/ EP2015/065398 and EP16194230.

The &#39;cryoWriter&#39; instrument and the protocols required to prepare sample grids for NS- and cryo-EM from nL sized total sample volumes and completely avoid the classical paper-blotting step are presented. Microfluidic principles and a micromechanical system are combined in the cryoWriter to make this possible.
Our experience shows that when the miniaturized methods presented in this manuscript are used, the parameter space for EM-grid preparation is larger than for classical methods, and under tighter user control. Importantly, the increased reproducibility achieved makes it possible to pre-screen with sample buffer system, complemented with a readily available test protein, to determine the optimum parameters before the actual experiment is performed. This keeps consumption of the sample of interest to the absolute minimum and is highly recommended. Critical steps for both NS- and cryo-EM grid preparation are: (i) Priming of the pump system; for sub-nanoliter volume dispensing, the system liquid (water) must be degassed and bubble free. (ii) Precise control of the glow discharge or plasma cleaning step; the surface characteristics of the EM grid are crucial for reproducible results. (iii) Sample conditioning; the time required for conditioning, e.g., with NS, depends on the buffer type, salt content and concentration (Figure 3), as well as on the nozzle geometry of the microcapillary24. (iv) Evaporation rates for NS-EM preparations for quantitative EM; the coffee-ring effect can prohibit the quantitative analysis of NS-EM preparations, and must be suppressed by slow evaporation rates controlled by the DP-stage.
Different aspects of the presented grid preparation methods can be freely combined allowing the development of versatile protocols for specific samples. Typical examples would be the removal of a substance that prevents high-resolution EM, e.g., glycerol, by a conditioning step prior to grid preparation for cryo-EM; the introduction of mediator molecules, such as ligands, by conditioning before the grids are prepared; or the examination of single cell lysate by NS- or cryo-EM (Figure 6).
The use of microfluidics and minimal sample amounts in the presented methods completely removes the need for paper-blotting steps. This is a great advantage, because paper blotting is a harsh treatment for proteins, potentially contaminating the sample with unwanted ions and inherently leading to massive sample loss. On the other hand, effects potentially caused by the air-water interface of the thin sample film formed when cryo-EM samples are prepared in the classical manner are not avoided when the cryoWriter is used. Grids suitable for cryo-EM can be prepared with a less than 0.2 s wait time between sample application and vitrification (data not shown). However, as proteins travel a few tenths of a nanometer in a few nanoseconds by diffusion, there is still enough time for them to collide with the air-water interface of a 100 nm thick sample film several times. However, the amount of proteins sticking to the air-water interface might be significantly reduced by these short time gaps and might prevent protein denaturation or restricted particle orientation. Another promising approach that might protect sensitive proteins from the air-water interface is to cover the sample film by low molecular weight surface-active substances. These compounds could be rapidly introduced by a conditioning step in the cryoWriter before grid preparation. The high surface-to-volume ratio of microfluidic systems is a further limitation of the cryoWriter, as sample can potentially be lost by unspecific adsorption to the microcapillary surface and disturb quantitative analysis by particle counting. The problem is addressed in two ways: First, the sample does not travel long distances within the microcapillary. Indeed, the nanoliter sample volume remains at the capillary tip throughout the processing. Second, the surface to volume ratio is further reduced by using microcapillaries with relatively large inner diameters, e.g., 180 &#181;m. Third, the surfaces of the microcapillaries can be easily passivated if necessary, e.g., by treating them with commercially available polylysine ethanol glycols (PLL-PEG).
The high-resolution analysis of proteins by the single particle approach used in EM only requires 100,000 to a few million images of individual protein particles. This means that microfluidic techniques can provide enough protein complexes for the structural investigation. A miniaturized immuno-precipitation method for the fast isolation of protein complexes (around 1 hour) from minimal cell amounts (approx. 40,000 cells) was developed earlier28. This method will now be directly linked to the miniaturized sample preparation stage of the cryoWriter. The final goal is to develop an integrated microfluidic pipeline for ultra-fast protein isolation and cryo-EM grid preparation that requires less than two hours in all. Furthermore, as demonstrated by Figure 6, the minute amount and volume of material needed for sample preparation and the almost lossless conditioning and grid preparation procedure achieved using the cryoWriter, make it possible to study the protein complexes of individual cells. Together, the miniaturized immuno-precipitation method and the cryoWriter lay the foundation of a new proteomics method called &#34;single cell visual proteomics&#34;, as we recently demonstrated for heat-shock experiments24. Data analysis algorithms geared to the analysis of &#34;visual proteomics&#34; images are currently being tested.

Hardware and software for the structural analysis of protein complexes by transmission electron microscopy (TEM) has massively advanced during recent years. The improvements made paved the way to a &#34;resolution revolution&#34;1,2 and fundamentally changed structural research. The revolution started with the advent of cryo-electron microscopy (cryo-EM)3,4, allowing the preparation of biological samples under close to physiological conditions while decreasing radiation sensitivity and preventing sample evaporation in the high vacuum of the transmission electron microscope5. In the following years, incremental technological progress gradually increased the resolution achievable. Among these innovations were the application of field-emission guns6,7, and, more recently, improved data analysis algorithms, such as maximum likelihood methods8,9. Direct electron detector cameras10,11,12,13, movie-mode imaging and the accompanying software developments14,15,16,17, provided the final breakthrough required to achieve atomic resolution for biological samples by single particle analysis (for a review see Cheng, Grigorieff, et al.18). The importance of cryo-EM was recently recognized by the award of the Nobel prize for chemistry to three of the pioneers.
To image a biological sample by TEM, the method used to load the EM grid with sample (subsequently referred to as &#34;grid preparation&#34;) must ensure that the resulting sample layer (i) is thin enough (&#60;100 nm) to avoid extensive noise by inelastic or multiply scattered electrons, (ii) withstands the high vacuum of the electron microscope, and (iii) protects the biomolecules from radiation damage. Two main methods are used to fulfill these perquisites: Negative stain(NS)19,20 procedures (Figure 1A) adsorb the sample to a thin carbon film, embed the biomolecules in amorphous heavy metal and then allow the assembly to dry in air. This is simple and quick, and the loaded EM grids (subsequently referred to as &#34;sample grids&#34;) are easy to store and can be kept for extended periods of time (generally years). In TEM, the preparations exhibit high contrast due to the NS and tolerate higher electron doses than cryo-preparations, but the resolution is limited to approximately 20 &#197;. Cryo-EM procedures (Figure 1B) employ holey carbon supports. A thin film of the sample solution is spanned across the holes and the EM grid is plunged into a cryogen, usually liquefied ethane, to rapidly cool it below -150&#176;C. The result is an amorphous, vitrified, 50 to 100 nm-thick film of the solution in the support holes. This thin, amorphous film withstands the high vacuum in the electron microscope and, in the ideal case, preserves biological structures in their native state. The procedure allows biological samples to be imaged at high-resolution. However, the sample grid must be kept at temperatures below -150 &#176;C at all times to avoid devitrification. It can be imaged using relatively high electron doses due to the low temperature, but the contrast and signal-to-noise ratio is nevertheless low. Therefore, averaging techniques are employed to increase contrast and, provided the sample is imaged from different angles, a high-resolution three-dimensional (3D) map can be reconstructed. The most commonly used and highly successful method for 3D reconstruction as of now is the single particle approach. For a recent review, see Cheng at al.18.
Negative stain TEM (NS-EM) is important for screening and quality control, when high-contrast is needed or when only limited amounts of sample are available (adsorption to the carbon film generally concentrates the sample). Single particle cryo-EM is the gold-standard method if high-resolution 3D reconstructions of the protein structure are aimed for.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57310/57310fig1.jpg" /> 
Figure 1: Principles of TEM grid preparation and comparison between the classical (panel A, B) and a microfluidic approach (panel C, D). A) Classical NS-EM grid preparation: About 3 &#181;L of sample are pipetted by hand onto an EM grid covered with a continuous carbon film (subsequently referred to as an &#39;NS-EM grid&#39;) (i). After incubation for approx. 10 s, filter paper is used to blot away the excess liquid from the side (ii), leaving the adsorbed biomolecules in a thin water film. Subsequently, the protein is incubated in a heavy metal salt solution, e.g., 2% uranyl acetate, for 20 s (iii), and again the liquid is removed by blotting from the side using filter paper (iv). Finally, the EM-grid is left to dry in air. B) Classical cryo-EM grid preparation: About 3 &#181;L of sample are pipetted by a hand onto a holey carbon film. To form a thin sample film, the surplus liquid is removed by paper-blotting face-on from one or both sides (ii). Finally, the grid is rapidly plunged into liquid ethane for vitrification (iii). C) NS-EM grid preparation using the cryoWriter setup: A 5 nL volume is aspirated from the sample stock using a microcapillary (i). For sample conditioning, the microcapillary tip is immersed into the conditioning solution, e.g., 2% ammonium acetate. Ions and small molecules are exchanged by diffusion (ii). Note that the dimensions of the microcapillary ensure that the whole process is diffusion driven. Proteins have much lower diffusion constants than salt ions and are not significantly lost24. Finally, the sample is dispensed onto the grid and allowed to dry (iii). D) Principles of cryo-EM grid preparation using the cryoWriter-based method: An EM grid covered with a holey carbon film is placed on the surface of a temperature-controlled platform and held by tweezers. The temperature of the platform is controlled at an offset from the dew point temperature of the grid environment. The grid is moved relative to the microcapillary containing the sample and the microcapillary is lowered until it is a few micrometers above the grid. Subsequently, a few nanoliters of sample are dispensed from it while the stage is moved in a spiral pattern; excess liquid is re-aspirated (i). After EM grid priming, the microcapillary is withdrawn and the grid remains on the temperature controlled platform (subsequently referred as dew point (DP) stage) for a short time to allow a controlled amount of sample to evaporate. For plunge freezing, the grid is rapidly withdrawn from the stage using the tweezers (ii), flipped by 90&#176; into the vertical position, and plunged into a cryogen bath (iii) (subsequently referred to as &#39;pick-and-plunge&#39; mechanism). <a href="/files/ftp_upload/57310/57310fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Unfortunately, the grid preparation methods used for NS and cryo-EM have not improved significantly since they were invented. Current drawbacks are the high sample consumption (approx. 3 &#181;L of 1 mg/mL protein) and the large amount (&#62; 99%) of sample lost (Figure 1A,B). Furthermore, the classical method used to prepare grids for cryo-EM is a harsh procedure for proteins: First, it involves an extensive face-on paper-blotting step (Figure 1B, ii), and, second, the protein is exposed to the air-water interface for a significant amount of time21. Here, an alternative method for sample pre-conditioning, sample grid preparation and post-processing (grid drying or vitrification) for NS-EM (Figure 1C) or cryo-EM (Figure 1D) is presented. The in-house built setup, called &#34;cryoWriter&#34;, uses miniaturized sample handling technology and microfluidic principles to aspirate, condition and dispense sample, avoiding paper blotting completely and providing alternative methods to thin samples for cryo-EM. It significantly reduces sample consumption and improves user-control over sample preparation as a whole. Furthermore, the method allows novel experimental applications; such as the preparation of isolated biological components of individual cells in an approach called &#34;single cell visual proteomics&#34;22,23,24,25.

<table><tbody><tr><td>&lt;strong&gt;Liquid handling&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Microcapillary tip</td><td>New Objective</td><td>FS360-100-30-N-20</td><td>SilicaTips 30 &amp;micro;m tip ID, 100 &amp;micro;m ID , pk. 20</td></tr><tr><td></td><td></td><td>FS360-150-30-N-20</td><td>SilicaTips 30 &amp;micro;m tip ID, 150 &amp;micro;m ID , pk. 20</td></tr><tr><td>Conductive sleeve</td><td>New Objective</td><td>CONGAS-1&amp;nbsp;</td><td>Conductive Elastomer for HV contact, 12&quot; long</td></tr><tr><td>Fused silica tubing</td><td>BGB-Analytik</td><td>TSP-150375</td><td>TSP Standard FS Tubing, 150 &amp;micro;m ID, 363 &amp;micro;m OD, 5 meter</td></tr><tr><td>PressFit Connector</td><td>BGB-Analytik</td><td>2525LD</td><td>Deact. PressFit Connector 0.25 to 0.25 mm ID, pk. 25&amp;nbsp;</td></tr><tr><td>Syringe 10 &amp;micro;l</td><td>BGB-Analytik</td><td>HA-80001</td><td>Hamilton 1701 LT - Luer Tip (needle not included)&amp;nbsp;</td></tr><tr><td>Syringe pump controller</td><td>Cetoni</td><td>A3921000093</td><td>neMESYS controller</td></tr><tr><td>Syringe pump dosing unit</td><td>Cetoni</td><td>A3921000095</td><td>neMESYS dosing unit</td></tr><tr><td>&lt;strong&gt;Temperature control&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dew point sensor</td><td>Meltec</td><td>UFT75-AT</td><td>Humidity/Temperature sensor, dew point calculation, USB and embedded DLL</td></tr><tr><td>Temperature sensor</td><td>Sensorshop24.ch</td><td>LS7-PT1000-1.0-3L</td><td>Surface mountable temperature sensor Pt1000</td></tr><tr><td>Peltier controller</td><td>Cooltronic</td><td>TC2812&amp;nbsp;</td><td>PID temperature controller for activation of Peltier driven systems with constant voltage output</td></tr><tr><td>Peltier element</td><td>Distrelec.ch</td><td>PE-127-14-15-S</td><td>40x40 mm</td></tr><tr><td>Water-cooling block</td><td>-</td><td>-</td><td>Water cooling block for 40x40 mm peltier element, copper base</td></tr><tr><td>Water pump</td><td>Digitec.ch</td><td>294877</td><td>XSPC X20 750 Dual 5.25 Bay Reservoir Pump Black V4</td></tr><tr><td>Water heat exchanger block</td><td>-</td><td>-</td><td>Water heat exchanger block with 2 fans to cool down circulating water</td></tr><tr><td>Small water coolers</td><td>PCHC.ch</td><td>223050</td><td>Alphacool MCX ram copper edition 2 pcs</td></tr><tr><td>Small peltier elements</td><td>Distrelec.ch</td><td>PE-031-10-15-S</td><td>Laird 15x15 mm 3.4 A 8.1 W 3.8 V 74 &amp;deg;C, 2 pcs</td></tr><tr><td>&lt;strong&gt;Mechanics&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Linear stage z-axis</td><td>Dyneos AG</td><td>M-404.2PD</td><td>High-load precision translational stage, 50 mm travel range, DC motor</td></tr><tr><td>Stage controller</td><td>Dyneos AG</td><td>C-863</td><td>Mercury Servo Controller</td></tr><tr><td>Microscope xy-axis stage</td><td>Prior Scientific</td><td>H117EIL5</td><td>Motorized stage for Zeiss Axiovert 200 inverted microscope</td></tr><tr><td>Small permanent magnets</td><td>Supermagnete</td><td>S-05-08-N</td><td>Rod magnet &amp;Oslash; 5 mm, height 8.47 mm, neodymium, N45, nickel-plated, pk. 10</td></tr><tr><td>Small electromagnet</td><td>Schramme</td><td>EG1025A02/110</td><td>Electromagnet 24V 220 N 150 N 2,5 W</td></tr><tr><td>Controller electromagnet</td><td>Conrad.ch</td><td>MST-1630.001 7</td><td>Electromagnet control PCB board</td></tr><tr><td>Solenoid hub</td><td>Distrelec.ch</td><td>HD8286-R-F-24V100%</td><td>Kuhnke Solenid Hub 30 mm 16 N 2 N 16 W</td></tr><tr><td>Mini tweezers</td><td>Electron Microscopy Sciences</td><td>0302-M5S-PS</td><td>Dumont Type 5 mini, super thin tips</td></tr><tr><td>Various optomechanical parts</td><td>Thorlabs</td><td>-</td><td>-</td></tr><tr><td>Various mechanical parts</td><td>Workshop Biozentrum, University Basel</td><td>-</td><td>Custom designed adapter plates, holders, etc. see attached CAD files</td></tr><tr><td>&lt;strong&gt;Optics&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Laser diode</td><td>Thorlabs</td><td>CPS780S</td><td>780 nm laser diode module 2.5 mW</td></tr><tr><td>Photo detector</td><td>Thorlabs</td><td>PDA100A-EC</td><td>Si Switchable Gain Detector, 320-1100 nm, 2.4 MHz BW, 100 mm2</td></tr><tr><td>&lt;strong&gt;EM grids&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DURASIN FILM TEM</td><td>emsdiasum.com</td><td>DTF-03523</td><td>30 nm DuraSiN&amp;trade; silicon nitride membranes for TEM, pack of 5</td></tr><tr><td>Quantifoil Cu 200 mesh R 2/1</td><td>Quantifoil Micro Tools GmbH</td><td></td><td></td></tr><tr><td>Quantifoil Cu 200 mesh R 1.2/1.3</td><td>Quantifoil Micro Tools GmbH</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Buffers / Neg. stain&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PBS</td><td>Sigma</td><td>D8537&amp;nbsp;SIGMA</td><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline</td></tr><tr><td>NanoVan 2%</td><td>nanoprobes.com</td><td></td><td>Negative stain vanadium based</td></tr><tr><td>NanoW 2%</td><td>nanoprobes.com</td><td></td><td>Negative stain tungsten based</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>openBEB</td><td></td><td></td><td>The openBEB 2 framework is avaible upon request, version 3 will be downloadable</td></tr><tr><td>CryoWriter control software (openBEB plugin)</td><td></td><td>Avaible upon request, extra fees for 3rd party driver software might apply.</td><td></td></tr></tbody></table>

miniaturized sample preparation for transmission electron microscopy

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein complexes to atomic resolution. However, protein isolation techniques and sample preparation methods for EM remain a bottleneck. A relatively small number (100,000 to a few million) of individual protein particles need to be imaged for the high-resolution analysis of proteins by the single particle EM approach, making miniaturized sample handling techniques and microfluidic principles feasible.
A miniaturized, paper-blotting-free EM grid preparation method for sample pre-conditioning, EM grid priming and post processing that only consumes nanoliter-volumes of sample is presented. The method uses a dispensing system with sub-nanoliter precision to control liquid uptake and EM grid priming, a platform to control the grid temperature thereby determining the relative humidity above the EM grid, and a pick-and-plunge-mechanism for sample vitrification. For cryo-EM, an EM grid is placed on the temperature-controlled stage and the sample is aspirated into a capillary. The capillary tip is positioned in proximity to the grid surface, the grid is loaded with the sample and excess is re-aspirated into the microcapillary. Subsequently, the sample film is stabilized and slightly thinned by controlled water evaporation regulated by the offset of the platform temperature relative to the dew-point. At a given point the pick-and-plunge mechanism is triggered, rapidly transferring the primed EM grid into liquid ethane for sample vitrification. Alternatively, sample-conditioning methods are available to prepare nanoliter-sized sample volumes for negative stain (NS) EM.
The methodologies greatly reduce sample consumption and avoid approaches potentially harmful to proteins, such as the filter paper blotting used in conventional methods. Furthermore, the minuscule amount of sample required allows novel experimental strategies, such as fast sample conditioning, combination with single-cell lysis for &#34;visual proteomics,&#34; or &#34;lossless&#34; total sample preparation for quantitative analysis of complex samples.

The &#34;cryoWriter&#34; setup was developed (depicted in Figure 2) in order to test the miniaturized EM grid preparation procedures proposed in Figure 1C,D. Figure 2A shows an overview of the various components mounted to an inverse fluorescence microscope. A cell-culturing module is installed on the left side of the microscope; a module for EM grid preparation is located on the right. The cell-culturing module (Figure 2B) allows the growth of adherent eukaryotic cells and live-cell imaging of the cell culture by the light microscope. Individual cells are lysed by the combined action of osmotic shock, electroporation and aspiration of the cell content into a microcapillary (Figure 2B, Figure 6A)24,25. The aspirated lysate sample can then be used to prepare grids for NS- or cryo-EM. Alternatively, a stock protein solution in a PCR tube can be the sample source. The microcapillary (Figure 2B) employed is connected to a high-precision pump system allowing sample volumes to be aspirated and dispensed with sub-nL precision. As detailed in the protocols, all sample processing is performed within this microcapillary or on the EM grid itself without significant sample transport. For example, the same microcapillary is used to lyse individual eukaryotic cells, aspirate the lysate, condition it, and finally dispense aliquots onto EM grids. The grid preparation module consists of a movable DP-stage that allows the temperature of the EM grid placed on it to be precisely controlled (Figure 2C). For NS-TEM, the prepared sample grid can then simply be removed from the cold stage and allowed to dry in air at room temperature. However, the so-called coffee-ring effects that can then result need to be avoided for quantitative TEM where protein &#39;particles&#39; are counted. To do so, grids are dried slowly on the DP-stage using a gradually increasing temperature gradient to slow down liquid evaporation. For cryo-EM, the temperature of the grid is kept close to the dew point; a positive offset of approximately 8 &#176;C is chosen, allowing the controlled evaporation of sample liquid for thin film stabilization and thinning, which can be monitored by a sensor if necessary26. After the selected thinning time, a pick-and-plunge mechanism is activated and the sample is vitrified (Figure 2C). Note that this plunging mechanism is not needed for NS-EM grids, which are stored at room temperature.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57310/57310fig2.jpg" /> 
Figure 2: Overview of the cryoWriter setup.&#160;A) Overview of the cryoWriter setup mounted on an inverse light-microscope (1).&#160;B)&#160;Inset of area indicated on the left side in panel A. Cell culturing compartment (2), with a microcapillary (3) for sample manipulation and cell lysis positioned above a miniaturized PDMS-based cell culture plate (4).&#160;C)&#160;Inset of area indicated on the right side in panel A. &#39;Pick-and-plunge&#39; mechanism. A holey carbon film EM grid (5) is mounted between the tips of tweezers (6) and positioned horizontally in direct contact with the temperature-controlled stage (7), referred as the dew-point stage (DP-stage) in the main text. The stage temperature is tightly controlled via a PID controller and a water-cooled Peltier element, keeping it at or close to the dew point temperature, depending on the ambient environment. The DP-stage (7) is mounted on a motorized xy axis to move the grid relative to the microcapillary. The microcapillary itself is mounted on a z-stage and can be lowered until it is very close to the surface of the EM grid and used to dispense nanoliter-sized volumes onto the sample support covering it (a continuous thin carbon layer for NS-EM or a holey carbon film for cryo-EM). Note that liquid uptake and dispensing is performed using a high-precision pump system (8). The dispensed liquid can be distributed by moving the grid relative to the microcapillary in a spiral pattern. For cryo-EM preparation, the pick-and-plunge freezing mechanism (9) rapidly transfers the sample-loaded grid into liquid ethane (10) for rapid cooling and sample vitrification.&#160;<a href="/files/ftp_upload/57310/57310fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 3&#160;shows representative results obtained for NS-EM grids prepared using the cryoWriter setup. The tip of the microcapillary was loaded with 5 nL of sample from a stock solution and dipped into a reservoir of NS solution (2% methylamine tungstate) for several minutes to allow diffusive exchange of NS and salt ions (for a theoretical discussion see Arnold, et al.24). Afterwards, the conditioned sample was dispensed onto the thin carbon film of a NS-EM grid and dried. Figure 3A shows the use of a slot grid in the same way to visualize the complete droplet, as required for quantitative TEM. In order to avoid the coffee ring effect, the freshly glow-discharged grid was initially held at the dew-point temperature (no water evaporation) and then slowly warmed up on the DP-stage. Note, that for most applications (e.g., quality control of the sample or structural analysis) this slow-drying process is not needed. High-quality NS-preparations are obtained without it, as shown in Figure 3B,C. Conditioning times for phosphate-free low salt buffers are around 3 min, e.g., with low-salt Tris-buffer (20 mM Tris-HCl pH 7.4 with 50 mM NaCl), as shown in Figure 3B using tobacco mosaic virus (TMV) as sample. Figure 3C presents a worst-case scenario as the TMV was in PBS buffer (2.7 mM KCl, 1.5 mM KH2PO4, 136.9 mM NaCl, 8.9 mM Na2HPO4&#183;7H2O, pH 7.4). Phosphate ions form transient crystals with the heavy-metal ions of NS (see Figure 5C), lengthening the conditioning time required (7 min). Other heavy metal salts can also be used with the grid preparation module, e.g., 2% methylamine vanadate or ammonium molybdate (see also Arnold, et al.24). However, uranyl acetate is not suitable; the crosslinking effect of this stain leads to aggregates if the protein sample is conditioned in solution, before adsorption to a carbon film (see Figure 5E)23.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57310/57310fig3.jpg" /> 
Figure 3: Typical results for NS grids prepared using the cryoWriter setup as indicated in&#160;Figure 1C. A)&#160;Overview image of a 3 nL droplet dispensed on a slot grid after conditioning with 2% methylamine tungstate.&#160;B)&#160;TMV in 20 mM TRIS buffer. The inset shows a 3x enlargement of the indicated region. Adapted from Arnold, et al.24&#160;(further permissions related to the material excerpted should be directed to the ACS).&#160;C)&#160;Tobacco mosaic virus (TMV) in PBS buffer. The inset shows a 3x enlargement of the indicated region. Adapted from Arnold, et al.24&#160;(further permissions related to the material excerpted should be directed to the ACS). Scale bars: A, 100 &#181;m; B, 50 nm; C, 80 nm.&#160;<a href="/files/ftp_upload/57310/57310fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Typical results obtained for cryo-EM grids prepared using the cryoWriter setup are depicted in Figure 4. Panel 4A shows a grid atlas of the area covered by vitrified sample. Panel 4B shows the homogeneity of the vitreous ice in a selected grid slot. In both cases, the sample was in 25 mM HEPES-KOH pH 7.5, 50 mM NaCl buffer containing 0.05% Fos 14 detergent. Many samples and buffers were tested and a comparable high quality vitreous ice was obtained, but the conditions required are buffer dependent (see also discussion of Figure 5). Panel 4C shows apoferritin particles and a bacteriophage in Tris-HCl buffer (20 mM Tris-HCl, 50 mM NaCl; pH 7.4) imaged at high defocus to increase contrast. Panel 4D shows a 200 kDa membrane protein stabilized by amphipoles.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57310/57310fig4.jpg" /> 
Figure 4: Typical results for cryo-EM grids prepared using the cryoWriter setup as indicated in&#160;Figure 1D. The samples and the buffers vary in the examples shown. All samples were loaded on holey carbon films.&#160;A)&#160;Collage of overview images (&#34;grid atlas&#34;) of a sample containing a 150 kDa membrane protein, the periphery of the vitreous ice is indicated by white arrows.&#160;B)&#160;Enlarged grid slot from a grid prepared with the same buffer, showing the holey carbon film with vitreous ice. Some holes are not filled with sample buffer as indicated by white arrows.&#160;C)&#160;Carbon hole with vitrified sample containing apoferritin protein complexes and bacteriophages. Inset: twofold enlargement showing the tail of a bacteriophage. The white asterisk indicates the carbon film. Note that the image was recorded with high defocus to increase contrast.&#160;D)&#160;A 200 kDa membrane protein reconstituted in amphipols. Inset: a 2x enlargement of the indicated region shown with increased contrast. Scale bars: A, 100 &#181;m; B, 10 &#181;m; C and D, 80 nm.&#160;<a href="/files/ftp_upload/57310/57310fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The cryoWriter setup allows systematic screening for optimal EM-grid preparation conditions; an example is shown in Figure 5A (apoferritin in 25 mM HEPES-KOH pH 7.5, 50 mM NaCl, 0.05% Fos 14). In this experiment, the vitreous ice &#34;thinning&#34; temperature was varied, but the thinning time (i.e., the time gap between sample application and plunge freezing) remained constant (1 s). At low offset temperatures (e.g., 8 K), the sample layer was too thick. At higher offset temperatures, the vitreous ice in the holes was thinner (10 K, 12 K), until at some stage (above 18 K) the grid became completely dry (not shown). In the results presented here, an offset of 12 K lead to a large homogeneous area of vitreous ice as indicated by the black arrows. Such optimization experiments can be performed with the buffer of the target sample using &#34;test&#34; proteins (such as apoferritin). The best conditions found are then applied to the target sample. Furthermore, grids with parameters far from the optimum can often be recognized during the preparation procedure and do not need to be screened in the electron microscope, saving significant time. Figure 5 also shows a gallery of typical cryo-EM (Panel B) and NS-EM (Panels C to E) artifacts specific to the cryoWriter setup. The cryo-EM grid shown in Panel 5B with TMV in PBS containing 0.1% decyl-&#946;-D-maltopyranoside (2.7 mM KCl, 1.5 mM KH2PO4, 136.9 mM NaCl, 8.9 mM Na2HPO4&#183;7H2O, pH 7.4, 0.1%DM) was excessively thinned. The background of the image is grainy because the salt concentration became too high. In general, the visual appearance of a sample does not seem to be a linear function of the salt concentration; grains suddenly become prominent when a threshold concentration is reached during the thinning process. Note that unwanted substances can be removed by a conditioning step prior to grid preparation, as described for NS-EM in protocol section 1.6. NS can cause other artifacts. In the example shown in Panel 5C, PBS buffer (2.7 mM KCl, 1.5 mM KH2PO4, 136.9 mM NaCl, 8.9 mM Na2HPO4&#183;7H2O, pH 7.4) without sample was conditioned in 2% methylamine tungstate for 3 min. Precipitates and crystals are evident and exert extensive forces on the carbon surface leading to cracks. The precipitates only form in a certain PBS and NS concentration range and can be avoided by conditioning the sample for longer (compare Figure 3C). Panel 5D shows the periphery of a dispensed NS sample droplet exhibiting a &#34;coffee ring&#34;. This would disturb quantitative, total sample analysis and can be avoided by slowing down the drying process, i.e., by keeping the EM grid at the dew point temperature during sample application, and then gradually increasing the temperature to dry it (see Figure 3A). Panel 5E (apoferritin in 20 mM HEPES, pH 7.0, conditioned 3 min) shows the crosslinking activity of 2% uranyl acetate stain, which cannot be used to condition protein samples before they are adsorbed to carbon film supports.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57310/57310fig5.jpg" /> 
Figure 5: Systematic changes and artifacts observed when the cryoWriter set-up was used to prepare grids for NS- and cryo-EM.&#160;A)&#160;Systematic vitreous ice thickness variation; optimization of grid preparation for cryo-EM. The offset temperature of the DP-stage was varied (8 K to 12 K) keeping the thinning time constant (1 s). The arrows indicate the periphery of the sample layer.&#160;B)&#160;Salt effects; too highly concentrated,&#160;i.e., the thinning step was too long. The inset depicts a 2x enlargement of the indicated region.&#160;C)&#160;Salt precipitates formed by PBS buffer in the presence of heavy metal salts. Samples containing PBS buffer must be conditioned longer than samples in other buffers. Here, PBS buffer without sample was conditioned in 2% methylamine tungstate for 3 min, a typical time for other sample buffers. Note the crack in the carbon film most probably due to the strong forces from the precipitates acting on the thin support during the drying process.&#160;D)&#160;&#39;Coffee ring&#39; effect.&#160;E)&#160;Apoferritin conditioned in 2% uranyl acetate for 3 min. Uranyl ions exhibit significant crosslinking activity and the apoferritin clusters form large aggregates. Scale bars: A, 80 &#181;m; B, 80 nm C, 12 &#181;m; D, 80 &#181;m; E, 200 nm.&#160;<a href="/files/ftp_upload/57310/57310fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The small amount and volume required for EM grid preparation using the cryoWriter setup enables new types of experiments. For example, the total contents of a single cell can be collected and prepared for NS- and cryo-EM. The procedure is indicated in Figure 6A. An adherent, eukaryotic cell (HEK 293) is lysed by simultaneous electroporation and sample aspiration (Panel 6A)25. A total volume of 3 nL is aspirated, which contains the cell lysate, and is retained in the microcapillary for further processing. For the NS-EM shown in Panel 6B, the cell-culturing medium was exchanged with PBS buffer (2.7 mM KCl, 1.5 mM KH2PO4, 136.9 mM NaCl, 8.9 mM Na2HPO4&#183;7H2O, pH 7.4) prior to cell lysis. The cell contents were aspirated in 3 nL of buffer and conditioned in a reservoir of NS as indicated in Figure 1C for 10 min. Afterwards, a 5 nL volume was dispensed onto the continuous carbon film of a NS-EM grid. Individual proteins, e.g., filamentous actin, and membrane patches with attached proteins can be recognized in the image. For the cryo-EM, shown in Figure6C, a volume of 3 nL was dispensed on a holey carbon EM grid without re-aspiration to remove liquid. The relatively thick film of sample formed was extensively thinned before vitrification. To do this, the DP-stage temperature was gradually increased, starting at the dew-point temperature. The thinning process was monitored by a real-time sensor-system until a pre-specified threshold was reached triggering the &#39;pick-and-plunge&#39; mechanism and sample vitrification (for details see Arnold, et al.26). Membrane structures and proteins can be recognized in the image.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57310/57310fig6.jpg" /> 
Figure 6: Single cell visual proteomics using the cryoWriter set-up. A) Lysis of a single adherent eukaryotic cell. The cell is grown (1, green) on an functionalized, ITO coated (red) glass-slide (2) in a miniaturized petri-dish (3)25. The ITO layer is electrically grounded. The cell is approached by the microcapillary (4), which is coated with platinum. An initial osmotic shock (not indicated) is given to facilitate lysis, which is performed by a series of electric pulses and by the shear forces exerted during the aspiration of the cell lysate. The process can be monitored by light microscopy; the objective lens of the microscope is indicated (5). For details, see our previous work24,25. B) NS-EM image of lysate from an individual HEK 293 cell. Filamentous actin and membrane patches with attached proteins are visible. Panel adapted from Arnold, et al.24 (further permissions related to the material excerpted should be directed to the ACS). C) Cryo-EM image of lysate from an individual HEK 293 cell. The inset shows a 2x enlargement of the indicated region where typical membrane structures with associated proteins are visible. Panel C adapted from Arnold, et al. 26 Scale bars: B, 50 nm; C, 80 nm. <a href="/files/ftp_upload/57310/57310fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Miniaturized Sample Preparation for Transmission Electron Microscopy at JoVE.com

Miniaturized Sample Preparation for Transmission Electron Microscopy

An instrument and methods for the preparation of nanoliter-sized sample volumes for transmission electron microscopy is presented. No paper-blotting steps are required, thus avoiding the detrimental consequences this can have for proteins, significantly reducing sample loss and enabling the analysis of single cell lysate for visual proteomics.

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein ...

miniaturized-sample-preparation-for-transmission-electron-microscopy

Universidad San Sebastian

Research

JoVE Journal

Biology

19.5K Views.  University of Basel. An instrument and methods for the preparation of nanoliter-sized sample volumes for transmission electron microscopy is presented. No paper-blotting steps are required, thus avoiding the detrimental consequences this can have for proteins, significantly reducing sample loss and enabling the analysis of single cell lysate for visual proteomics.

Video: Miniaturized Sample Preparation for Transmission Electron Microscopy

A Method for Obtaining Serial Ultrathin Sections of Microorganisms in Transmission Electron Microscopy

Observing cells and cell components in three dimensions at high magnification in transmission electron microscopy requires preparing serial ultrathin sections of the specimen. Although preparing serial ultrathin sections is considered to be very difficult, it is rather easy if the proper method is used. In this paper, we show a step-by-step procedure for safely obtaining serial ultrathin sections of microorganisms. The key points of this method are: 1) to use the large part of the specimen and adjust the specimen surface and knife edge so that they are parallel to each other; 2) to cut serial sections in groups and avoid difficulty in separating sections using a pair of hair strands when retrieving a group of serial sections onto the slit grids; 3) to use a 'Section-holding loop' and avoid mixing up the order of the section groups; 4) to use a 'Water-surface-raising loop' and make sure the sections are positioned on the apex of the water and that they touch the grid first, in order to place them in the desired position on the grids; 5) to use the support film on an aluminum rack and make it easier to recover the sections on the grids and to avoid wrinkling of the support film; and 6) to use a staining tube and avoid accidentally breaking the support films with tweezers. This new method enables obtaining serial ultrathin sections without difficulty. The method makes it possible to analyze cell structures of microorganisms at high resolution in 3D, which cannot be achieved by using the automatic tape-collecting ultramicrotome method and serial block-face or focused ion beam scanning electron microscopy.

This study presents reliable and easy procedures for obtaining serial ultrathin sections of a microorganism without expensive equipment in transmission electron microscopy.

Observing cells and cell components in three dimensions at high magnification in transmission electron microscopy requires preparing serial ultrathin ...

Engineering

Microcrystal Electron Diffraction of Small Molecules

A detailed protocol for preparing small molecule samples for microcrystal electron diffraction (MicroED) experiments is described. MicroED has been developed to solve structures of proteins and small molecules using standard electron cryo-microscopy (cryo-EM) equipment. In this way, small molecules, peptides, soluble proteins, and membrane proteins have recently been determined to high resolutions. Protocols are presented here for preparing grids of small-molecule pharmaceuticals using the drug carbamazepine as an example. Protocols for screening and collecting data are presented. Additional steps in the overall process, such as data integration, structure determination, and refinement are presented elsewhere. The time required to prepare the small-molecule grids is estimated to be less than 30 min.

Here, we describe the procedures developed in our laboratory for preparing powders of small molecule crystals for microcrystal electron diffraction (MicroED) experiments.

A detailed protocol for preparing small molecule samples for microcrystal electron diffraction (MicroED) experiments is described. MicroED has been ...

Biochemistry

Sample Preparation and Experimental Design for In Situ Multi-Beam Transmission Electron Microscopy Irradiation Experiments

There is a need to understand materials exposed to overlapping extreme environments such as high temperature, radiation, or mechanical stress. When these stressors are combined there may be synergistic effects that enable unique microstructural evolution mechanisms to activate. Understanding of these mechanisms is necessary for the input and refinement of predictive models and critical for engineering of next generation materials. The basic physics and underlying mechanisms require advanced tools to be investigated. The in situ ion irradiation transmission electron microscope (I&#179;TEM) is designed to explore these principles.
To quantitatively probe the complex dynamic interactions in materials, careful preparation of samples and consideration of experimental design is required. Particular handling or preparation of samples can easily introduce damage or features that obfuscate the measurements. There is no one correct way to prepare a sample; however, many mistakes can be made. The most common errors and things to consider are highlighted within. The I&#179;TEM has many adjustable variables and a large potential experimental space, therefore it is best to design experiments with a specific scientific question or questions in mind.
Experiments have been performed on large number of sample geometries, material classes, and with many irradiation conditions. The following are a subset of examples that demonstrate unique in situ capabilities utilizing the I3TEM. Au nanoparticles prepared by drop casting have been used to investigate the effects of single ion strikes. Au thin films have been used in studies on the effects of multibeam irradiation on microstructure evolution. Zr films have been exposed to irradiation and mechanical tension to examine creep. Ag nanopillars were subjected to simultaneous high temperature, mechanical compression, and ion irradiation to study irradiation induced creep as well. These results impact fields including: structural materials, nuclear energy, energy storage, catalysis, and microelectronics in space environments.

Sample preparation techniques are outlined with specific considerations for in situ ion irradiation TEM experiments. Ion species, energy, and fluence are discussed with methods for how to select and compute them. Finally, procedures for conducting an experiment are described and accompanied by the representative results.

There is a need to understand materials exposed to overlapping extreme environments such as high temperature, radiation, or mechanical stress. When these ...

Optimizing Sample Preparation for Cryogenic Electron Microscopy

Cryogenic electron microscopy (cryo-EM) has revolutionized structural biology by enabling the study of macromolecular structures in near-native conditions, suspended in vitreous ice. This technique allows for the high-resolution visualization of proteins and other biomolecules without the need for crystallization, offering significant insights into their function and mechanism. Recent advancements in single-particle analysis, coupled with improved computational data processing, have made cryo-EM an indispensable tool in modern structural biology. Despite its growing adoption, cryo-EM faces persistent challenges that can limit its effectiveness, particularly uneven particle distribution. This issue often leads to poor resolution and reduced accuracy in reconstructed protein structures. This article outlines a simple, practical approach to address this challenge, using the small heat-shock protein from Methanocaldococcus jannaschii (MjsHSP16.5) as an example. The method optimizes sample preparation to minimize preferential adsorption, ensuring more homogeneous particle distribution and higher-quality protein cryo-EM structures. This technique offers valuable guidance for researchers aiming to overcome similar challenges in structural studies.

This protocol presents a practical method using Methanocaldococcus jannaschii (MjsHSP16.5) as an example to address uneven particle distribution through sample preparation optimization, providing a reference for researchers to efficiently elucidate macromolecular structures using cryogenic electron microscopy (cryo-EM).

Cryogenic electron microscopy (cryo-EM) has revolutionized structural biology by enabling the study of macromolecular structures in near-native ...

Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae

The Drosophila neuromuscular junction (NMJ) has emerged as a valuable model system in the field of neuroscience. The application of confocal microscopy at the Drosophila NMJ enables researchers to acquire synaptic information, encompassing both quantitative data on synapse abundance and detailed insights into their morphology. However, the diffuse distribution and limited visual range of the TEM present challenges for the ultrastructural analysis. This study introduces an innovative and efficient sample preparation method that surpasses the conventional approach. The procedure begins by placing a metal mesh at the base of a flat-bottomed bottle or test tube, followed by positioning fixed larvae samples onto the mesh. An additional mesh is placed over the samples, ensuring that they are positioned between the two meshes. The fixed samples are thoroughly dehydrated and infiltrated before proceeding with the embedding procedure. Then embedding of the samples in epoxy resin is performed in a flat sheet manner, which allows for the preparation of muscles for positioning and sectioning. Benefiting from these steps, all the muscles of Drosophila larvae can be visualized under light microscopy, therey facilitating subsequent positioning and sectioning. Excess resin is removed after locating the 6th and 7th muscles of body segments A2 and A3. Serial ultra-thin sectioning of the 6th or 7th muscle is performed.

Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae

This report provides a new sample preparation procedure for visualizing neuromuscular junctions in Drosophila Larvae. This method is more effective in preventing the curling of the samples compared to the traditional method and is particularly useful for Drosophila neuromuscular junction ultrastructural analysis.

The Drosophila neuromuscular junction (NMJ) has emerged as a valuable model system in the field of neuroscience. The application of confocal microscopy at ...

Neuroscience

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, especially cells in suspension such as hematopoietic stem cells (HSCs), remains limited due to the requirement of a high cell number during sample preparation. There are a few cytospin or monolayer approaches for TEM analysis from scarce samples, but these approaches fail to get significant quantitative data from the limited number of cells. Here, an alternative and novel approach for sample preparation in TEM studies is described for rare cell populations that enables quantitative analysis.
A relatively low cell number, i.e., 10,000 HSCs, was successfully used for TEM analysis compared to the millions of cells typically used for TEM studies. In particular, Evans blue staining was performed after paraformaldehyde-glutaraldehyde (PFA-GA) fixation to visualize the tiny cell pellet, which facilitated embedding in agarose. Clusters of numerous cells were observed in ultra-thin sections. The cells had a well preserved morphology, and the ultra-structural details of the Golgi complex and several mitochondria were visible. This efficient, easy and reproducible protocol allows sample preparation from a low cell number and can be used for qualitative and quantitative TEM analysis on rare cell populations from limited biological samples.

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It also describes an imaging method for studying the 3D structure of thick biological specimens by scanning transmission electron tomography. The sample preparation protocol is based on conventional methods in which the sample is fixed using chemical agents, treated with a heavy atom salt contrasting agent, dehydrated in a series of ethanol baths, and embedded in resin. The specific imaging conditions for observing thick samples by scanning transmission electron microscopy are then described. Sections of the sample are observed using a through-focus method involving the collection of several images at various focal planes. This enables the recovery of in-focus information at various heights throughout the sample. This particular collection pattern is performed at each tilt angle during tomography data collection. A single image is then generated, merging the in-focus information from all the different focal planes. A classic tilt-series dataset is then generated. The advantage of the method is that the tilt-series alignment and reconstruction can be performed using standard tools. The collection of through-focal images allows the reconstruction of a 3D volume that contains all of the structural details of the sample in focus.

This report describes a sample preparation protocol and specific imaging conditions for performing scanning transmission electron tomography of thick biological specimens.

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

Optimized Protocol for Retinal Organoid Sample Preparation for TEM

Preparing Retinal Organoid Samples for Transmission Electron Microscopy

Retinal organoids (ROs) are a three-dimensional culture system mimicking human retinal features that have differentiated from induced pluripotent stem cells (iPSCs) under specific conditions. Synapse development and maturation in ROs have been studied immunocytochemically and functionally. However, the direct evidence of the synaptic contact ultrastructure is limited, containing both special ribbon synapses and conventional chemical synapses. Transmission electron microscopy (TEM) is characterized by high resolution and a respectable history elucidating retinal development and synapse maturation in humans and various species. It is a powerful tool to explore synaptic structure in ROs and is widely used in the research field of ROs. Therefore, to better explore the structure of RO synaptic contacts at the nanoscale and obtain high-quality microscopic evidence, we developed a simple and repeatable method of RO TEM sample preparation. This paper describes the protocol, reagents used, and detailed steps, including RO fixation preparation, post fixation, embedding, and visualization.

Author Spotlight: Analyzing the Synaptic Ultrastructure in Mature Retinal Organoids Using TEM

This protocol provides an optimized and elaborate preparation procedure for retinal organoid samples for transmission electron microscopy. It is suitable for applications that involve the analysis of synapses in mature retinal organoids.

Retinal organoids (ROs) are a three-dimensional culture system mimicking human retinal features that have differentiated from induced pluripotent stem ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

Miniaturized Sample Preparation for Transmission Electron Microscopy

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Chapters in this video

A Method for Obtaining Serial Ultrathin Sections of Microorganisms in Transmission Electron Microscopy

Microcrystal Electron Diffraction of Small Molecules

Sample Preparation and Experimental Design for In Situ Multi-Beam Transmission Electron Microscopy Irradiation Experiments

Optimizing Sample Preparation for Cryogenic Electron Microscopy

Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Preparing Retinal Organoid Samples for Transmission Electron Microscopy

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging