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Interfacing Microfluidics with Negative Stain Transmission Electron Microscopy Nikita Mukhitov, John Michael Spear, Scott Stagg, and Michael G. Roper Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b03884 • Publication Date (Web): 07 Dec 2015 Downloaded from http://pubs.acs.org on December 16, 2015

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Analytical Chemistry

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Interfacing Microfluidics with Negative Stain

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Transmission Electron Microscopy

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Nikita Mukhitov §, John M. Spear §, Scott M. Stagg, Michael G. Roper*

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*To whom correspondence should be addressed:

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Michael G. Roper, Florida State University, 95 Chieftain Way, Tallahassee, FL, 32306

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E-mail: [email protected]

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§

Contributed to the work equally

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ACS Paragon Plus Environment


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Abstract

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A microfluidic platform is presented for preparing negatively stained grids for use in

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transmission electron microscopy (EM). The microfluidic device is composed of glass etched

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with readily fabricated features that facilitate the extraction of the grid post-staining and

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maintains the integrity of the sample. Utilization of this device simultaneously reduced

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environmental contamination on the grids and improved the homogeneity of the heavy metal

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stain needed to enhance visualization of biological specimens as compared to conventionally

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prepared EM grids. This easy-to-use EM grid preparation device provides the basis for future

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developments of systems with more integrated features, which will allow for high throughput

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and dynamic structural biology studies.

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Introduction

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Cryogenic electron microscopy (cryoEM) is quickly becoming a routine method in the

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determination of near atomic, high-resolution structures of biological molecules. However, for

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most samples before cryoEM data can be collected, the sample quality and heterogeneity

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must be first characterized using negative staining.1 The conventional workflow for negative

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staining of biological molecules consists of manual application of sample, removal of excess

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solution, and finally the addition of a heavy metal solution (for example, uranyl acetate,

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ammonium molybdate, or osmium tetroxide) (Figure 1A).

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contrast of the images by forming a strongly scattering shell around the biological sample,

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which gives biological molecules a light appearance on a dark background. It also

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encapsulates the sample, preserving the molecular structure during dehydration before the

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sample is inserted into the EM column. Because of these many roles, the thickness and

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uniformity of the stain is crucial for image quality and distinction of structural features.2

The stain acts to increase the

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Conventionally, EM grids are prepared by hand and, as such, variability is introduced

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due to user-to-user differences. The variability of the staining can have large effects on the

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final stained sample, ultimately hindering the resolution, image processing, and data analysis.1-

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manual sample handling and are thus prone to variations in the final result.2,3

While several reports have described procedures for optimized negative staining, all require

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Over the last two decades, microfluidics has shown its potential in offering reproducible,

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small volume, fluid control for the design of integrated lab-on-a-chip systems.6,7 This has

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resulted in the development of platforms that integrate multiple handling steps and minimize

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user-contributed variability. While microfluidics has previously been interfaced with other

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analytical platforms, it has only rarely been coupled with EM. In many cases, specialty devices

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were built for time-resolved cryoEM8-9 and for negative staining10-11; however, these required

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specialty instrumentation10-11 or fabrication methods8-9. 3



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The objective of this work was to develop a platform that could alleviate the variability

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associated with manual staining of EM grids. To achieve this, we developed a microfluidic

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system that enclosed the grid in a chamber where the delivery of the sample and drying was

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performed in a controlled fashion (Figure 1B). Images acquired from this system indicated

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reproducible stain thickness and image quality. We expect that this initial system will be

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further enhanced with the available suite of microfluidic tools. For example, subsequent

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iterations of this design with valves12, timers13-15, or other microfluidic features16,17 can be used

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for structural biology studies on dynamic systems or in high throughput applications.

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Experimental Methods

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Reagents. Nitric acid, hydrogen peroxide and hydrofluoric acid were obtained from VWR

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(Radnor, PA). Sodium hydroxide and ethanol were obtained from Sigma Aldrich (St. Louis,

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MO).

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(Morrisville, PA). For all solutions, ultrapure deionized water was used (Barnstead

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International, Inc., Dubuque, IA).

(Tridecafluoro-1,1,2,2-tetrahydrooctyl)

trichlorosilane

was

obtained

from

Gelest

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Microfluidic Fabrication. The fabrication workflow for the production of borosilicate glass

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microfluidic devices is shown in Figure 2. The extraction divot was etched to a depth of 45

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µmusing conventional methods.18 The channels and grid chamber were then fabricated using

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a second etch to a depth of 50 µm. The same process was repeated for another piece of

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glass, producing the mirror image of the design features to serve as the bottom complement.

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All dimensions of the channels were verified using a Mitutoyo SJ-411 Surface Profiler

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(Kanagawa, Japan). Fluid access holes were drilled with a 1.1 mm diamond-tipped drill bit

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(Crystalite Corp., Lewis Center, OH). The finished top slide was then fitted with a reservoir

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(Idex, Lake Forest, IL) using epoxy.

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For surface modification, the glass was cleaned by submerging in 5 M NaOH for 10

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minutes. The surface was rinsed with water and dried with N2. Subsequently, the slides were

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oxidized in a plasma cleaner (Harrick, Ithaca, NY) for 2 minutes. Immediately after, the slides

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were placed in a vacuum desiccator and (tridecafluoro-1,1,2,2-tetrahydrooctyl) trichlorosilane

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was deposited using a method previously described.19 Afterwards, the slides were rinsed with

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water, dried with N2, and stored in clean petri dishes at room temperature until use.

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Kvβ2.1 Expression and Purification. Full length rat Kvβ2.1 (GenBankTM accession number:

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CAA54142) was expressed and purified as previously reported.20 Briefly, the PCR verified

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pQE70 expression vector was transformed into BL21-DE3 cells using standard molecular

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techniques and Kvβ2.1 purified using the Talon Co2+ His-Pur resin (BD Biosciences, East

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Rutherford, NJ). Eluted protein was purified using a Superose 6 10/300 column (GE Life

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Sciences, Pittsburgh, PA) using an ÄKTA FPLC (GE Life Sciences). Purified protein was

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concentrated to 1.9 mg/ml using a 5000 molecular weight cut off spin concentrator (Thermo,

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Waltham, MA) and stored at -80°C until further use. The final storage buffer contained the

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following: 20 mM Tris pH 8.0, 150 mM KCl, 1 mM 2-mercaptoethanol, and 10% (w:v) glycerol.

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All reagents used were purchased from Amresco (VWR, Radnor, PA).

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Prior to staining, Kvβ2.1 was thawed on ice and centrifuged at 16,000X g for 20 minutes

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at 4°C to remove any aggregated or precipitated materials. The protein was diluted to 0.095

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mg/ml using dilution buffer (20 mM Tris pH 8.0, 150 mM KCl, 1 mM 2-mercaptoethanol) prior to

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use in microfluidic or conventional sample preparation.

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Conventional Sample Preparation. For conventional preparation of negative stain EM grids, 3 µl of diluted Kvβ2.1 was applied to continuous carbon coated GC400 copper mesh grids (Electron Microscopy Sciences, Hatfield, PA) that were rendered hydrophilic by glow discharge in 3:1 (v:v) Ar/O using a Solarus 950 advanced plasma system (Gatan, Pleasanton, CA). Grids were hand blotted using filter paper (Ted Pella, Redding, CA), then immediately stained using 3 µl of 2% (w:v) uranyl acetate (Electron Microscopy Sciences). Excess stain was then hand blotted using filter paper (Ted Pella) and the grids were air dried and stored at room temperature in a sealed desiccator before imaging.

Microfluidic Sample Preparation. The bottom glass microfluidic slide was placed into an aluminum manifold built in-house (Figure 3). A carbon coated, copper grid was rendered hydrophilic in the same manner as described above and placed into the device chamber. Eight drops (~20 µL each) of dilution buffer were distributed around the non-etched parts of the glass slides. The top slide was aligned using the manifold and lowered to the bottom slide, displacing the buffer and creating a thin sealing layer. The top of the manifold was attached and screwed down to seal the device. Sample (20 µL) was loaded in the inlet and a vacuum was applied at the outlet to fill the chamber with sample.

Alternatively, sample could be

delivered to the inlet using a syringe with appropriate fitting. The vacuum was removed and sample was left in the chamber for 10 seconds after which 50 µL of uranyl acetate stain was loaded into the inlet and carried through with vacuum. After 10 seconds, compressed air was blown into the inlet and used to dry the grid. The air also purged the thin film of buffer between the slides, enabling the device to be opened and the grid extracted via the divot with a pair of forceps. The grids were stored at room temperature in a sealed desiccator before imaging.

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Electron Microscopy and Particle Classification. EM micrographs of Kvβ2.1 were collected on a CM-120 BioTwin (Phillips, Hillsboro, OR) operating at 120 keV using a Tem-Cam F224 slow scan CCD camera (Tietz, Freising, Germany) with a magnification that resulted in a nominal pixel size of 2.88 angstroms per pixel at the specimen level. EM micrographs were uploaded to the Appion processing suite.21 Kvβ2.1 particles were picked in a semi-automatic fashion using the template picker FindEM.22

Two dimensional (2D) class averages were

generated using the maximum likelihood alignment algorithm within the Xmipp package.23 3D volumes of Kvβ2.1 were generated using the Frealign reconstruction package.24 6038 particles were used for the final reconstruction for both the device-prepared and manually-prepared samples. Both reconstructions refined to 21 Å resolution as assessed by the FSC0.143 criterion.25 The Kvβ2.1 crystal structure (PDB ID: 1ZSX), back-filtered to 50 Å, was used for the initial model. Kvβ2.1 volumes were visualized using Chimera.26

Results and Discussion The method developed achieved reproducibility in the staining process by integrating all the sample preparation steps into a single device that housed the EM grid.

Further

advantages brought to light by this device were: (1) it could be operated by hand, (2) the device was reusable, and (3) the system is amenable for future integration with on-chip valves and plumbing. The device consisted of a chamber with support barriers around the edges that held an EM grid in place during delivery of sample and stain. The support barriers were used to prevent the grid from sliding during the application of the sample. It was found that in the absence of the support barriers, the grid slid in the chamber, tearing the thin layer of carbon film, ruining the sample.

The extraction divot also permitted easy grid extraction upon

disassembly of the device. On occasion, the grid would stick to the top glass slide again 7



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ruining the carbon film and grid. Having a chamber and divot etched also on the top slide alleviated this problem. A challenge encountered was how to seal the device to ensure the sample or stain would not wick out of the channels, but would still allow the device to be opened afterwards and allow removal of the grid. Reversible sealing of the microfluidic device was achieved by derivatizing the glass surface with a fluorosilane to render the glass both fluorophilic and hydrophobic. We initially attempted to seal the device with a fluorous liquid between the slides13,19, but large fibril-like structures were observed after EM images were acquired. By using a few drops of aqueous buffer between the glass surfaces in conjunction with an aluminum manifold to hold the pieces together (Figure 3), the seal was sufficient to prevent the sample from wicking between the slides as well as allowing a syringe pump to drive solution through the device, while still enabling the device to be opened after staining. In Figure 4, the general quality of the grids prepared by hand (top row) and the microfluidic device (bottom row) were compared using low magnification images.

In both

cases, five grids were prepared consecutively and a 3x3 square area from each grid is shown. The first benefit that was observed with the use of the microfluidic device was improvements to the cleanliness of the grids. Large uneven stain features, seen as dark black regions on these images, was commonplace in the conventional method, but was mostly absent from the grids made using the microfluidic system (Figure 4). A further zoomed-out view (80 X) of two grids is shown in the Electronic Supplementary Information (ESI), Figure S1. Variability was also observed with respect to the staining. The right-most image of the top row of Figure 4 shows striations of different stain thicknesses in the grids that were prepared conventionally. The thickness of the stain will dictate the contrast and signal to noise ratio of the images. The homogeneity of the images shown in Figure 4 was quantified by measuring the RSD of the gray scale values as described in the ESI. The RSD from the grids prepared by hand was 8


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24% (Figure S2A), while the RSD from those prepared by the device was 2% (Figure S2B). It should be pointed out that the conventionally made grids were still usable at higher magnifications by zooming in on regions that did not contain these artifacts. However, the contamination and stain variations consumed functional space on the grid and may make image processing and specimen classification more difficult. At higher magnifications, the quality of the images acquired following preparation in the device was equal, if not better, to those prepared conventionally.

A magnification series

comparing both preparations is shown in Figure 5 (top row, conventional preparation; bottom row, microfluidic preparation).

As a test sample, Kvβ2.1 was used as a model biological

specimen. Kvβ2.1 is a 320 kDa cytosolic complex that directly interacts with voltage-gated potassium channels.20 It was chosen for these studies due to its relatively small size as smaller specimens often are more difficult to observe in negative stain. Again, stain artifacts were evident in the low magnification image from the grid prepared by hand (Figure 5A, left image), and uneven staining was observed in the 28,000 X magnification image (Figure 5A, middle). Nonetheless, by selectively choosing the location for higher magnification images, stained Kvβ2.1 particles can be discerned (Figure 5A, right). When using the device for grid preparation (Figure 5B), the low magnification images showed negligible contamination and a consistent stain thickness. As the magnification increased, the Kvβ2.1 particles are seen as monodisperse and evenly stained yielding high contrast particles with consistent staining from image to image. To illustrate the viability of this approach for structural biology, 2D class averages and 3D reconstructions were made from the Kvβ2.1 negative stain images produced by hand or with the device (Figure 6). Reference-free 2D class averages from device-prepared sample were qualitatively more feature rich and showed more unique orientations (Figure 6C) than the averages from the manually prepared sample (Figure 6A). This observation was consistent 9



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with 3D reconstructions of the Kvβ2.1 particles (Figure 6B and 6D). Both device-prepared and manually-prepared samples refined to 21 Å resolution, but the contours of the device-prepared sample better matched a fitted Kvβ2.1 crystal structure with only 2,320 atoms outside of the EM density (Figure 6D) as opposed to 2,904 atoms in the manually-prepared sample (Figure 6B) when both reconstructions enclosed the same total volume. Together, these analyses revealed that the data collected from manual preparation were of lower quality and showed fewer features in the 3D reconstruction (Figure 6A and 6B) as compared to the data collected from the microfluidic system (Figures 6C and 6D). One possible interpretation for the better results from the device-prepared sample is that the microfluidic chamber may have protected Kvβ2.1 from flattening during the dehydration step of grid preparation. These results validate our device-based approach and provide a foundation for future integration with other microfluidic features.

Conclusion A simple microfluidic system is presented that allows reproducible staining of EM grids with the added benefit of reduced stain artifacts. High magnification images from grids prepared by the microfluidic system showed similar image qualities as those prepared by hand. With this methodology for housing the grid, opportunities abound for more integrated systems using elastomeric materials for incorporation of valving and other microfluidic features.

For example, this system can subsequently be complemented with gradient

generators or multi-analyte perfusion and reaction timers to study both multi-variable interactions as well as reaction kinetics. This proof of principle paves the way for future added layers of complexity that can be used to uniquely investigate structural biology dynamics.

Supporting Information 10


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The Supporting Information is available free of charge on the ACS Publications website. Low magnification (80 X) images of a grid prepared in the conventional manner and by the microfluidic device; description of the method used to measure the gray scale levels of Figure 4; surface plots of the gray scale levels of Figure 4.

Acknowledgements This work was supported in part by a grant from the National Institutes of Health (R01 GM108753) to SMS, and a Developing Scholar Award from Florida State University (MGR). The authors declare the following competing financial interest(s): Authors NM, JMS, SMS, and MGR are coauthors on an invention disclosure related to portions of the work reported herein.

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Figure Captions

Figure 1. Workflow for the preparation of negative stain grid for EM. [A] The procedure for conventional sample preparation by hand is shown.

A series of five steps is used to

produce the final stained sample, but the large number of steps involving manual handling leads to variable qualities in the final sample.

Additionally, the grid is exposed to the

environment and is prone to contamination. [B] The procedure using a microfluidic platform for the sample preparation is shown. Upon loading the grid in the chamber, the sample is applied and stained within the device, minimizing variations in preparation steps. Because the grid is preserved in the device throughout the preparation stage, there is less chance for environmental contamination.

Figure 2. Device fabrication. [A] First the extraction divot was etched into the glass with subsequent etching of the grid chambers and channels. The divot allowed for an efficient means to extract the grids post-staining. [B] The top and side view of the final, fabricated device is shown. The use of the support barriers kept the grid in place during delivery of solutions.

Figure 3. Completed view of device. A final image of the EM negative stain preparation device is shown.

A metal manifold was used to align and clamp the device together.

Reservoirs permitted incorporation of pumps for automated delivery of reagents. A US quarter is shown on the left for scale.

Figure 4. Macroscopic stain features. Low magnification (130 X) images of macroscopic stain features with and without the sample prep device. Each panel represents a separately 14


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prepared grid. (Top) When prepared by hand, contamination with particulates and debris was noted. The three images on the right show striations in the stain thickness. (Bottom) When prepared with the microfluidic device, the grid was predominantly clean and only infrequent particulates were observed. Stain thickness was even throughout the grid as determined by the homogeneous image contrast.

Figure 5. Magnification series of Kvβ2.1 particles. High magnification images of samples prepared by hand [A] and with the microfluidic device [B]. The final stained images at 45,000 X magnification are comparable, with a slightly improved stain coverage when using the microfluidic device. These images are representative of the five grids that were prepared by hand and the device.

Figure 6. Comparison of 2D class averages and 3D reconstructions from TEM images acquired using manual preparation and the microfluidic device. [A] Reference-free 2D class averages from images collected on a manually prepared grid. [B] 3D reconstruction of Kvβ2.1 from the manually prepared grid. The Kvβ2.1 crystal structure (Blue, ribbon representation, PDB ID: 1ZSX) was fitted to the EM density map for comparison purposes. [C] Reference-free 2D class averages from images collected on a grid prepared within the microfluidic device. [D] 3D reconstruction from the device-prepared grid also fitted with the Kvβ2.1 crystal structure. The surface contours (gray) for both reconstructions were adjusted so that both reconstructions enclosed the same volume.

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Figure 1.

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Figure 2.

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Figure 3.

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Figure 4.

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Figure 5.

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Figure 6.

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For TOC Only.

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Interfacing Microfluidics with Negative Stain ... - ACS Publications

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