Patent Publication Number: US-10788404-B2

Title: Microscope sample preparation device

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of U.S. application Ser. No. 15/263,949, filed Sep. 13, 2016, now U.S. Pat. No. 9,958,362, which claims priority to U.S. provisional application No. 62/236,368, filed Oct. 2, 2015. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     This invention relates, generally, to the field of microscopy. More specifically, it relates to preparation of samples for electron microscopy. 
     2. Brief Description of the Prior Art 
     Preparation of high quality samples is critical for structure determination of biomolecules. Sample preparation for negative stain EM is typically done by hand and consists of a series of blotting steps of both the sample and heavy metal stain. The stain is used to introduce contrast to the images and to lock the native structure of the protein into place. 
     Conventional negative staining of samples on EM grids is the primary method used by most EM labs to evaluate their samples and can be the only method for specimen preparation of small or highly heterogeneous samples. There are almost as many protocols for making negative stain grids as there are EM labs, and researchers adhere to their own protocols that have been developed for years. For example, one protocol, as depicted in  FIG. 1 , might call for retrieving EM grid  102  using tweezers  104 , hand pipetting microliter sample  103  onto the carbon-coated copper grid  102 , hand blotting sample  103  with absorbent filter paper  106  to remove most of the liquid, hand pipetting a microliter volume of heavy metal stain  105  onto EM grid  102 , and again hand blotting EM grid  102  with absorbent paper  106  to remove excess stain. Alternately, other labs advocate transferring a grid between different drops of sample, water, and stain and then blotting. 
     There are several disadvantages to the conventional method of preparing an EM sample by hand. First, the grid and sample are open to the environment, and thus, subject to unwanted contamination. Second, the sample is exposed to air, which prevents the use of air-sensitive samples. Third, there is a large amount of variability within a single EM grid, between grids stained by a single user, and even more variability between users. 
     The significance of the variability is so great that the preparation is frequently compared to an art-form. Typically, this problem is overcome by arbitrarily sampling regions of the grid until one can be found where the specimen subjectively looks the best. However, this irreproducibility can lead to bias, staining artifacts, and poor signal-to-noise, which can degrade image resolution and information content. 
     A few examples of microfluidic systems have been published for sample preparation of cryo-EM samples or for negative-stained samples (Jain et al., 2012; Kemmerling et al., 2012; Lu et al., 2009; Lu et al., 2014). However, these were highly specialized devices which would need to be redesigned for specific samples. Additionally, the designs relied on sample spraying techniques that can be disruptive to the structure of macromolecular complexes. Proteins are often prone to denaturation at the air/water interface, and spraying techniques are limited to samples that are relatively insensitive to the interface, such as well-behaved samples like ribosomes and GroEL. Ultimately, these protocols offered no quantitative assessment of the sample. Id. 
     Collectively the technologies mentioned above may have some merit to improve throughput and reproducibility of sample preparation, but they required the utilization of robotics thus hindering the methods&#39; translation. Moreover, these advances are auxiliary additions to the same preparation workflow. 
     Accordingly, what is needed is a device and method for preparing a microscope sample that remove or minimize user variation and prevent exposure of the sample to air. However, in view of the art considered as a whole at the time the present invention was made, it was not obvious to those of ordinary skill in the field of this invention how the shortcomings of the prior art could be overcome. 
     All referenced publications are incorporated herein by reference in their entirety. Furthermore, where a definition or use of a term in a reference, which is incorporated by reference herein, is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply. 
     While certain aspects of conventional technologies have been discussed to facilitate disclosure of the invention, Applicants in no way disclaim these technical aspects, and it is contemplated that the claimed invention may encompass one or more of the conventional technical aspects discussed herein. 
     The present invention may address one or more of the problems and deficiencies of the prior art discussed above. However, it is contemplated that the invention may prove useful in addressing other problems and deficiencies in a number of technical areas. Therefore, the claimed invention should not necessarily be construed as limited to addressing any of the particular problems or deficiencies discussed herein. 
     In this specification, where a document, act or item of knowledge is referred to or discussed, this reference or discussion is not an admission that the document, act or item of knowledge or any combination thereof was at the priority date, publicly available, known to the public, part of common general knowledge, or otherwise constitutes prior art under the applicable statutory provisions; or is known to be relevant to an attempt to solve any problem with which this specification is concerned. 
     BRIEF SUMMARY OF THE INVENTION 
     The long-standing but heretofore unfulfilled need for a device and method for preparing an EM sample that remove or minimize user variation and prevent exposure of the sample to air is now met by a new, useful, and nonobvious invention. 
     The present disclosure is directed to a microfluidic sample preparation device, preferably for electron microscopy. Various embodiments may allow for sealing of an EM grid, facile and reproducible delivery of sample, followed by delivery of subsequent solutions that may be negative stains or other biological samples. According to various embodiments, the EM grid may be contained in a grid chamber using a plurality of support barriers and may be gently and easily removed with an extraction divot disposed at least partially below the grid chamber. The fluid may be directed to the grid using channels integrated into the platforms of the microfluidic system. Single or multiple grids may be housed in a platform, which may allow for high throughput testing. For example, a device with nine grids may require less than 1 μL of sample per grid. This may allow more screening in circumstances where sample quantity is limited. 
     Various embodiments comprise a device to deliver air-sensitive samples to an EM grid via an air tight chamber. This technology fills a niche for which no similar technology currently exists. Traditionally, EM staining is a tedious and time consuming task that offers little reproducibility. The conventional staining method is done in a manual fashion in an open environment, which may introduce contamination, is not viable for air-sensitive samples, and may be plagued by user-to-user variations. 
     Various embodiments may comprise two platforms, which may be comprised of etched pieces of glass, aligned to one another forming an internal chamber sized to house the EM grid. Integrated microfluidic channels allow the sample to be delivered to the grid in an automated fashion. Timing may be introduced using automated or integrated valves allowing time-dependent snapshots of the sample. 
     The long-standing but heretofore unfulfilled need for an EM sample preparation device capable of sealing of an EM grid from exposure to air, facile and reproducible delivery of sample, followed by delivery of subsequent solutions that may be negative stains or other biological samples, and methods for its use, are now met by a new, useful, and nonobvious invention. 
     These and other important objects, advantages, and features of the invention will become clear as this disclosure proceeds. 
     The invention accordingly comprises the features of construction, combination of elements, and arrangement of parts that will be exemplified in the disclosure set forth hereinafter and the scope of the invention will be indicated in the claims. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       For a fuller understanding of the invention, reference should be made to the following detailed description, taken in connection with the accompanying drawings, in which: 
         FIG. 1  depicts the conventional method of preparing a sample for EM imaging highlighting the degree of manual interaction required to complete the preparation. 
         FIG. 2  is an overhead view of an embodiment of a bottom platform. 
         FIG. 3  is an overhead view of the embodiment provided in  FIG. 2  with the addition of a microscope grid. 
         FIG. 4  is an embodiment of the novel method of preparing an EM sample. 
         FIG. 5A  is a section view of section line A-A in  FIG. 2  with the inclusion of the top platform. 
         FIG. 5B  is a section view of section line A-A in  FIG. 3  with the inclusion of the top platform. 
         FIG. 6A  depicts an example of functionalized top and bottom platforms using fluoropolymer. 
         FIG. 6B  depicts an example of functionalized top and bottom platforms using alkane chains. 
         FIG. 6C  depicts an example of functionalized top and bottom platforms using biotin-streptavidin reaction. 
         FIG. 7  is an exemplary embodiment of a mechanical clamp for securing the top and bottom platforms together. 
         FIG. 8  is an embodiment of a bottom platform comprising multiple grid chambers. 
         FIG. 9  provides several embodiments of a bottom platform having multiple grid chambers (top two rows) and fluidic timers (bottom row). 
         FIG. 10  is an exemplary embodiment of bottom platform illustrating the possibility of combining multiple sets of grid chambers with each set coupled to a microfluidic timer. 
         FIG. 11  is an exemplary embodiment of bottom platform illustrating the use of a gradient generator with multiple grid chambers. 
         FIG. 12  is an exemplary embodiment of bottom platform illustrating the use of a gradient generator with multiple grid chambers and an additional sample inlet. 
         FIG. 13  depicts the use of an embodiment of a bottom platform with a separate gradient generator platform. 
         FIG. 14  depicts an exemplary method for manufacturing the platforms. The photomasks on the left half are used to fabricate the platforms shown on the right using photo lithography. In order to obtain two different depths, separate exposure and etching steps are implemented. First an extraction divot is etched into the glass and subsequently the grid chambers and channels are added. This may allow for the integration of plumbing and an extraction pathway. 
         FIG. 15  illustrates low magnification (130×) images of artifacts resulting from conventional manual preparation (top row) and from preparation according to the present invention (bottom row). 
         FIG. 16  provides a comparison between the samples prepared by hand (row A) and the samples prepared by the present invention (row B) by way of high magnification images. 
         FIG. 17  illustrates two-dimensional reference free class average of Kvβ2.1 particles picked from micrographs prepared using the sample preparation device of the present disclosure. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     In the following detailed description of the preferred embodiments, reference is made to the accompanying drawings, which form a part thereof, and within which are shown by way of illustration specific embodiments by which the invention may be practiced. It is to be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the invention. 
     The present invention includes a novel device and method for preparing a sample for EM imaging. The present invention provides an innovative approach to 1) create a robust and reproducible method for negative staining of EM grids; 2) automate preparation of multiple samples simultaneously; and 3) integrate quantitative assessments of sample stability. The technologies described herein are suitable for applications across the field of EM and will have a significant impact on a multitude of other biological systems. 
     As shown in  FIG. 2 , an embodiment of the novel apparatus includes bottom platform  102  having inlet  104 , outlet  106 , and grid chamber  108  disposed between inlet  104  and outlet  106 . Grid chamber  108  is in fluid communication with inlet  104  and outlet  106  through inlet channel  110  and outlet channel  112 , respectively. Bottom platform  102  further includes a plurality of grid support barriers  114  disposed in grid chamber  108  and extraction divot  116  located at least partially within the outer perimeter of grid chamber  108 . Bottom platform  102  also preferably includes at least one alignment marker  118 . 
     Bottom platform  102  is preferably flat and may be comprised of generally any material including, but not limited to glass, metals, ceramics, plastics, silicon, and elastomers. Fabrication may be achieved through known processes for creating microfluidic devices including, but not limited to lithography, 3D printing, hot embossing, and milling. The fabrication method is at least partially dependent on the structural features of the bottom platform and the material included in the bottom platform. 
     As depicted in  FIG. 3 , EM grid  120  is intended to rest in grid chamber  108  within the perimeter created by support barriers  114 . The exemplary embodiment shows grid chamber  108  as hexagonal in shape, but any shape is contemplated. Support barriers  114  are strategically arranged to create a perimeter of a proper size for receiving and securing EM grid  120  within grid chamber  108 . The support barriers  114  minimize grid movement while a pressure-driven fluid flow passes through grid chamber  108 . The fragile nature of EM grid  120  and its carbon coating require that EM grid  120  remain generally static as the pressurized flow overtakes EM grid  120 . It was discovered that the absence of support barriers  114  resulted in EM grid  120  sliding around grid chamber  108  upon application of fluid, thus causing the carbon film to tear. 
     In the depicted embodiment, support barriers  114  are arranged in a generally circular fashion. The overall pattern/arrangement of barriers  114  is dependent on the shape of EM grid  120 , and thus, may be arranged in a different pattern to secure EM grid  120  within grid chamber  108 . 
     In an embodiment, the arrangement of barriers  114  must include a gap between barriers  114  sufficiently sized to account for extraction divot  116  disposed within the gap. Extraction divot  116  allows for easy removal of the fragile EM grid  120  using sharp-tipped forceps or a similarly designed device. Extraction divot  116  further aids in preventing EM grid  120  from sticking to bottom platform  102  when a user attempts to remove EM grid  120  from grid chamber  108 . 
     Referring now to  FIG. 4 , bottom platform  102  is designed to receive top platform  122 . In its simplest form, top platform  122  is a simple barrier secured in overlying relation to bottom platform  102 . Top platform  122  includes inlet aperture  124  and outlet aperture  126  to provide a fluid passage to the inlet  104  and outlet  106 , respectively. Upon loading EM grid  120  into chamber  108 , all the externally conducted steps shown in  FIG. 1  can be performed in a temporarily sealed environment minimizing the exposure and the variability resulting from manual EM grid handling. EM grid  120  is preferably sealed into chamber  108  using bottom platform  102  and top platform  122  to ensure accurate and reproducible flow patterns for sample preparation 
     An embodiment of the novel method of EM sample preparation, using an embodiment of the novel device, is illustrated in  FIG. 4 . The sample is prepared according to the five general steps executed from left to right. In the first step, EM grid  120  is deposited within support barriers  114  located in grid chamber  108  in bottom platform  102 . Top platform  122  is secured in overlying relation to bottom platform  102  with inlet and outlet apertures  124 ,  126  respectively aligned with inlet  104  and outlet  106 . In the second step, sample  103  is inserted into the system through inlet  124  using a fluid application device, such as pipette  128 . The third step includes inserting stain  105  into the system using a fluid application device, such as pipette  128 . The fourth step comprises drying the system using a gas inserted into aperture  124 . Once EM grid  120  is adequately dried, top platform  122  is removed. EM grid  120  is removed from bottom platform  102  in the final step. Through the use of the microfluidic platforms, all the preparation steps are integrated into a single system overcoming the irreproducibility obstacles prevalent in EM sample preparation. 
     In experimental testing, EM grid  120  would often stick to the top platform  122  when top platform  122  was removed in step five above. The fragility of the EM grid  120  became an issue when attempting to remove EM grid  120  from top platform  122 . As a result, a preferred embodiment of the present invention, as depicted in  FIGS. 5A-5B , includes top platform  122  having etching in its lower surface that is similar to the etching in the upper surface of bottom plate  102 .  FIG. 5  provide a sectional view along section line A-A shown in  FIGS. 2-3  and illustrates how fluid, depicted by arrows  130 , passes into the system through inlet aperture  124  and then out of the system through outlet aperture  126 . The figures depict inlet and outlet channels  110 ,  112  as having a depth generally equal to the depth of grid chamber  108 , but the channels can be any size relative to the size of the grid chamber. 
     In an embodiment, the top platform is simply an inverted bottom platform to reduce manufacturing efforts. In yet another embodiment, top platform  122  simply includes an extraction divot disposed in the lower surface to allow a user to remove an EM grid stuck to the top platform using an extraction tool. 
     To ensure proper alignment of the top and bottom platforms  122 ,  102 , an embodiment of the present invention may include each platform having an alignment marker  118  (See e.g.  FIGS. 8-11 and 14 ). In an embodiment, the alignment marker on the top platform is designed to interact with the alignment marker on the bottom platform to prevent respective movement of the two platforms in the transversal and longitudinal directions. The alignment markers are disposed at least on the upper surface of the bottom platform and on the lower surface of the top platform. 
     As is discussed below in the experiment section, there was a substantial benefit from sealing the top and bottom platforms. Therefore, a preferred embodiment includes a sealed system to ensure accurate and reproducible flow patterns and also to protect the sample from the air-water interface. At the end of the sample preparation, however, the EM grid must be removed for visualization by EM. Therefore, a reversible sealing method for the system is required. 
     The system may employ functionalized materials for sealing the system. The different substrates can be functionalized in a panel of ways to provide different sample solvent/matrix compatibility. For example, functionalization methods include, but are not limited to fluorine functionalized surfaces ( FIG. 6A ), alkane functionalized surfaces ( FIG. 6B ), biotin-streptavidin ( FIG. 6C ), carbon coating by sputtering, plasma oxidized, and carbon coated and plasma oxidized. It should be noted that the plasma oxidation rendered the surface hydrophilic and led to rapid transfer of solution between slides. This strategy can be used to accelerate and guide the passage of fluid through the channels. 
     Alternatively, the bottom and top platforms may be temporarily sealed using mechanical devices, including, but not limited, to a binder clip, clamps, manifolds that have a built in screw/clamping system, magnets, or a device integrated into the top and bottom platforms. In an embodiment, a gasket is disposed between the sandwiched top and bottom platforms to seal the grid chamber and channels. 
     A particular example of a mechanical clamping device is provided in  FIG. 7 . The device includes base platform  202  having recess  204  in which bottom and top platforms  102 , 122  (not shown) are intended to rest. Clamping arms  206  force platforms  102 ,  122  together when in the securing configuration shown. Clamping arms  206  may be opened by rotating the top assembly arm  208  in an outboard direction, which removes the clamping force on the platforms  102 ,  122 . Bottom and top platforms  102 , 122  can then be easily removed from recess  204 . 
     Fluid (sample, stain, rinse, etc.) delivery and control can take on various forms depending on the type of sample being prepared and the goals of the preparation. The method shown in  FIG. 4  relies on pipette  128  for the delivery of the sample and stain to the grid chamber  108 . Other manual fluid delivery options include, but are not limited to, a syringe pump, a surface tension/capillary action driven fluid, a vacuum at the outlet aperture  126 , peristatic pumps, and tilting the system to rely on gravity to drive the fluid from inlet aperture  124  to outlet aperture  126 . Many of these pressure-driven systems would be implemented “off-chip,” but some could also be implemented directly within the microfluidic system using micro-fabrication techniques. Some of these micro-fabrication techniques require multiple layers of the device, which is compatible with this system. 
     Fluid delivery may, alternatively, be automated. These automated measure include, but are not limited to, “on-chip” valves or pumps, electroosmotic flow—voltage driven, and the use of a pre-filled tube having pre-measured segments of the sample, stain, rinse, etc. 
     In an embodiment, the fluid delivery mechanisms are coupled to the inlet aperture, and preferably also the outlet aperture, to maintain a sealed environment and ensure direct application into the system. The coupling can be achieved according to any method known by a person having ordinary skill in the art for securing a fluid delivery system to a microfluidic platform, including, but not limited to, press fitting tubes into the apertures, securing nano-port fitting in the apertures, and bonding a threaded reservoir into the apertures and then interfacing the reservoir with tubing and fittings. 
     The removal of manual handling and manual application of fluids during EM sample preparation, which is now possible with the present invention, opens the field of EM sample preparation to both high throughput production, microfluidic timers, and microfluidic gradient generators. It should be noted that the microfluidic features can be on the same device or a separate platform. A separate device having the microfluidic features is may be desirable for easily interchanging the preparation platforms. 
     Referring now to  FIG. 8 , an embodiment of the present invention provides a high throughput system for simultaneous preparation of multiple EM samples and multi-screening from the same sample. Bottom platform  102  includes twelve separate grid chambers  108  each with their own inlet  104  and outlet  106 . Obviously alignment of bottom platform  102  and top platform (not shown) is critical when several grid chambers  108  are disposed in each platform. Thus, each platform includes alignment marker  118 . Preferably alignment marker  118  on bottom platform  102  is designed to interconnect with an alignment marker on the top platform to prevent lateral and longitudinal movement between the two platforms. 
     Referring now to  FIG. 9 , an embodiment may include multiple grid chambers in fluidic communication and arranged in series or in parallel to create microfluidic timers for fluid delivery/fluid interaction and/or to increase the production rate of EM samples. The first two rows of bottom platforms  102  provides various examples of how multiple fluidly coupled grid chambers might be arranged. The third row of bottom platforms  102  depicts exemplary embodiment of microfluidic timers used with a single grid chamber  108 . As illustrated in the first two rows, each set of fluidly coupled grid chambers includes a single inlet  104  and a single outlet  106 . The grid chambers  108  may be arranged in any configuration and may be coupled through various intermediate channels  132 . Furthermore, each grid chamber  108  preferably includes a plurality of support barriers  114  for securing an EM grid within a particular grid chamber  108 . Likewise, each grid chamber  108  preferably includes an extraction divot  116  for removing an EM grid from a particular grid chamber  108 . These fluidly coupled grids significantly improve the speed at which samples can be prepared compared to the conventional manual preparation. 
     The third row of bottom platforms  102  depicts exemplary embodiments of microfluidic timers used with a single grid chamber. Two inlets  104  may be used to create timed reactions, which occur while the injected fluids pass through inlet channel  110  to grid chamber  108 . By using different length inlet/mixing channels  110 , the time for the sample to reach grid chambers  108  and for the reaction to occur can vary. 
     Referring to  FIG. 10 , an embodiment of platform  102  may include grid chambers  108  in series with each series coupled to a microfluidic timer comprising two inlets  104  converging to inlet channel  110 . This arrangement produces different incubation and mixing times and allows for complexes and reactions to reach different states before entering each sequential grid chamber. The time points of reaction can be recorded in a “snap-shot” by sequentially applying the sample to the series of grids to capture time-dependent processes. Likewise, fixative could be delivered at specific time increments to trap complexes at different stages of activity. 
     An embodiment, as shown in  FIG. 11 , may employ a gradient generator to mix a panel of different conditions to study their effect on a structure of a complex/molecule. This can also be used to study time-dependent assembly mechanisms and reactions. A multitude of samples can be created by adjusting the variable parameters, which include, but are not limited to, the number of inputs and complex gradients, the mixing times and reaction times, the flow rates, and the amount of grids that can be screened. 
     The embodiment of bottom platform  102  provided in  FIG. 11  includes a gradient between inlets  104  and grid chambers  108 . As shown, fluid A is inserted into the left inlet and fluid B is inserted into the right inlet. The gradient creates varying concentrations of the two fluids in each grid chamber  108 . The leftmost grid chamber has a low concentration of fluid B and a high concentration of fluid A. The reverse is true at the rightmost grid chamber and varying concentrations can be found in the grid chambers between the two outer grid chambers. 
     An embodiment shown in  FIG. 12  includes an additional stain delivery inlet  134  to apply stain directly to the grid instead of flowing it through the gradient channels. The embodiment may also include soft lithography valving (V 1 , V 2 , VS 1 -VS 12 ), which are used to shunt the flow only for the desired paths. This prevents backflow of stain for the sample delivery and allows the devices to be reused. 
     In an embodiment as shown in  FIG. 13 , bottom platform  102  may be separate from gradient platform  140 . Fluids are first inserted into gradient inlets  144 , and after mixture, the fluid exists gradient outlets  146 . Outlets  146  are fluidly coupled (not depicted) to inlets  104  for each grid chamber  108  in bottom platform  102 . A separate gradient platform allows for simplistic and efficient replication of use. 
     Experimental Research 
     The reagents used in the experiment included nitric acid, hydrogen peroxide, hydrofluoric acid, sodium hydroxide, ethanol, and (tridecafluoro-1,1,2,2-tetrahydrooctyl) trichlorosilane. Ultrapure deionized water was used for all solutions and sample preparation, and KvBeta was recombinantly expressed in and purified using known methods. 
     Fabrication. In order to get multiple depth steps in a single platform, the microfabrication steps were repeated twice on the same wafer ( FIG. 14 ). First, borofloat photoresist wafers with a layer of AZ1500 positive photoresist on a chrome layer were exposed to 18 mW cm −1  collimated UV radiation for 15 seconds through mylar, patterned photomask  150  containing extraction divot  116 . The exposed photoresist was removed with AZ 400K Developer, diluted 1:3 in H 2 O. The bottom chrome layer was then developed with a chrome etchant solution. The exposed glass was then etched in a 5:1:3 (v:v:v) mixture of H2O:HNO3:HF to a 40 μm depth. For the second step, the wafer was aligned with photomask  152  containing the design with the channels  110 ,  112  and grid chamber  108 . The features were developed and etched again to a depth of 110 μm. This produced a channel depth of 70 μm with an extraction divot of 40 μm. The total chamber volume was 3 μL. 
     The same process was repeated for another chip, developing the mirror reflection of the design features, this would serve as the top complement platform. All dimensions of the channels were verified using a P-15 stylus profilometer. Fluid access holes were drilled with a 1.1 mm diamond-tipped drill bit, after which the remaining photoresist and chrome were removed. The finished top platform was then fitted with a nanoport that was attached using epoxy. 
     For the surface modification, the glass was cleaned by submerging in 5 M NaOH for 10 minutes. The surface was rinsed with water and dried with N 2 . Subsequently the platforms were oxidized in a plasma cleaner for 2 minutes. Immediately after, the platforms were placed in a vacuum desiccator and (tridecafluoro-1,1,2,2-tetrahydrooctyl) trichlorosilane was deposited using a known method. Subsequently, the platforms were rinsed with water, dried with N 2 , and stored in clean petri dishes at room temperature until use. 
     Sample Preparation. The bottom glass platform was placed into the aluminum manifold. A carbon coated, copper grid was rendered hydrophilic using a plasma cleaner and gently placed into the device chamber. Several 20 μL drops of buffer (20 mM Tris pH 8.0, 150 mM KCl, 1 mM 2-mercaptoethanol) were distributed around the non-etched parts of the glass platforms. The top platform was aligned using the manifold and lowered on top of the bottom platform, in process displacing the buffer and creating a thin sealing layer. The top of the manifold was attached and screwed down to seal the device. 20 μL of sample was loaded in the inlet and a vacuum was applied at the outlet to fill the chamber with sample. Alternatively, the sample could be delivered to the inlet using a syringe with appropriate fitting. The vacuum was removed and the sample was left in the chamber for 10 seconds after which 50 μL of uranium 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 platforms, enabling the device to be opened and the grid extracted via the divot with a pair of forceps. 
     Electron Microscopy and Reconstruction. EM micrographs of Kvβ2.1 were collected on a CM-120 BioTwin operating at 120 keV at room temperature with a nominal pixel size of 2.88 angstroms per pixel equipped with a Tem-Cam F224 slow scan CCD camera. EM micrographs were uploaded to the Appion processing suite. Kvβ2.1 particles were picked in a semi-automatic fashion using the template picker FindEM. Two dimensional (2D) class averages were generated using the maximum likelihood alignment algorithm within the Xmipp package. 
     Results and Discussion. Reproducibility in the staining process was attained by integrating all the sample preparation steps into a single device that housed the EM grid ( FIG. 4 ). Further advantages realized through various embodiments include, but are not limited to: (1) simplicity of use and no requirement for further accessories, (2) easily fabricated and reusable and (3) the system is amenable for integration of further “on-chip” valving and plumbing. 
     After grid  120  was placed in the chamber, it was confined by support barriers  114  to prevent sliding and flow induced friction that could tear the carbon film containing the sample. In absence of support barriers, EM grid  120  became torn upon application of the sample. The support barriers preserved the grid integrity completely. Extraction divot  116  also permitted easy grid access and extraction. Upon disassembling of the device, grid  120  would occasionally stick to top platform  122 . To address this problem, a divot  116  and set of barriers  114  were etched into top platform  122  as well, permitting extraction without sticking. 
     In order to interface microfluidics with EM preparation, reversible sealing of the grid inside a device is required. This was achieved by silanizing the glass surface with (tridecafluoro-1,1,2,2-tetrahydrooctyl) trichlorosilane, producing an eight carbon long, fluorinated moieties on the surface. This rendered the glass both fluorophilic and hydrophobic. The fluorophilic surfaces of the two glass platforms interact with each other and form a non-covalent interaction, sufficient to seal the chip reservoir and channels. The fluorophilic surfaces may prevent the sample from wicking in between the platforms, yet the force is sufficient to incorporate syringe pump integration. The platforms appeared to seal better when a film of buffer was introduced between the platforms. This is believed to be due to an alignment of the fluorinated chains that might be in a collapsed state when dehydrated. In some embodiments, longer alkane chains with higher amount of fluorination may be used to strengthen the glass bond while maintaining reversible sealing. 
     Besides the improvement in the reproducibility of the staining, the cleanliness of the grids was improved and found to be free of particulate contamination. When making grids, contamination known as “crud” is the norm yet it consumes functional space on the grid and has the potential to interfere with the sample application and staining. By incorporating all the steps into a single device as shown in  FIG. 4 , contamination was effectively eliminated. 
     The contamination of the grids was compared between the hand prepared grids and the grids prepared using the device of the present invention, which is depicted in  FIG. 15 . All images were taken from separately prepared grids, however, the grids were prepared using the same sample and on the same day. The second row of images corresponds to the grids prepared using the present invention. As is shown, the grids are predominantly clean, only infrequent particulates are observed, and the stain thickness is even throughout the grid. The top row of images corresponds to grids prepared by hand under the prior art methodology. When prepared by hand, abundant contamination with particulates and debris are evident. In addition, vast differences between hand prepared grids, including striations of different stain thicknesses produced during the drying process, illustrate the variability in the staining process. In contrast, the grids, produced using the device of the present disclosure, were reproducibly prepared, with similar stain thickness and in absence of contamination. Clearly the present invention aids in sample preparation and will help transition this technology to non-expert users. 
     The collective quality of the images acquired following preparation in the device of the present invention was equal if not better to those prepared by hand. A magnification series comparing both preparations is shown in  FIG. 16 . The sample contamination is evident in the low magnification image prepared by hand (first image in row A). Nonetheless, stained KvBeta particles can be discerned at the higher magnifications. When using the device of the present invention, depicted in row B, the particles appear to be more evenly distributed with a denser stain. As the magnification increases, the Kvβ2.1 particles are seen as monodisperse and evenly stained yielding strong signal to noise. The cleaner surfaces provided with the device may increase the available area for imaging, minimize variability, and increase reproducibility of the staining methods. The final stain images at 52 k magnification are comparable, with slightly improved stain coverage when using the device. 
     To illustrate the viability of this approach for structural biology, a 2D class average of the Kvβ2.1 complex was performed ( FIG. 17 ). The reconstruction reveals structural motifs comparable to methods performed by hand and validates the coupling of microfluidics with EM sample preparation. 
     Glossary of Claim Terms 
     Fluid Delivery Mechanism: is a device configured to transfer fluid from one location to another location. 
     Gradient Generator: is a plurality of fluidic channels designed to increase or decrease the concentration of a fluid observed in passing from one gradient outlet to another gradient outlet. 
     Microfluidic Timer Channel: is a fluidic channel having an indirect extended route between an inlet and the grid chamber. 
     Platform: is a generally rigid material, such as glass. 
     Stain: is a fluid used to artificially highlight tissue, microorganisms, and other biological structures for viewing, typically under a microscope. 
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     Where a definition or use of a term in a reference, which is incorporated by reference herein, is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply. 
     The advantages set forth above, and those made apparent from the foregoing description, are efficiently attained. Since certain changes may be made in the above construction without departing from the scope of the invention, it is intended that all matters contained in the foregoing description or shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense. 
     It is also to be understood that the following claims are intended to cover all of the generic and specific features of the invention herein described, and all statements of the scope of the invention that, as a matter of language, might be said to fall therebetween.