Abstract:
This disclosure provides systems, methods, and apparatus related to liquid flow cells for microscopy. In one aspect, a device includes a substrate having a first and a second oxide layer disposed on surfaces of the substrate. A first and a second nitride layer are disposed on the first and second oxide layers, respectively. A cavity is defined in the first oxide layer, the first nitride layer, and the substrate, with the cavity including a third nitride layer disposed on walls of the substrate and the second oxide layer that define the cavity. A channel is defined in the second oxide layer. An inlet port and an outlet port are defined in the second nitride layer and in fluid communication with the channel. A plurality of viewports is defined in the second nitride layer. A first graphene sheet is disposed on the second nitride layer covering the plurality of viewports.

Description:
RELATED APPLICATIONS 
       [0001]    This application claims priority to U.S. Provisional Patent Application No. 62/034,597, filed Aug. 7, 2014, which is herein incorporated by reference. This application is related to U.S. patent application Ser. No. 14/524,866, filed Oct. 27, 2014, which is herein incorporated by reference. 
     
    
     STATEMENT OF GOVERNMENT SUPPORT 
       [0002]    This invention was made with government support under Contract No. DE-AC02-05CH11231 awarded by the U.S. Department of Energy, under Award No. HDTRA1-13-1-0035 awarded by the Defense Threat Reduction Agency (DTRA), and under Grant No. EEC-0832819 awarded by the National Science Foundation (NSF). The government has certain rights in this invention.  
     
    
     TECHNICAL FIELD 
       [0003]    This disclosure relates generally to liquid flow cells for microscopy and more particularly to liquid flow cells for transmission electron microscopy. 
       BACKGROUND 
       [0004]    Transmission electron microscopy (TEM) provides ultrahigh resolution imaging of samples, surpassing the diffraction limit offered by optical microscopy. Due to the ultrahigh vacuum environment associated with TEM, the imaging of liquid samples or samples (e.g., nanoparticles or biological samples) suspended in a liquid (i.e., in situ liquid TEM) is not straightforward. In recent years, the development of in situ flow cells generally comprising a thin spacer sandwiched between two chips with thin (less than about 100 nanometer (nm)) silicon nitride membranes has led the field of in situ liquid TEM. The liquid sample is isolated from the ultrahigh vacuum environment by the spacer and is imaged through the silicon nitride membrane. Metallic contacts can be used to produce electrical biases across the liquid sample. 
         [0005]    In situ liquid TEM imaging has found applications in a wide number of fields and has been used to study chemical and electrochemical processes, biological structures, and nanoparticle growth and dynamics, all at the nanoscale. However, due to the thickness and material properties of silicon nitride, some of the in situ flow cells suffer from decreased resolution and contrast, charging effects, and increased damage to the sample from electrons scattered by the silicon nitride. The low thermal conductivity of silicon nitride can also result in heating of the sample. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0006]    Details of one or more embodiments of the subject matter described in this specification are set forth in the accompanying drawings and the description below. Other features, aspects, and advantages will become apparent from the description, the drawings, and the claims. Note that the relative dimensions of the following figures may not be drawn to scale. 
           [0007]      FIG. 1  shows an example of a flow diagram illustrating a manufacturing process for a liquid flow cell. 
           [0008]      FIGS. 2A-2I  show examples of schematic illustrations of a liquid flow cell at various stages in the manufacturing process. 
           [0009]      FIG. 2J  shows an example of a cross-sectional schematic illustration of a liquid flow cell. 
           [0010]      FIG. 3  shows an example of a flow diagram illustrating a manufacturing process for a liquid flow cell having a top plate. 
           [0011]      FIGS. 4A-4G  show examples of schematic illustrations of a liquid flow cell having a top plate at various stages in the manufacturing process. 
           [0012]      FIG. 5  shows an example of a top-down schematic illustration of a liquid flow cell including electrodes. 
           [0013]      FIG. 6  shows an example of a cross-sectional schematic illustration of a liquid flow cell including two graphene sheets. 
       
    
    
     DETAILED DESCRIPTION 
       [0014]    Reference will now be made in detail to some specific examples of the invention including the best modes contemplated by the inventors for carrying out the invention. Examples of these specific embodiments are illustrated in the accompanying drawings. While the invention is described in conjunction with these specific embodiments, it will be understood that it is not intended to limit the invention to the described embodiments. On the contrary, it is intended to cover alternatives, modifications, and equivalents as may be included within the spirit and scope of the invention as defined by the appended claims. 
         [0015]    In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. Particular example embodiments of the present invention may be implemented without some or all of these specific details. In other instances, well known process operations have not been described in detail in order not to unnecessarily obscure the present invention. 
         [0016]    Various techniques and mechanisms of the present invention will sometimes be described in singular form for clarity. However, it should be noted that some embodiments include multiple iterations of a technique or multiple instantiations of a mechanism unless noted otherwise. 
       Introduction 
       [0017]    Graphene has been shown to be a good membrane material for in situ liquid TEM due to its electron transparency, electrical properties, and thermal properties. Graphene liquid cells previously developed, consisting of pockets of solution trapped between two sheets of graphene, have enabled atomic resolution imaging of samples in situ, and show a complete absence of the charging effects seen in silicon nitride (Si 3 N 4 ). 
         [0018]    Besides its high electron transparency, another advantage of using graphene layers is the surface inertness of graphene. This inertness is expected to allow the encapsulation of free floating biological samples without them being nonspecifically absorbed onto a liquid cell surface. Additionally, recent work suggests that graphene membranes may have a stabilizing effect when imaging biological samples, which may reduce damage to specimens due to the electron beam. 
         [0019]    Despite these advantages, the current fabrication methods of graphene liquid cells may not allow for liquid flow and may lack contacts for in situ electrochemical studies. Further, current fabrication methods may require a great deal of expertise. The resulting graphene liquid flow cell also may be non-reusable. Difficulty in getting 100% intact graphene sheets over large areas makes simply replacing silicon nitride with graphene in current liquid flow cell designs impractical. Further, the low bending stiffness of graphene compared to tensioned silicon nitride results in increased liquid thickness under vacuum, which may negate the advantage offered by the atomically thin graphene. This leaves silicon nitride liquid flow cells as a currently viable option for in situ liquid TEM studies. 
         [0020]    Described herein are embodiments of a graphene on nitride (e.g., silicon nitride) flow cell and methods of fabrication thereof. Such a flow cell combines advantages offered by both graphene and silicon nitride. The channel in such a flow cell may be capable of sustained liquid flow and can be electrical contacted. By fully integrating nanofluidic channels (e.g., with widths of about 500 nanometers (nm) to 2 microns) fabricated within a single substrate, the bowing of the liquid flow cell under vacuum may be significantly reduced (e.g., to about 30 nm or less). This may enable improved resolution and contrast in TEM images. Such liquid flow cells also may be reusable. Further, the fabrication methods described herein can be performed prior to the introduction of a liquid sample to the liquid flow cell and may require little or no assembly by the user. 
         [0021]    The flow region in embodiments of the liquid flow cells described herein may include a nanofluidic channel etched in an oxide layer (e.g., silicon oxide) which is sandwiched between thin (e.g., about 50 nm thick) silicon nitride layers. The imaging region of a liquid flow cell may include patterned perforations (e.g., having dimensions of about 60 nm to 500 nm, or about 300 nm) in silicon nitride sealed by graphene. These graphene covered holes may serve as viewports for electron imaging. Prior to graphene sealing, the graphene covered holes may serve as etch vias for defining the nanofluidic channel in the silicon oxide. 
       Methods and Devices 
       [0022]      FIG. 1  shows an example of a flow diagram illustrating a manufacturing process for a liquid flow cell.  FIGS. 2A-2I  show examples of schematic illustrations of a liquid flow cell at various stages in the manufacturing process shown in  FIG. 1 . In the method of fabricating a liquid flow cell, patterning techniques, including masking as well as etching processes, may be used to define the shapes of the different components of the liquid flow cell. 
         [0023]    Starting at block  105  of the method  100  shown in  FIG. 1 , a first oxide layer is formed on a first surface of a substrate and a second oxide layer is formed on a second surface of the substrate. In some embodiments, the first oxide layer and the second oxide layer are formed simultaneously. In some embodiments, the first oxide layer and the second oxide layer are formed in two separate process operations. In some embodiments, the substrate comprises a semiconductor. In some embodiments, the substrate comprises silicon. In some embodiments, the substrate comprises a double side polished semiconductor wafer. When the substrate comprises silicon, the oxide layer may be silicon oxide and the silicon oxide may be thermally grown on the silicon substrate. In some embodiments, the oxide may be deposited on the substrate using a chemical vapor deposition (CVD) technique or a physical vapor deposition (PVD) technique. In some embodiments, the substrate is about 150 microns to 450 microns thick, or about 300 microns thick. In some embodiments, dimensions of the first surface and the second surface of the substrate are about 2 millimeters (mm) to 8 mm by about 2 mm to 8 mm, or about 4 mm by about 6 mm. In some embodiments, the first and the second oxide layers are each about 50 nm to 300 nm thick, or about 200 nm thick. 
         [0024]    At block  110  of the method  100 , a first nitride layer is deposited on the first oxide layer and a second nitride layer is deposited on the second oxide layer. In some embodiments, the first nitride layer and the second nitride layer are deposited simultaneously. In some embodiments, the first nitride layer and the second nitride layer are deposited in two separate process operations. In some embodiments, the first and the second nitride layers are deposited with a CVD process or a low pressure CVD process. In some embodiments, the first and the second nitride layers comprise silicon nitride. When silicon nitride is deposited with low pressure CVD, the silicon nitride layer may be a layer having a high tensile stress. A layer of silicon nitride having a high tensile stress may be beneficial in later fabrication operations and may be less susceptible to buckling than a layer of silicon nitride having a low tensile stress. In some embodiments, the first and the second nitride layers are each about 20 nm to 60 nm thick, or about 40 nm thick. 
         [0025]      FIG. 2A  shows an example of a cross-sectional schematic illustration of a partially fabricated liquid flow cell  200  at this point (e.g., up through block  110 ) in the process  100 . The liquid flow cell  200  includes a substrate  202 , a first oxide layer  204 , a second oxide layer  206 , a first nitride layer  214 , and a second nitride layer  216 . 
         [0026]    Returning to  FIG. 1 , at block  115 , a portion of the first nitride layer and the first oxide layer is removed. In some embodiments, the portion of the first nitride layer and the first oxide layer removed has dimensions of about 200 microns to 500 microns by about 200 microns to 500 microns, about 400 microns square, or about 200 microns by about 400 microns. In some embodiments, this is performed by etching the first nitride layer and then etching the first oxide layer. In some embodiments, etching the first nitride layer and etching the first oxide layer is performed with a dry etching process. 
         [0027]      FIG. 2B  shows an example of a cross-sectional schematic illustration of the partially fabricated liquid flow cell  200  at this point (e.g., up through block  115 ) in the process  100 . The liquid flow cell  200  includes a region  220  where the first oxide layer  204  and the first nitride layer  214  have been removed. 
         [0028]    Returning to  FIG. 1 , at block  120 , a cavity is formed in the substrate to expose the second oxide layer. For example, a first side of the second oxide layer may be disposed on the substrate, and a portion of the substrate may be removed to expose the first side of the second oxide layer. In some embodiments, the cavity is formed by etching the substrate to expose the second oxide layer. In some embodiments, etching the substrate is performed with a wet etching process (e.g., a potassium hydroxide (KOH) etch or a tetramethylammonium hydroxide (TMAH) etch when the substrate comprises silicon). A KOH etch will etch the second oxide layer, so the etch duration may be timed when using a KOH etch so as to not etch the second oxide layer too much. A TMAH etch will not etch the second oxide layer. 
         [0029]      FIGS. 2C and 2D  show examples of schematic illustrations of the partially fabricated liquid flow cell  200  at this point (e.g., up through block  120 ) in the process  100 .  FIG. 2C  shows an example of a top-down schematic illustration of the liquid flow cell  200 , and  FIG. 2D  shows an example of a cross-sectional schematic illustration of the liquid flow cell  200  through line  1 - 1  of  FIG. 2C . The liquid flow cell  200  includes a cavity  224  in the substrate  202  exposing the second oxide layer  206 . For example, a first side of the second oxide layer  206  may be disposed on the substrate  202  and the cavity  224  in the substrate  202  may expose the first side of the second oxide layer  206 .  FIG. 2C  shows the area of the cavity  224  proximate the second oxide layer  206  as projected onto the second nitride layer  216 . 
         [0030]    Returning to  FIG. 1 , at block  125 , a third nitride layer is deposited on the first nitride layer, substrate walls of the cavity, and the second oxide layer (e.g., the first side of the second oxide layer). In some embodiments, the third nitride layer is deposited with a CVD process or a low pressure CVD process. In some embodiments, the third nitride layer comprises silicon nitride. In some embodiments, the first nitride layer, the second nitride layer, and the third nitride layer all comprise the same nitride (e.g., silicon nitride). In some embodiments, the third nitride layer is about 20 nm to 60 nm thick, or about 40 nm thick. In some embodiments, the third nitride layer is thick enough so that it forms a continuous layer. In some embodiments, the third nitride layer is also deposited on the second nitride layer, adding to the thickness of the second nitride layer. 
         [0031]      FIG. 2E  shows an example of a cross-sectional schematic illustration of the partially fabricated liquid flow cell  200  at this point (e.g., up through block  125 ) in the process  100 . The liquid flow cell  200  includes a third nitride layer  232  disposed on the second oxide layer  206  (e.g., the first side of the second oxide layer  206 ) and walls of the substrate  202  that define the cavity  224 . The third nitride layer  232  is not shown as being disposed on the first nitride layer  214  as in some embodiments, the first nitride layer  214  and the third nitride layer  232  may comprise the same nitride and together may form a nitride layer. 
         [0032]    Returning to  FIG. 1 , at block  130 , an inlet port, an outlet port, and a plurality of viewports between the inlet port and the outlet port are formed in the second nitride layer. At least some of the plurality of viewports are positioned to be opposite the cavity; that is, at least some of the plurality of viewports overlie (or underlie, depending on the orientation of the liquid flow cell) the cavity. In some embodiments, the inlet port, the outlet port, and the plurality of viewports are formed in the second nitride layer with a lithography process (e.g., electron beam lithography) and a reactive ion etch (RIE) process. In some embodiments, the inlet port, the outlet port, and the plurality of viewports are formed in the second nitride layer using ion milling. In some embodiments, the inlet port, the outlet port, and the plurality of viewports have a circular shape. In some embodiments, the inlet port, the outlet port, and the plurality of viewports have dimensions (e.g., a diameter) of about 60 nm to 500 nm, about 60 nm to 120 nm, or about 80 nm to 100 nm. Viewports with smaller diameters may be less susceptible to the graphene covering the viewports (see block  140 ) bowing into or out of the viewports under vacuum. In some embodiments, dimensions of the inlet port and the outlet port are larger than dimensions of a view port. In some embodiments, the plurality of viewports includes about 10 viewports to 100 viewports, about 20 viewports to 90 viewports, or about 50 viewports. In some embodiments, a spacing between viewports of the plurality of viewports is about 250 nm to 1 micron. 
         [0033]      FIGS. 2F and 2G  show examples of schematic illustrations of the partially fabricated liquid flow cell  200  at this point (e.g., up through block  130 ) in the process  100 .  FIG. 2F  shows an example of a top-down schematic illustration of the liquid flow cell  200 , and  FIG. 2G  shows an example of a cross-sectional schematic illustration of the liquid flow cell  200  through line  1 - 1  of  FIG. 2F . The liquid flow cell  200  includes an inlet port  242 , an outlet port  244 , and a plurality of viewports  246  defined in the second nitride layer  216 .  FIG. 2G  shows the area of the cavity  224  as projected onto the second nitride layer  216 , with at least some of the plurality of viewports positioned over the cavity  224 . 
         [0034]    Returning to  FIG. 1 , at block  135 , a portion of the second oxide layer is removed to form a channel from the inlet port to the outlet port. In some embodiments, this is performed with an etching process. In some embodiments, the etching process is a wet etching process (e.g., a hydrofluoric (HF) acid etch). In some embodiments, the plurality of viewports provide access to the second oxide layer between the inlet port and the outlet port. 
         [0035]      FIG. 2H  shows an example of a cross-sectional schematic illustration of the partially fabricated liquid flow cell  200  at this point (e.g., up through block  135 ) in the process  100 . The liquid flow cell  200  includes a channel  248  in the second oxide layer  206  between the inlet port  242  and the outlet port  244 . The channel  248  is defined by the third nitride layer  232  and the second nitride layer  216  as shown in  FIG. 2H . Into and out of the plane of the page of  FIG. 2H , the channel  248  also is defined by second oxide layer  206 . The channel  248  also underlies the plurality of viewports  246 . In some embodiments, the channel  248  has cross-sectional dimensions of about 50 nm to 300 nm (e.g., a thickness of the second oxide layer  206  that was removed) by about 500 nm to 2 microns (e.g., a width of the channel defined by the second oxide layer  206  into and out of the plane of the page of  FIG. 2H ), or about 200 nm (e.g., a thickness of the second oxide layer  206  that was removed) by 1 micron (e.g., a width of the channel defined by the second oxide layer  206  into and out of the plane of the page of  FIG. 2H ). In some embodiments, a length of the channel  248  between the inlet port  242  and the outlet port  244  is about 50 microns to 1.5 mm. In some embodiments, a channel  248  with larger cross-sectional dimensions will aid in the flow of the liquid in the channel. In some embodiments, a channel  248  with smaller cross-sectional dimensions (e.g., a smaller thickness through which an electron beam travels) will provide for higher resolution TEM. 
         [0036]    Returning to  FIG. 1 , at block  140 , a graphene sheet is positioned on the second nitride layer (e.g., the graphene sheet being positioned on a second side of the second nitride layer, with a first side of the second nitride layer being disposed on a second side of the second oxide layer) to cover the plurality of viewports. In some embodiments, the graphene sheet comprises a single layer of graphene. In some embodiments, the graphene sheet comprises multiple layers of graphene. 
         [0037]    For example, in some embodiments, the graphene is grown on copper using a CVD process. A thin poly(methyl methacrylate) (PMMA) solution may be deposited (e.g., spin coated) on the graphene. In some embodiments, a 10 nm to 20 nm thick layer of PMMA is deposited on the graphene. The PMMA may increase the structural integrity as well as the flexibility of the graphene and aid in ensuring successful transfer of the graphene onto the second nitride layer. 
         [0038]    Then, the copper may be etched (e.g., with a sodium persulphate solution). The graphene may be washed in several deionized (DI) water baths. In some embodiments, the graphene is transferred onto the second nitride layer with the graphene contacting the second nitride layer. In some embodiments, the transfer process is a wet transfer process or a dry transfer process. When the transfer process is a wet transfer process, critical point drying may be used. In some embodiments, the PMMA is then removed (e.g., using an acetone solution or forming gas). In some embodiments, the graphene seals the plurality of viewports. 
         [0039]    In some embodiments, in addition to covering the plurality of viewports, the graphene may cover the inlet port and/or the outlet port. When the graphene covers the inlet port or the outlet port, the graphene is patterned to remove the graphene from the inlet port or the outlet port before the PMMA is removed. Also, when electrodes are disposed on the second silicon nitride layer (see  FIG. 5 ), the graphene may be patterned so that the graphene does not contact an electrode. 
         [0040]      FIG. 2I  shows an example of a cross-sectional schematic illustration of the liquid flow cell  200  at this point (e.g., up through block  140 ) in the process  100 . The liquid flow cell  200  includes a substrate  202 , the substrate  202  having a first surface and a second surface. A first oxide layer  204  is disposed on the first surface of the substrate  202  and a second oxide layer  206  is disposed the second surface of the substrate  202 . A first nitride layer  214  is disposed on the first oxide layer  204  and a second nitride layer  216  is disposed on the second oxide layer  206 . A cavity  224  is defined in the first oxide layer  204 , the first nitride layer  214 , and the substrate  202 . The cavity  224  includes a third nitride layer  232  disposed on walls of the substrate  202  that define the cavity  224 . A channel  248  is defined in the second oxide layer  206 . The channel  248  is also defined by the second nitride layer  216 , the third nitride layer  216 , and a graphene sheet  252 . An inlet port  242  and an outlet port  244  are defined in the second nitride layer  216  and in fluid communication with the channel  248 . A plurality of viewports  246  are defined in the second nitride layer  216  and positioned between the inlet port  242  and the outlet port  244 . At least some of the plurality of viewports are positioned opposite the cavity  224 . The graphene sheet  252  is disposed on the second nitride layer  216  and covers the plurality of viewports  246 . In some embodiments, the graphene sheet  252  comprises a monolayer of graphene. In some embodiments, the graphene sheet  252  comprises a few layers of graphene. 
         [0041]    The liquid flow cell  200  shown in  FIG. 2I  may be used for TEM when the TEM sample holder is configured to accept the liquid flow cell  200 . For example, the TEM sample holder may have a tube for liquid delivery to the inlet port  242  and a tube to accept liquid from the outlet port  244 . As another example, a portion of the liquid flow cell  200  including the inlet port  242  could be isolated from the vacuum of a TEM with polymer gaskets (e.g., a fluoropolymer elastomer gasket). Similarly, a portion of the liquid flow cell  200  including the outlet port  244  could be isolated from the vacuum of a TEM with polymer gaskets. Liquid could then be introduced to the portion of the liquid flow cell  200  including the inlet port  242 . The positions and orientations of the different components of a liquid flow cell  200  can be altered so that the liquid flow cell is able to be used with a specific TEM sample holder. 
         [0042]    The liquid can be imaged with the liquid flow cell  200 , with an electron beam being transmitted though the graphene sheet  252 , the liquid, and the third nitride layer  232 . When using the liquid flow cell  200  for TEM, liquid could be continuously input into the inlet port  242  such that there is a flow of liquid through the channel  248  when an image is generated. Alternatively, liquid could be input into the inlet port  242  so that it fills the channel  248  and then an image generated. The liquid may not be exposed to the ultrahigh vacuum of a TEM due to the liquid being confined to the liquid flow cell while it is being imaged. 
         [0043]    The orientation of the liquid flow cell  200  in a microscope may depend on the sample being imaged and on the configuration of the TEM sample holder. For example, if a biological sample is being imaged in a TEM, the liquid flow cell  200  may be oriented so that the electron beam impinges on the third nitride layer  232 , interacts with the liquid, and then impinges the graphene sheet  252 . The third nitride layer  232  may diffuse the energy of the electron beam and aid in preventing damage to the sample. 
         [0044]    Variations of and/or additions to the method  100  are possible. For example, a flow cell may be fabricated by depositing a nitride layer on both sides of a substrate, depositing an oxide layer on one of the nitride layers, and then depositing a nitride layer on the oxide layer. The oxide layer between the two nitride layers can be used to fabricate a channel with viewports, similar to the liquid flow cell  200  shown in  FIG. 2 . A fabrication method of a liquid flow cell having a nitride layer/oxide layer/nitride layer may include process operations similar to process operations described with respect to the method  100  described in  FIG. 1 . 
         [0045]      FIG. 2J  shows an example of a cross-sectional schematic illustration of a liquid flow cell  260  fabricated with such a process. The liquid flow cell  260  includes a substrate  262 , the substrate  262  having a first surface and a second surface. A first nitride layer  264  is disposed on the first surface of the substrate  262  and a second nitride layer  266  is disposed on the second surface of the substrate  262 . A first side of an oxide layer  268  is disposed on the second nitride layer  266 . A third nitride layer  270  is disposed on a second side of the oxide layer  268 . A cavity  274  is defined in the first nitride layer  264  and the substrate  262 . In some embodiments, the substrate  262  and the second nitride layer  266  define the cavity  274 ; that is, in some embodiments, the second nitride layer  266  defines a surface of the cavity  274 . A channel  280  is defined in the oxide layer  268 . A width of the channel defined by the oxide layer  268  into and out of the plane of the page of  FIG. 2J . The channel  280  is also defined by the first nitride layer  264 , the second nitride layer  266 , and a graphene sheet  294 . An inlet port  282  and an outlet port  284  are defined in the third nitride layer  270  and in fluid communication with the channel  280 . A plurality of viewports  290  are defined in the third nitride layer  270  and positioned between the inlet port  282  and the outlet port  284 . At least some of the plurality of viewports  290  are positioned opposite the cavity  274 . The graphene sheet  294  is disposed on the third nitride layer  270  and covers the plurality of viewports  290 . Dimensions of the liquid flow cell  260  may be similar to dimensions of the liquid flow cell  200  described in  FIGS. 2A-2I . 
         [0046]    As another example, in some embodiments, the liquid flow cell  200  may need to be thicker for it to fit into a TEM sample holder. In some embodiments, thickness of the liquid flow cell  200  may be increased by joining the liquid flow cell  200  to a second plate.  FIG. 3  shows an example of a flow diagram illustrating a manufacturing process for a liquid flow cell having a second plate. Specifically,  FIG. 3  shows an example of a flow diagram illustrating a manufacturing process for a second plate and joining the second plate to a liquid flow cell as fabricated with the method  200  shown in  FIG. 2 . Portions of the manufacturing process for a second plate may be similar to the manufacturing process for a liquid flow cell.  FIGS. 4A-4G  show examples of schematic illustrations of a liquid flow cell having a top plate at various stages in the manufacturing process. 
         [0047]    Starting at block  305  of the method  300  shown in  FIG. 3 , a first oxide layer is formed on a first surface of a second substrate and a second oxide layer is formed on a second surface of the second substrate. In some embodiments, the second substrate comprises a semiconductor. In some embodiments, the second substrate comprises silicon. In some embodiments, the second substrate comprises a double side polished semiconductor wafer. When the second substrate comprises silicon, the oxide layer may be silicon oxide and the silicon oxide may be thermally grown on the silicon substrate. In some embodiments, the oxide may be deposited on the substrate using a CVD technique or a PVD technique. In some embodiments, the substrate is about 150 microns to 450 microns thick, or about 300 microns thick. In some embodiments, dimensions of the first surface and the second surface of the substrate are about 2 mm to 8 mm by about 2 mm to 8 mm, or about 2 mm by about 2 mm. In some embodiments, the first and the second oxide layers are each about 50 nm to 300 nm thick, or about 200 nm thick. 
         [0048]    At block  310  of the method  300 , a first nitride layer is deposited on the first oxide layer of the second substrate and a second nitride layer is deposited on the second oxide layer of the second substrate. In some embodiments, the first and the second nitride layers are deposited with a CVD process or a low pressure CVD process. In some embodiments, the first and the second nitride layers comprise silicon nitride. In some embodiments, the first and the second nitride layers are each about 20 nm to 60 nm thick, or about 40 nm thick. 
         [0049]      FIG. 4A  shows an example of a cross-sectional schematic illustration of a partially fabricated second plate  400  at this point (e.g., up through block  310 ) in the method  300 . The second plate  400  includes a substrate  402 , a first oxide layer  404 , a second oxide layer  406 , a first nitride layer  414 , and a second nitride layer  416 . 
         [0050]    Returning to  FIG. 3 , at block  315 , a portion of the first nitride layer and the first oxide layer of the second substrate is removed. In some embodiments, the portion of the first nitride layer and the first oxide layer removed has dimensions of about 200 microns to 500 microns by about 200 microns to 500 microns, about 400 microns square, or about 200 microns by about 400 microns. In some embodiments, this is performed by etching the first nitride layer and then etching the first oxide layer. In some embodiments, etching the first nitride layer and etching the first oxide layer is performed with a dry etching process. 
         [0051]      FIG. 4B  shows an example of a cross-sectional schematic illustration of the partially fabricated second plate  400  at this point (e.g., up through block  315 ) in the method  300 . The second plate  400  includes a region  420  where the first oxide layer  404  and the first nitride layer  414  have been removed. 
         [0052]    Returning to  FIG. 3 , at block  320 , a cavity is formed in the second substrate to expose the second oxide layer. For example, a first side of the second oxide layer may be disposed on the substrate, and a portion of the substrate may be removed to expose the first side of the second oxide layer. In some embodiments, the cavity is formed by etching the substrate to expose the second oxide layer. In some embodiments, etching the substrate is performed with a wet etching process (e.g., a KOH etch or a TMAH etch when the substrate comprises silicon). 
         [0053]      FIGS. 4C and 4D  show examples of schematic illustrations of the partially fabricated second plate  400  at this point (e.g., up through block  320 ) in the method  300 .  FIG. 4C  shows an example of a top-down schematic illustration of the second plate  400 , and  FIG. 4D  shows an example of a cross-sectional schematic illustration of the second plate  400  through line  1 - 1  of  FIG. 4C . The second plate  400  includes a cavity  424  in the second substrate  402  exposing the second oxide layer  406 . For example, a first side of the second oxide layer  406  may be disposed on the substrate  402  and the cavity  424  in the substrate  402  may expose the first side of the second oxide layer  406 .  FIG. 4C  shows the area of the cavity  424  proximate the second oxide layer  406  as projected onto the second nitride layer  416 . 
         [0054]    Returning to  FIG. 3 , at block  325 , a polymer material is deposited on the second nitride layer of the second plate. In some embodiments, the polymer material is deposited so that it covers the area of the cavity  424  proximate the second oxide layer  406  as projected onto the second nitride layer  406 . In some embodiments, the polymer material is a photoresist (e.g., an epoxy-based negative photoresist, such as SU-8, or an epoxy-based photoresist). In some embodiments, the polymer material is deposited with a spin coating process. In some embodiments, a thickness of the polymer material is about 2 microns to 8 microns, or about 5 microns. 
         [0055]      FIG. 4E  shows an example of a cross-sectional schematic illustration of the partially fabricated second plate  400  at this point (e.g., up through block  325 ) in the method  300 . The second plate  400  includes a polymer material  432  disposed on the second nitride layer  416 . 
         [0056]    Returning to  FIG. 3 , at block  330 , a portion of the polymer material, the second nitride layer, and the second oxide layer are removed to form a through hole. In some embodiments, this performed by first patterning the polymer material and then the second oxide layer is removed (e.g., using a buffered oxide etch (BOE)) and the second nitride layer is removed (e.g., using a RIE process or ion milling) to form the though hole. In some embodiments, the second oxide layer and the second nitride layer are removed with a dry etching process. 
         [0057]      FIG. 4F  shows an example of a cross-sectional schematic illustration of the partially fabricated second plate  400  at this point (e.g., up through block  330 ) in the process  300 . The second plate comprises the second substrate  402  having a first surface and a second surface. The first oxide layer  404  is disposed on the first surface of the second substrate  402  and the second oxide layer  406  is disposed on the second surface of the second substrate  402 . The first nitride layer  414  is disposed on the first oxide layer  404  and the second nitride layer  416  is disposed on the second oxide layer  406 . The polymer material  432  is disposed on the second nitride layer  416 . The second plate  400  defines a through hole  438  through the first nitride layer  414 , the first oxide layer  404 , the second substrate  402 , the second oxide layer  406 , the second nitride layer  416 , and the polymer layer  432 . 
         [0058]    Returning to  FIG. 3 , at block  335 , the second plate is joined with a liquid flow cell using the polymer material. In some embodiments, the graphene sheet of a liquid flow cell fabricated according to the method  200  shown in  FIG. 2  is joined to the second nitride layer of the second plate with the polymer material. In some embodiments, the graphene sheet and the second nitride layer of a liquid flow cell fabricated according to the method  200  shown in  FIG. 2  are joined to the second nitride layer of the second plate with the polymer material. The cavity of the substrate of the liquid flow cell is aligned with the though hole of the second substrate. Heat and pressure may be used to join the liquid flow cell to the second plate. 
         [0059]      FIG. 4G  shows an example of a cross-sectional schematic illustration of the liquid flow cell  200  and the second plate  400  at this point (e.g., up through block  335 ) in the method  300 . The second plate  400  is joined to the liquid flow cell  200  with the polymer material  432 . For example, as shown in  FIG. 4G , in some embodiments, the graphene sheet  252  of the liquid flow cell  200  and the second nitride layer  416  of the second plate  400  are joined with the polymer material  432 . In some embodiments, the nitride layer (i.e., the nitride layer having the graphene sheet disposed thereon) and the graphene sheet  252  of the liquid flow cell  200  and the second nitride layer  416  of the second plate  400  are joined with the polymer material  432 . In some embodiments, the nitride layer (i.e., the nitride layer having the graphene sheet disposed thereon) of the liquid flow cell  200  and the second nitride layer  416  of the second plate  400  are joined with the polymer material  432 . The through hole  438  in the second plate  400  is aligned with the cavity  224  of the liquid flow cell  200 . That is, the liquid flow cell  200  and the second plate  400  are aligned so that an electron beam can be transmitted through the graphene sheet  252 , a liquid in the channel, and the third nitride layer of the liquid flow cell. 
         [0060]    In some embodiments, the liquid flow cell  200  and the second plate  400  define an inlet channel  605  connected to (or in fluid communication with) the inlet port  242  and an outlet channel  610  connected to (or in fluid communication with) the outlet port  244 . In some embodiments, the polymer material  432  is disposed proximate a perimeter of the cavity  224  (as projected on a surface) in the liquid flow cell  200  and a perimeter of the through hole  438  (as projected on a surface) in the second plate  400 . 
         [0061]    A liquid flow cell can be further modified. For example, in some embodiments, a liquid flow cell includes electrodes so that electrical contact can be made with the liquid at the inlet port and at the outlet port of the liquid flow cell. Such electrodes may be used to apply a voltage or a current to a liquid when imaging the liquid or to aid in or cause particles in the liquid to flow due to electrophoresis. Such electrodes also may be used to apply a voltage or a current to a liquid to aid in or cause the liquid to flow. 
         [0062]      FIG. 5  shows an example of a top-down schematic illustration of a liquid flow cell including electrodes. The liquid flow cell  500  shown in  FIG. 5  may have a similar cross-section as the liquid flow cell  200  shown in  FIG. 2I . The liquid flow cell  500  includes a first electrode  510  and a second electrode  520  disposed on a silicon nitride layer  505  of the liquid flow cell  500 . Also shown are an inlet port  530 , an outlet port  535 , and a graphene sheet  552  covering a plurality of viewports (not shown). The first electrode  510  includes a first contact pad  512  proximate an edge of the second nitride layer  505 , a first probe  514  at an edge of the inlet port  530 , and a first lead  516  connecting the first contact  512  pad and the first probe  514 . Similarly, the second electrode  520  includes a second contact pad  522  proximate the edge of the second nitride layer  505 , a second probe  524  at an edge of the outlet port  535 , and a second lead  526  connecting the second contact pad  522  and the second probe  524 . In some embodiments, the first electrode  510  and the second electrode  520  comprise gold or platinum. In some embodiments, the first electrode  510  and the second electrode  520  are about 20 nm to 60 nm thick, or about 40 nm thick. In some embodiments, the first electrode  510  and the second electrode  520  are about 1 mm to 4 mm long, or about 2 mm to 3 mm long. 
         [0063]    With the first electrode  510  and the second electrode  520 , a bias or current can be applied across a liquid in a channel of the liquid flow cell  500  from the edge of the liquid flow cell. Other configurations of electrodes are possible, and the configuration may depend on the configuration of the TEM sample holder. 
         [0064]    When fabricating a liquid flow cell according to the method  100  shown in  FIG. 1 , electrodes could be formed at many different points in the method  100 . For example, in some embodiments, electrodes could be deposited and patterned after block  125  or after block  140 . 
         [0065]    A liquid flow cell can also be modified so that an electron beam impinges a first graphene sheet, a liquid, and a second graphene sheet, and the electron beam does not interact with a silicon nitride layer. Such a liquid flow cell may provide higher resolution imaging. 
         [0066]      FIG. 6  shows an example of a cross-sectional schematic illustration of a liquid flow cell including two graphene sheets. The liquid flow cell  600  shown in  FIG. 6  may be similar to the liquid flow cell  200  shown in  FIG. 2I , with the third nitride layer defining a second plurality of viewports and a second graphene sheet covering the plurality of viewports. The liquid flow cell  600  includes a substrate  602 , a first oxide layer  604 , a second oxide layer  606 , a third nitride layer  632 , and a second nitride layer  616 . The third nitride layer  632  is disposed on the walls of the substrate  602  that define a cavity  624 . A first nitride layer is not shown in  FIG. 6  because in the manufacturing process of a liquid flow cell  600 , the first nitride layer disposed on the first oxide layer  604  may have the third nitride layer  632  disposed thereon, forming a single nitride layer, as explained with respect to block  125  of the method  100  shown in  FIG. 1 . 
         [0067]    An inlet port  642 , an outlet port  644 , and a plurality of viewports (not indicated) are defined in the second nitride layer  616 . A second plurality of viewports (not indicated) are defined in the third nitride layer  632 . In some embodiments, the second plurality of viewports defined in the third nitride layer  632  have similar dimensions and spacing as the plurality of viewports defined in the second nitride layer  616 . A channel  648  is defined in the second oxide layer  606  (e.g., a width of the channel defined by the second oxide layer  606  into and out of the plane of the page of  FIG. 6 ). The channel  648  is also defined by a second graphene sheet  654 , the third nitride layer  632 , the second nitride layer  616 , and a first graphene sheet  652 . The first graphene sheet  652  is disposed on the second nitride layer  616  and covers the plurality of viewports. The second graphene sheet  654  is disposed on the third nitride layer  632  and covers the second plurality of viewports. In some embodiments, the first graphene sheet  652  comprises a monolayer of graphene. In some embodiments, the first graphene sheet  652  comprises a few layers of graphene. In some embodiments, the second graphene sheet  654  comprises a monolayer of graphene. In some embodiments, the second graphene sheet  654  comprises a few layers of graphene. 
         [0068]    The liquid flow cell  600  may be fabricated with a method similar to the method  100  shown in  FIG. 1  with some additional process operations. For example, in some embodiments, at block  130  of the method  100 , a plurality of viewports is defined in the second nitride layer  616 . A second plurality of viewports also may be defined in the third nitride layer  632 . Then, in some embodiments, after block  135  of the method  100  (i.e., after the channel  648  is formed in the second oxide layer  606 ), the second graphene sheet  654  is positioned on the third nitride layer  632  to cover the second plurality of viewports defined in the third nitride layer  632 . 
         [0069]    The second graphene sheet  654  may be transferred to the liquid flow cell  600  with a wet transfer process or a dry transfer process. When the second graphene sheet  654  is put into position to seal the second plurality of viewports defined in the third nitride layer  632 , the graphene sheet may be in contact with the third nitride layer  632  and be suspended over the cavity  624 . To aid in ensuring the second graphene sheet  654  seals the viewports, a liquid (e.g., water) may be disposed between the second graphene sheet  654  and the third nitride layer  632 . This may allow the second graphene sheet  654  suspended over the cavity  624  to move into the cavity  624 . As the liquid evaporates, capillary action may pull in the second graphene sheet  654  onto the third nitride layer  632 . For example, the partially fabricated liquid flow cell may be heated to about 50° C. to 150° C., or about 100° C., for about 5 minutes to 25 minutes, or about 15 minutes, to evaporate the liquid and aid in ensuring that the viewports defined in the third nitride layer  632  are sealed. 
         [0070]    Further modifications of a flow cell are possible. For example, in some embodiments, a liquid flow cell includes multiple channels, two or more inlet ports, and/or two or more outlet ports. For example, a liquid flow cell may include two inlet ports and one outlet port. A channel from each inlet port may meet or join in the liquid flow cell and form one channel that connects to the outlet port. Observations of reactions that occur when two liquids mix could be made with such a liquid flow cell. 
         [0071]    While embodiments of liquid flow cells described herein have been described as being used primarily in transmission electron microscopy, the liquid flow cells may also be used for other types of microscopy. For example, embodiments of the liquid flow cell may be used in conjunction with optical microscopy or near field optical microscopy, either in the transmission mode or reflection mode, due to the optical transparency of the materials of the liquid flow cell. This may provide the capability of performing correlative studies of biological samples with florescent tags using optical microscopy and electron microscopy and generate more detailed information regarding the structure, conformation, and dynamics of the biological samples. 
         [0072]    A benefit of the fabrication methods described above is the use of silicon nitride membranes with small graphene viewports. A reduction in the bowing of graphene may contribute to a uniformly thin cross section of a channel for the flow of liquids and may be useful for subsequent analysis in both optical and electron microscopy. 
         [0073]    Embodiments of the liquid flow cells described herein may be used for many different experiments. For example, a liquid flow cell may be used in experiments involving the chemical transformation of nanocrystals. Controlling such transformations has emerged as a versatile approach for the design of heterostructured nanocrystals with programmable structural, morphological, and compositional complexity. Study of the controlled dissolution of pre-made metal nanocrystals can generate valuable knowledge about the chemical reactivity and relative stability of different surface facets, which has implications not only for the chemical design of hybrid nanocrystals through controlled overgrowth, but also for the application of metal nanocrystals in catalysis and plasmonics. For example, a liquid flow cell may be used in experiments aimed at better understanding the dissolution of anisotropic gold nanocrystals with tailored surface facets under an electric bias. 
         [0074]    As another example, a liquid flow cell may be used in experiments involving visualizing DNA permeation into cells under electric pulses. The science of relevance here is that electric pulses of sufficient strength cause an increase in the trans-membrane potential difference, which enables the permeation of DNA molecules. This phenomenon has been widely used in medical deliveries of complex DNA drugs into target cells and in selected gene expression, yet the delivery mechanism has not yet been visualized and interpreted. The detailed in situ record of DNA conformational change can give understanding of the mechanism involved, thereby enabling better engineering of this process for targeted drug delivery. 
       CONCLUSION 
       [0075]    The liquid flow cells described herein allow for unprecedented resolution and contrast in TEM studies. Understanding electrochemical processes at the atomic scale has broad implications in catalysis, battery technology, and fuel cells. Further, the liquid flow cells may make possible new areas in biological research. 
         [0076]    In the foregoing specification, the invention has been described with reference to specific embodiments. However, one of ordinary skill in the art appreciates that various modifications and changes can be made without departing from the scope of the invention as set forth in the claims below. Accordingly, the specification and figures are to be regarded in an illustrative rather than a restrictive sense, and all such modifications are intended to be included within the scope of invention.