Patent Document

BACKGROUND OF THE INVENTION 
       [0001]    1. Field of the Invention 
         [0002]    The invention relates to specimen holders for imaging and analysis and particularly to in situ holders capable of exciting a specimen for dynamic experimentation. 
         [0003]    2. Description of the Prior Art 
         [0004]    In situ is a Latin phrase meaning “in the place.” The ability to observe dynamic processes directly, close to their natural state as they undergo changes is vital for the advancement of research in many modern day applications. Various examples of in situ holders for transmission electron microscopy (TEM) include cooling holders, heating holders, nano indentation holders, straining holders, biasing holders and environmental cell holders. Holders are also utilized in a variety of other imaging and analysis devices. 
         [0005]    Observations that require the presence of controlled environmental fluids around the specimen at elevated temperatures is one challenging aspect of in situ imaging and analysis. Fluids referred herein may include liquids, gases or plasma. An electron beam, such as that utilized by a TEM to create a specimen image, interacts strongly with matter. This leads to electron beam broadening which is detrimental to image resolution. To avoid extraneous scattering of the electron beam, it is desirable to have a very low-pressure beam path within the TEM column, optimally a high vacuum environment. A specimen immersed in fluids, opposes this basic principle. This limits the in situ environment height adjacent the specimen to less than a few hundred microns, making it extremely difficult to incorporate a heating system within the confined space of the holder necessary to create such an environment. Apart from this, high temperatures also give rise to specimen drift because of asymmetrical thermal expansion of the holder and consequential displacement of the specimen within the TEM. The presence of these fluids, therefore, decreases the resolution of the microscope images, limiting the amount of useful information that can be gathered. 
         [0006]    In situ experimentation that requires the presence of fluids is carried out with the help of an environmental cell (E-Cell) that is part of the specimen holder. Typically, such a cell consists of two thin film windows, which completely seal a confined space around the specimen to provide a controlled atmosphere for in situ experimentation. The basic requirement for such a device is to contain the fluid within the cell so that the main microscope vacuum remains undisturbed. 
         [0007]    Traditionally, heating holders employed resistive heating to elevate the specimen temperature.  FIGS. 1   a  and  1   b  illustrate the basic principle of resistive heater  100  utilized within a TEM specimen holder. Here, an electrical current is passed through a resistance coil  102 , which is either wrapped around, as in  FIG. 1   a , or placed near the specimen cup  104 , as in  FIG. 1   b , containing a TEM specimen  106  as shown. This generates heat within the coil, which in turn heats the specimen either by conduction, as in  FIG. 1   a  or by radiation, as in  FIG. 1   b . This heating technology is highly constrained when used for in situ heating of a specimen within an environmental cell that contains fluids. The major limitations of these prior art systems include several factors. First, resistive heating requires a complex heating mechanism, which comprises many parts, including resistance coils, radiation shields, electrical connectors and insulating blocks. This complete assembly occupies a large volume. This technology, when incorporated within an in situ holder, would increase the fluid path length of the cell and dramatically reduce the image resolution of the specimen. Resistive heaters, furthermore, have a limited working life. The heating coils may last only a few hours, especially in the presence of gaseous oxidizing or reducing environments. Conduction and radiation from the heating coils not only heat the specimen but also the entire surrounding region of the environmental cell as well as the microscope goniometer or adjacent parts of other imaging or analysis devices. This introduces significant specimen drift, thereby limiting image resolution. It is also not possible to use resistive heating coils for the localized heating of the specimen. 
         [0008]    Resistive heating is also limited by the maximum specimen temperature that can be achieved, generally limited to the range of 1,000° C. to 1,200° C. Moreover, the use of electrical current for heating can generate an electromagnetic field around the specimen, which may interfere with the electron imaging beam, limiting the image resolution and causing beam drift. Lastly, in light of the inefficient heating mechanism, the time required to attain steady state specimen temperatures is significant. Most dynamic processes occur within a fraction of a second. This makes the use of resistive heating technology difficult for most modern imaging applications. 
         [0009]    Some current TEM holders employ microelectromechanical systems (MEMS) for imaging the specimen at elevated temperatures as illustrated in  FIGS. 2   a  and  2   b . A MEMS-based system  120  utilizes two facing dies made from a silicon substrate; bottom die  122  and top die  124 , with a central hole  126 . Membranes  128 , typically constructed of silicon nitride cover the central hole  126  of each die. These membranes form a gas-flow channel  129  with an inlet  130  and an outlet  132 . This nanoreactor membrane contains an embedded heater  134  in the form of a thin platinum wire  136 , as shown in  FIG. 2   b . The heating capability of up to 500° C. is derived from local electrical resistive heating of the platinum wire  136 . The compact design of the MEMS based system provides a fluid path length of less than 10 microns and a stable and rapid specimen heating environment of less than one second. 
         [0010]    There are, however, many shortcomings associated with such devices. The MEMS based TEM holders are designed to image small specimens such as particles. These holders cannot be used to observe a standard TEM specimen having a diameter of 3 mm. The specimen viewing area in a MEMS based holder is limited to a few square microns. In the MEMS based system the particle specimen is in contact with the silicon nitride membrane. This limits its use to a specimen temperature of less than 1,200° C. Lastly, the reaction chamber in a MEMS based holder is often limited to one time use. 
         [0011]    One approach devised to carry out in situ gas flow experiments is the use of an environmental transmission electron microscope (ETEM). This is a term coined for TEM&#39;s modified to include a differentially pumped E-Cell. This ETEM consists of radial holes incorporated in the objective lens pole pieces for the first stage of differential pumping. The regular sample area of the ETEM is the controlled environment volume. Differential pumping systems are connected between apertures using appropriate vacuum pump technology. This permits higher gas pressure in the sample region, while maintaining high vacuum conditions in the remainder of the TEM column. A conventional reactor-type gas manifold system enables inlet of flowing gases into the ETEM, and a sample stage with a furnace allows samples to be heated. 
         [0012]    The use of ETEM for in situ experiments has many disadvantages: (i) the high installation and operating cost of ETEM, especially to carry out only a dedicated set of experiments; (ii) ETEM can be used only to circulate gases over the specimen; (iii) since the ETEM does not have a completely sealed E-Cell, the gas pressure around the specimen is typically lower than 1 atmosphere, thus failing to replicate real life conditions, as it is necessary to observe gas-solid reactions at or close to atmospheric pressures; (iv) the gas path length within the ETEM is considerably large; (v) the gas circulation within the ETEM may contaminate the region around the objective lens pole pieces, which, apart from affecting the image resolution, could also affect the results of the next specimen analysis, which may require a different gaseous environment; (vi) it does not include a specimen heating system and heating has to be carried out using a standard heating specimen holder; and (vi) the partial pressure of any residual gas contained in the microscope column may negatively impact the experimental results. 
         [0013]    Laser-induced breakdown spectroscopy (LIBS) is a type of atomic emission spectroscopy which utilizes a highly energetic laser pulse as the excitation source. LIBS operates by focusing the laser onto a small area at the surface of the specimen; when the laser is discharged it ablates a very small amount of material, in the range of picograms to nanograms, which instantaneously generates a plasma plume with temperatures of about 10,000-20,000 K. At these temperatures, the ablated material dissociates into excited ionic and atomic species. During this time, the plasma emits a continuum of radiation which does not contain any useful information about the species present, but within a very small timeframe the plasma expands at supersonic velocities and cools. At this point the characteristic atomic emission lines of the elements can be observed. O. Bostanjoglo and E. Endruschat, in “Kinetics of Laser-induced Crystallization of Amorphous Germanium Films”, Phys. Stat. Sol. (a), 91, 17 (1985), and H. Domer and O. Bostanjoglo, in “High-speed transmission electron microscope”, Rev. Sci. Instrum., 74 (10), 4369-4372, (2003) disclose an attached a Q-switched Nd-YAG laser system to a TEM to investigate the crystallization of amorphous Ge films by time resolved microscopy. A. Takaoka, N. Nakamura, K. Ura, H. Nishi, and T. Hata disclose, in “Local Heating of Specimen with Laser Diode in TEM”, J. Electron Microsc., Vol. 38, No. 2, 95-100, 1989, heating specimens locally to a temperature greater than 1000° C. by introducing a laser diode and small lens system into the vacuum space in the TEM. Some prominent laboratories have modified commercial TEM&#39;s by setting up an elaborate network of laser optics in order to pulse the electron beam as well as ablate the specimen. V. A. Lobastov, R. Srinivasan, and A. H. Zewail disclose, in “Four-dimensional ultrafast electron microscopy”, PNAS, Vol. 102, No. 20, 2005, a diode-pumped mode-locked Ti:Sapphire laser oscillator to develop a 4D ultra fast electron microscope. Here the laser is used to generate ultra fast electron pulse derived from a train of femtosecond pulses and concurrently heat the specimen and induce melting of metals. Similarly, T. LaGrange et. al., disclose, in “Single-shot dynamic transmission electron microscopy”, Appl. Phys. Lett., 89, 044105, 2006, the modification of a commercial JEOL2000 TEM and designed a dynamic transmission electron microscope (DTEM) with the help of an Nd-YAG laser system. This DTEM is used for vast arrays of applications including the in situ analysis of Nano wire catalysis and growth. 
         [0014]    D. Shindo et. al., in “Development of a multifunctional TEM specimen holder equipped with a piezodriving probe and a laser irradiation port”, J. Electron Microsc., Vol. 58, No. 4, 245-249, 2009, disclose the development of a specimen holder to introduce laser irradiation onto the specimen to study various photo-induced phenomena. Even though this holder has the capability of introducing a laser beam onto the specimen, it does not have a provision for an E-Cell to observe the dynamic reactions between specimen and fluids, nor does it have the ability to focus or adjust the laser beam. 
         [0015]    Many of these references highlight the importance of lasers in the field of imaging and analysis. They describe various forms of TEM&#39;s that have been modified to focus a laser beam onto the specimen. They do not, however, have any provision for a self contained specimen holder which permits the adjustment of the beam and the selective the flow of fluids over the specimen in a controlled environment. Moreover, the custom installation and operating cost of such modified TEMs are usually very high and the modifications are made to carry out very specific sets of experiments. 
         [0016]    There remains a need, therefore, for an optimized in situ holder for the dynamic observation at elevated temperatures in the presence or absence of fluids. Such an in situ holder should have the capability of introducing a beam of electromagnetic radiation through the specimen holder and should be compatible with most major commercially available TEM&#39;s. It should be portable and should not involve any modification to the installed microscopes for in situ microscopy. 
         [0017]    The holder should be designed such that it can accept a wide range of specimens, including a 3 mm diameter disk, particles dispersed on a grid or FIB lamellae contained on a support grid, and further should incorporate a compact heating design in order to minimize the fluid path length within the environmental cell. It should provide the ability to heat the specimen in the presence of fluids to a temperature in excess of 2,000° C., while providing the capability to heat a localized region of the specimen in order to limit the amount of heat radiated and conducted from the hot specimen to the surrounding region of the environmental cell and microscope components, reducing specimen drift and minimizing the amount of energy required to reach the desired specimen temperature. Finally, it should provide the capability for thermal cycling of the specimen with a short time interval, while incorporating high steady state specimen temperatures in a small time duration. 
       SUMMARY OF THE INVENTION 
       [0018]    A specimen holder is disclosed having the benefits of previously known in situ heating and environmental cell holders, while allowing for high temperature localized heating of the specimen in the presence or absence of fluids, using electromagnetic radiation. The holder includes an optical assembly to focus electromagnetic radiation onto the specimen to optically excite the specimen in a well defined and limited area. The optical components of this holder can also be adapted for applications such as cathodoluminescence detection, x-ray fluorescence and photoluminescence. 
         [0019]    For heating applications, an external source of electromagnetic radiation, such as a laser having a fixed wavelength, is attached to the holder using a standard connector. The laser beam enters the holder through a collimator that helps maintain a parallel laser beam path as it travels along the length of the holder barrel. A converging lens module is assembled near the specimen end of the holder barrel. The spot diameter of the laser beam at the focus point on the specimen can be varied by translating this converging lens module to locally heat the specimen in a limited manner. The converged laser beam is further reflected onto the specimen with the help of a minor or a polished surface on the holder tip. The angular position of the mirror is such that the laser beam nominally strikes the center of the specimen. The dimension and position of the minor can also be varied based on the application to obtain the desired irradiation effect on the specimen. Few examples of such minors include convex, concave and spherical mirrors. 
         [0020]    When laser irradiation is used, as in the preferred embodiment, a radial, symmetric heating zone is generated on the specimen. This allows for uniform expansion of the specimen at high temperatures, minimizing specimen drift from uneven thermal expansion or contraction across the specimen. The maximum temperature that can be attained by the specimen is limited largely by the material properties of the specimen and the laser power, thus creating a potential for applications in an extraordinary range of fields including catalysis, chemical vapor deposition, and molecular beam epitaxy. 
         [0021]    At any given time, imaging and analysis is carried out on a small specimen region. Thus, it is not required to heat the entire specimen. Spot heating of the specimen also reduces the energy and the time required to achieve a given steady state specimen temperature. Heat radiated from the specimen increases exponentially with the increase in the heated surface area. Hence, spot heating of the specimen reduces the heat radiation exponentially. This keeps the surrounding region of the holder close to ambient temperatures and reduces drift, which in turn enhances resolution. Localized heating of the specimen also reduces the heat conduction to other parts of the holder therefore minimizing thermal drift of the device as a whole. 
         [0022]    The electromagnetic radiation may be modulated using a computer program. This provides dynamic thermal cycling of the specimen between ambient and elevated temperatures. Further, a pulsed laser can be attached to the holder to provide pulses of energy as small as a few nanoseconds for specific applications. 
         [0023]    Unlike a MEMS heating holder, this holder can be used with standard TEM specimen types, as well as non-traditional specimens such as cones, pillars and lamellae. 
         [0024]    The environmental cell on the specimen holder provides a controlled atmosphere for in situ observations and analysis. This cell preferentially consists of a pair of thin windows separated by spacers. The specimen is placed between the thermally insulating spacers. A particular O-ring sealing mechanism provides the user with the flexibility to choose the desired fluid path length. Since there is no heating element present within the E-Cell, the holder can be used with a fluid path length as small as the specimen thickness. An optimum fluid path length can be selected based on the required specimen temperature and the acceptable image resolution. The inlet and outlet conduits allow the entrance and the exit of fluids from the environmental cell. 
         [0025]    The holder, together with its particular features and advantages, will become more apparent from the following detailed description and with reference to the appended drawings. 
     
    
     
       DESCRIPTION OF THE DRAWINGS 
         [0026]      FIGS. 1   a  and  1   b  are diagrammatic representations of prior art TEM resistance heating devices. 
           [0027]      FIGS. 2   a  and  2   b  are diagrammatic representations illustrating selective components of a prior art MEMS heating device. 
           [0028]      FIG. 3   a  is a top isometric view of the in situ holder tip assembly in accordance with an embodiment of the present invention. 
           [0029]      FIG. 3   b  is a bottom isometric view of the in situ holder tip assembly in accordance with an embodiment of the present invention. 
           [0030]      FIG. 4  is an isometric view of the specimen holder assembly. 
           [0031]      FIG. 5  is an exploded view illustrating the window and the spacer assembly within the environmental cell as viewed from the bottom. 
           [0032]      FIG. 6  is a diagrammatic representation of a small fluid path length within the E-cell. 
           [0033]      FIG. 7  is a diagrammatic representation of a second fluid path length within the E-cell. 
           [0034]      FIG. 8  is a diagrammatic isometric view of the holder tip illustrating the laser converging and reflection optics of the present invention. 
           [0035]      FIG. 9  is a diagrammatic sectional view of the environmental cell illustrating the laser beam striking the specimen. 
           [0036]      FIG. 10  is a schematic view of the complete in situ holder describing the complete optical assembly of the present invention. 
           [0037]      FIG. 11  is a diagrammatic functional illustration of the in situ holder integrated with a gas delivery and imaging system of a transmission electron microscope. 
       
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
       [0038]    The present invention will be described in detail with respect to its preferred embodiment which is a specimen holder for a transmission electron microscope.  FIG. 4  shows an isometric view of the complete in situ holder assembly  1 . Assembly  1  is of a conventional design, which, as known to those skilled in the art, may take a variety of forms to accommodate various microscopes produced by various manufacturers. The mechanical needs of each device result in varying geometries of barrel  50  with respect to length, diameter and location of components. Generally, assembly  1  is comprised a holder tip  2  which contains the E-Cell  4  and the primary operative components of the assembly. Holder tip  2  is supported and restrained at the appropriate location within the microscope by barrel  50  which may be designed with a variety of diameters and lengths. At least one O-ring  48  is disposed along the length of barrel  50  to seal the internal environment of the microscope from ambient air. A laser assembly  500 , or other electromagnetic radiation beam generator (as shown in  FIG. 10 ) is disposed near the non-operative end of barrel  50  and is positioned to be located outside of the microscope environment when in use. A electromagnetic radiation source connection  80  and fluids connectors  82  are provided to supply laser irradiation and the in situ environment, in accordance with common practice of those skilled in the art. It is to be specifically noted that the laser may be substituted with any appropriate electromagnetic radiation beam generator, including X-rays and visible light. 
         [0039]    Referring now to  FIGS. 3   a  and  3   b , specimen holder tip  2  includes E-Cell  4 . Lid  6  is slidably disposed on main body  12  of holder tip  2 . Main body  12  is provided with a track  11  which is adapted to receive lid  6  and constrain its limited slidable displacement. Displacement of lid  6  provides access to E-Cell  4  on the underside of main body  12 . A travel limiting stop  20  is provided at one end of main body  12  to restrict the travel of lid  6  in the open, or loading position, as shown in  FIG. 5 . Lid  6  may be slidably displaced from a position engaging stop  20  to a position engaging tangs  10  in the closed, or operative position, as illustrated in  FIGS. 3   a  and  3   b . Tangs  10  receive and restrain lid  6  in the operative position, as will be more fully described below. Travel of lid  6  in the closed position is further limited by travel stop  20 A. E-Cell  4  is a cylindrical cavity, nominally 3.1 mm in diameter and 650 μm deep to accommodate a standard 3 mm diameter specimen disk. 
         [0040]    E-cell assembly  42  is placed within the cavity while in the open position as more fully described with reference to  FIG. 5 . Once the E-Cell  4  is loaded, lid  6  is displaced to the operative position. Tangs  10  are resilient armatures having restraining profiles at the movable ends. Tangs  10  may therefore be displaced inwardly by applying manual pressure in a direction perpendicular to the longitudinal axis of main body  12 . In order to relieve lid  6  from restraint by tangs  10  in the operative position, tangs  10  are depressed and lid  6  is slidably displaced (to the right in  FIGS. 3   a  and  3   b ) to contact travel stop  20 . In order to engage lid  6  with tangs  10  for restraint in the operative position, lid  6  is merely slidably displaced (to the left in  FIGS. 3   a  and  3   b ) until tangs  10  engage a locking interface provided on lid  6  (not shown). A clamping mechanism for more securely engaging lid  6  to main body  12  when in the operative position is provided by clamp  6   a  which is slidably engaged with main body  12  along track  11 , as will be more fully described below. 
         [0041]    Main body  12  is provided with a series of recesses and conduits to accommodate fluid conduits  22  which will not be described further as being within the ambit of one skilled in the art. Fluid inlet and outlet conduits  22   a  and  22   b , respectively, are a means for the environmental fluid to enter and exit E-cell  4 . Although  FIGS. 3   a  and  3   b  illustrate cylindrical fluid inlet and outlet conduits, one of skill in the art will recognize that other appropriately shaped conduits will serve the purpose of supplying fluid to the specimen. Appropriate fluid connections are provided throughout holder assembly  1  to fluidly communicate with connectors  82  in a conventional manner. 
         [0042]    Mirror retainer assembly  14  is utilized to receive and support minor  15 , which adapted to reflect the laser beam onto the specimen, as more fully discussed below. Minor  15  is bonded to minor retainer  14  at a precise, preselected angle or may be dynamically adjustable by external control. Minor retainer  14  is removably affixed to main body  12  by mounting screw  18 . 
         [0043]    Referring now to  FIGS. 3 ,  5  and  9 , E-Cell components  42  are assembled as a precisely sized unit having a particular height dimension to assist in maintaining a vacuum seal between lid  6  and main body  12 . Main body  12  is provided with a mounting surface  100  disposed at the lower portion (as shown in  FIG. 5 ) of E-Cell cavity  101 . Mounting surface  100  is further provided with an O-ring receiving recess  102 , as shown in  FIG. 9 , of conventional design. O-ring  44   a  is located within this recess  102 . It is intended that the orifice within the E-Cell components  42  provides clear access for a laser beam to engage the specimen, as will be described more fully below. The first of the E-Cell assembly components, window frame  30   a , constructed of silicon, is mounted within the E-Cell cavity  101  immediately adjacent mounting surface  100  and in sealing engagement with O-ring  44   a . Window frame  30   a  is provided with a orifice  31  which is sized and shaped in any one of a variety of geometric shapes and is preferably square in two dimensions and frustopyramidal in three dimensions, with the larger end facing the incoming laser beam. An electron and electromagnetic radiation transparent membrane may be deposited on the orifice  31  and window frame  30   a  is presented as an integrated whole which is fluid impermeable. It is specifically noted that use of the membrane may be eliminated in certain applications to increase image resolution. Spacer  36   b  is mounted immediately adjacent window frame  30   a  and is disposed having an orifice  36   c  centrally located therein corresponding to orifice  31  of window frame  30   a . Orifice  36   c  is generally larger in dimension than orifice  31 . Specimen  38  is mounted immediately adjacent to spacer  36   b  and is typically a 3 mm diameter disk which has been appropriately thinned at the central point  82  for TEM imaging and analysis. Specimen  38  is optimally provided with an outer rim thickness of up to 200 μm. To obtain an electron transparent region, the specimen is thinned at the central region from a few nanometers to tens of nanometers. Other types of specimens can be particles dispersed onto a grid or FIB lamellae attached to a support structure. Spacer  36   a  and window frame  32   a  are provided with orifices  36   d  and  31   a , respectively, and are mounted similarly to the corresponding spacer  36   b  and window frame  30   a . The total assembly height is optimally 650 μm which corresponds to the E-cell cavity  101  depth. Spacers  36   a  and  36   b  act as thermal insulators and help obtain the desired fluid path length above and below the specimen, and further provide the interior space within the E-Cell  4  which contains the environmental fluid, as supplied to E-Cell cavity by fluid inlet conduit  22   a  and evacuated by fluid outlet conduit  22   b  in a conventional manner. E-cell cavity  101  is nominally designed to incorporate window frames having thicknesses ranging from about 75 μm to about 325 μm. The window membrane material must be electron transparent, able to withstand high temperature, pressure differentials in and around the chamber, and should not react with the fluid present within the chamber and may comprise, for example, silicon nitride, silicon oxide or amorphous silicon as dictated by user requirements. The thickness of window membranes  31 ,  31   a  is limited by the cell pressures desired within the E-Cell  4 . In one preferred embodiment, window membranes  31  and  31   a  are constructed from silicon nitride deposited on a silicon substrate using low-pressure vapor deposition techniques (LPCVD). It has been shown that a pair of 15 nm thick silicon nitride membranes are able to withstand a pressure differential of up to one atmosphere. Diffused scattering of the electrons passing through the membrane increases with increasing thickness, degrading the attainable resolution. The thickness therefore should be minimized. 
         [0044]    E-Cell assembly  4  is restrained within E-Cell cavity  101  by the action of lid  6 . Lid  6  is provided with an O-ring receiving recess  102   a , corresponding to recess  102  in main body  12 , for receiving and restraining O-ring  44   b . O-ring  44   b  provides a sealing engagement between lid  6  and window frame  32   a . This sealing engagement, when lid  6  is in the operative position, causes E-Cell  4  to be restrained as a unit within E-Cell cavity  101  for imaging and analysis. Additional sealing of the E-Cell cavity is provided by O-ring  44   c , disposed between main body  12  and lid  6 . Additionally, clamp  6   a  is slidingly engaged with lid  6  to more securely depress lid  6  into engagement with E-Cell  4 . Clamp  6   a  is provided with a wedge shaped armature  6   b  which is interposed between lid  6  and main body  12 . Once lid  6  is engaged with tangs  10  in the operative position, clamp  6   a  is slidingly displaced along track  11  (as shown in  FIG. 3   a ) such that armature  6   b  is increasingly interposed between lid  6  and main body  12  and its increasing height causes lid  6  to be pressed more completely against main body  12  on the side opposite armature  6   b . This causes lid  6  to more fully compress O-rings  44   a, b  and  c . It is to be specifically noted that those skilled in the art may utilize any sealing methodology other than O-rings to provide an enclosed environment for the E-Cell cavity  101  and other aspects of the holder assembly. This sealing mechanism provides the user with the flexibility of establishing a wide range of fluid lengths. The external height of the E-cell  4  is only 2.3 mm which is compatible with the objective pole pieces of most major commercially available TEMs. 
         [0045]    In certain embodiments, the thickness of the components of the E-cell  4  may be adjusted to achieve a particular fluid path length above and below the specimen. However, the total height of the E-cell assembly  4  should not exceed 650 μm +/−25 μm. Tables 1 and 2 illustrate two different configurations of the E-cell  4  components to achieve a path length of 250 μm and 10 μm respectively. This assembly is illustrated in  FIGS. 6 and 7 . 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 Example of Variable E-cell Assembly Height 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Window Frame Thickness 
                 320 μm × 2 = 640 μm 
               
               
                   
                 Specimen Disk Thickness 
                 10 μm × 1 = 10 μm 
               
               
                   
                 TOTAL THICKNESS 
                 650 μm 
               
               
                   
                 FLUID PATH LENGTH 
                  10 μm 
               
               
                   
                 CONFIGURATION 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 Example of Variable E-cell Assembly Height 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Window Frame Thickness 
                 200 μm × 2 = 400 μm 
               
               
                   
                 Specimen Disk Thickness 
                 200 μm × 1 = 200 μm 
               
               
                   
                 Top Spacer Thickness 
                 25 μm × 1 = 25 μm 
               
               
                   
                 Bottom Spacer Thickness 
                 25 μm × 1 = 25 μm 
               
               
                   
                 TOTAL THICKNESS 
                 650 μm 
               
               
                   
                 FLUID PATH LENGTH 
                 250 μm 
               
               
                   
                 CONFIGURATION 
               
               
                   
                   
               
             
          
         
       
     
         [0046]    Table 1 shows an E-cell  4  configuration for use with thin specimens. In this embodiment, the spacers are not utilized in the assembly. The fluid path length totally comprises the specimen thickness of 10 μm. Table 2 shows an E-cell  4  configuration for use with thicker specimens. As illustrated, the fluid path length is 250 μm, corresponding to the combined height of the specimen  38  and the top and bottom spacers  36   a  and  36   b . The major contributor to the relatively large fluid path length is the specimen  38  thickness. As illustrated in  FIG. 6  the fluid path length configuration is 10 μm and in  FIG. 7  the fluid path length configuration is 250 μm. The unique E-Cell  4  sealing mechanism provides the user with the flexibility to choose the desired fluid path length ranging from the specimen thickness to 500 μm. A fluid path length can be selected based on the required specimen temperature and the acceptable image resolution. If a very high specimen temperature is desired, it is recommended to have a greater fluid path length to minimize the negative effects of radiation. 
         [0047]    The use of a laser in the present system allows for high precision, localized heating of the TEM specimen. The laser optical components for this holder are illustrated in FIGS.  3  and  8 - 10 . A standard laser connector  80 , for example a SMA  905  laser connector, is provided at the handle  54  of the holder body ( FIGS. 4 and 10 ). Such laser connectors are well known in the art and therefore will not be explained in detail here. The desired laser  500  is connected to the holder using the SMA connector  80 . The laser beam  70  then enters a collimator  78 . Collimator  78  helps produce a parallel laser beam and prevents it from diverging as it travels along the length of the holder barrel towards the specimen tip  2 . The converging lens module  72  located at the holder tip  2  focuses the beam to a fine spot. Referring specifically to  FIGS. 3   a  and  3   b , converging lens module  72  is illustrated, having a slidable lens body  72 A which is disposed within barrel  50  such that it may be displaced along the longitudinal axis of the holder assembly  1  or may be angularly displaced to permit translation of laser beam  70  across the face of specimen  38 . An actuation rod (not shown) is inserted in port  176  and controls the longitudinal movement of lens body  72 A. This movement changes the focus and/or position of the laser beam and therefore the beam diameter at the point of contact with specimen  38 . A fluid line conduit  175  is located within lens body  72 A to permit the displacement of lens body  72 A without interference with the passage of the environmental fluid into E-Cell  4 . Lens  174  is partially visible in  FIG. 3   b  and comprises at least one movable element which is utilized to focus the laser beam. Lens body  72 A is laterally displaced with respect to main body  12  and is resiliently affixed thereto by springs  177 . The small diameter laser beam  70  strikes the laser mirror  15  and is reflected to a precise location on the specimen  38  within E-cell  4 . Depending on the wavelength of the electromagnetic radiation and the focal length of the converging lens, the focused beam spot size at the specimen can be varied from a few to hundreds of microns. The collimator  78 , converging lens module  72  and the mirror  15  are precisely aligned so that the laser beam  70  clears the window frame  30   a  through orifice  31  and strikes the specimen in the vicinity of the center point  82 . The window membrane is transparent to the laser beam  70  and does not absorb or reflect it. As a result, a radial symmetric heating zone is generated on the specimen. This allows for uniform expansion of the specimen at high temperatures, thus minimizing specimen drift. 
         [0048]    The laser optics of the present invention, i.e., collimator  78 , converging lens  72  and minor  15 , act together to precisely focus laser beam  70  onto the E-cell  4  to attain high specimen temperatures. The maximum temperature that can be attained on the specimen is limited largely by the material properties of the specimen and the laser  500  power, thus creating a potential for applications in an extraordinary range of fields. The inventors have found that less than 1 Watt of laser energy was required to raise the specimen temperature to 2,000° C. An additional advantage of the presently described specimen laser spot heating is the speed in which the steady state specimen temperature is achieved. Most specimen reactions occur instantly once a critical temperature is obtained. Standard TEM heating holders utilizing resistive heaters have a slow heating response time and it takes a considerable amount of time to reach a steady state specimen temperature. The laser optics utilized in the present holder achieves sub millisecond heating response times due to the small heating zone. As a result, steady state specimen temperature is achieved instantly. The laser beam  70  can easily be modulated to provide dynamic thermal cycling of the specimen between ambient and elevated temperatures. Pulsed lasers can be attached to the holder to provide pulses of energy within a time frame as small as few nano seconds. 
         [0049]    In addition, the laser heating system of the present invention is adjustable so that it may be used with a wide variety of specimens. The spot size of laser beam  70  may be adjusted by longitudinal displacement of lens body  72 A. This allows the flexibility of changing the laser power density. For example, it is possible to first melt a 10 μm hole in the specimen at high laser power density, thus locating the laser beam position within the microscope. The laser beam size may be increased to obtain the desired specimen temperature in the vicinity of the hole. 
         [0050]    Referring now to  FIG. 11 , the fluid flow assembly design has the provision of flowing up to four different gases simultaneously through the cell. The various gases are provided in conventional cylinders  205  which are each in fluid communication with mass flow controllers  207 . Mass flow controllers regulate the flow of gas under either manual or computer-operated control. A gas mixing chamber  209  is provided which combines the selected gases into a uniform composite which may be flowed to the holder assembly  1  through supply line  210 . Supply line  210  is affixed to the appropriate fluid connector  82  and subsequently to fluid supply conduit  22   a . The uniform gas mixture is then circulated into the E-Cell  4 . The continuous flow of gas is maintained with the help of the pressure differential generated between the inlet and the outlet ports of the holder by turbo molecular pump  215  mounted externally and the internal pressure of gas cylinder  205 . This pump in combination with diaphragm pump  220  and mass flow controllers  207  continuously flow the gas or gas mixture to supply the appropriate pressure within E-Cell  4 , by means of gas exhaust line  210   a  and gas supply line  210 . The primary consideration given towards the design of the gas flow system is the attainable pressure within E-cell  4 . Higher gas pressures can be achieved by switching off the differential pumps and maintaining a steady flow of gas into E-Cell  4 . The pressure inside E-Cell  4  may be varied by simultaneously pumping the cell and/or regulating the mass flow rate of the gases. Gas flow regulation as well as adjustments to the laser power may be either manually or computer controlled utilizing a standard computerized interface such as Labview, a program developed by National Instruments. 
         [0051]    Similarly, an external liquid circulation unit can be attached to the holder in a similar fashion to incorporate biological applications that require the flow of liquids through the cell. 
         [0052]    The terms and expressions which have been employed herein are used as terms of description and not as limitation, and there is no intention in the use of such terms and expressions of excluding equivalents of the features shown and described or portions thereof, it being recognized that various modifications are possible within the scope of the invention claimed. Although particular embodiments of the present invention have been illustrated in the foregoing detailed description, it is to be further understood that the present invention is not to be limited to just the embodiments disclosed, but that they are capable of numerous rearrangements, modifications and substitutions.

Technology Category: 5