Patent Abstract:
A method of forming extremely small pores in a substrate may be used to produce, for example, an apparatus for the study of biological molecules, by providing a small pore in a piezoelectric substrate having electrodes, the latter that may be energized to change the pore dimensions.

Full Description:
CROSS REFERENCE TO RELATED APPLICATION 
     This application is a continuation of U.S. application Ser. No. 12/614,237 filed Nov. 6, 2009 and hereby incorporated by reference in its entirety. 
    
    
     This invention was made with government support under 0520527 awarded by the National Science Foundation. The government has certain rights in the invention. 
    
    
     BACKGROUND OF THE INVENTION 
     The present invention relates to electrophysiology and “patch clamping” for investigating ionic and molecular transport through cellular membranes via ion channels and, in particular, to a substrate providing a set of nano to microscale pores that may be readily sealed to cellular membranes. Ion channel investigation using patch clamps often plays an important role in drug discovery and preliminary drug screening or evaluation, for example, by providing a model that shows an effect of a drug on ion channels. Doing so can be useful for either avoiding adverse effects or for creating a positive therapeutic effect for the treatment of ion channel related diseases. 
     Drug screening can require a large number of ion channel measurements. Accordingly, in current practice, planar patch clamps are preferable because they allow parallelization of multiple samples on a substrate, often referred to as a wafer, chip, or well-plate, and facilitate measurement automation. Each sample has a cell or cell wall that is positioned so that an ion channel in the cell or cell wall is aligned with a pore at the sample site. The cell or cell wall is sealed to the patch clamp substrate in a manner that allows a small amount of electrical current to be used in performing ion channel investigations, typically by way of an extremely high resistance seal between the patch clamp substrate and the cell wall (a gigaohm seal or gigaseal). Gigaohm seals achieved using on-chip patch clamp procedures usually have electrical resistance values of about 1 gigaohm, with resistance values of up to about 5 gigaohms being achieved in some relatively rare instances. 
     Planar patch clamp substrates can be made from, for example, silicon, Teflon®, PDMS (polydimethylsiloxane), PSG (phosphosilicate glass), or glass. While such materials prove suitable for many planar patch clamp implementations, a single crystal quartz (quartz) material can be a particularly desirable for making planar patch clamp substrates. Quartz exhibits particularly high electrical insulating properties and is piezoelectric. Its unique electrical characteristics allow it to be used as a patch clamp substrate by providing very low levels of background noise while performing ion channel investigations. Furthermore, quartz exhibits particularly good mechanical characteristics such as, for example, good hardness, thermal stability, and chemical stability characteristics. Despite a general recognition of quartz&#39;s suitability for use as a patch clamp substrate, many of its desirable characteristics, such as hardness, make fabricating (micromachining) the pores in a quartz substrate rather difficult and/or time consuming. 
     Traditionally, micromachining of quartz is performed using a combination of lithography and reactive ion etching (RIE). However, RIE techniques require multiple steps and are relatively slow processes. 
     Another method of micromachining quartz is by way of direct laser beam ablation. During direct laser beam ablation, a high power density, short pulse width femtosecond laser beam is irradiated directly onto quartz. The nonlinear interaction between the ultrafast laser pulses and quartz, which has a band gap of about 9 eV, results in a cyclic multiphoton absorption and electron excitation between the ground and excited states. During this process, the initial excited electrons induce an avalanche ionization and generate a microplasma which ablates the quartz. However, since quartz has a wide band gap, this approach is also slow and is limited in terms of pore diameter and material thickness that can be achieved. 
     Recently, numerous advances have been made in micromachining of pores in non-quartz substrates, for example, by utilizing nanosecond lasers, such as excimer lasers instead of femtosecond lasers. Excimer lasers, which emit ultraviolet (UV) light, have been successfully implemented in relatively fast drilling procedures in non-quartz materials. However, quartz has excellent optical transmission over a large spectrum, from UV to infrared (IR), whereby it is transparent to light(s) in this spectrum. Since quartz is transparent to and therefore substantially unaffected by UV light(s), it has been widely accepted that excimer lasers are not usable for micromachining quartz. 
     Furthermore, although various patch clamping and other techniques have been developed and, at least to some extent, standardized for successfully modeling and investigating ion channel function voltage-sensitive (or voltage-gated) ion channels, in-depth investigation of yet other types of ion channels, such as mechanosensitive ion channels, remains at least somewhat frustrating and/or impracticable. Accordingly, numerous molecular mechanisms and their functionalities within mechanosensitive ion channels remain unknown, whereby cellular responses to mechanical stimuli remain some of the least understood of the known sensory mechanisms. 
     SUMMARY OF THE INVENTION 
     The present invention provides an improved technique for the generation of nanoscale-sized pores, for example, pores having diameters of nearly 200 nm, using a laser through a substrate that is substantially transparent to the laser&#39;s emitted wavelength and therefore tends to be unaffected by the laser using previously known practices. In the technique, the substrate is backed by an energy absorbing material that has relatively high coefficients of thermal expansion and UV absorption. The laser light is transmitted through the substrate and focused adjacent a back side of the substrate, for example, at or immediately in front of or behind an interface defined between the substrate and energy-absorbing material. Doing so can increase a temperature of the energy-absorbing material which, in turn, heat the substrate from beyond its back side. Heating the energy-absorbing material in this manner increases the temperature of the substrate to an extent that melts the substrate, and correspondingly facilitates melting-type formation of small diameter smooth pores and can also lead to formation of a crater(s) at the back side of the substrate. According to some aspects of the invention, the crater can be formed by a shock wave that results from thermal expansion of the energy absorbing material, but in any event, is formed by ablation or other thermal related removal of material from the back side of the substrate. The pore and/or crater are therefore created by a sandwich drilling or sandwich drilling-like technique that can give the pore and/or crater, of the quartz chip, surface characteristics that are similar to surface characteristics of pipettes that have been fire-polished with open flame procedures. In other words, the pore(s) and/or crater(s) formed according to aspects of the present invention have surfaces that are substantially smooth, their surface irregularities having been removed or attenuated by surface tension induced flowing of melted material in such irregularities which tends to smooth the same. 
     Specifically then, the present invention provides a method of creating nanoscale holes by using steps including creating a multi-layered assembly that comprises a substrate material and an energy absorbing material. The substrate material receives the nanoscale hole and defines a front side and an opposing back side. The energy absorbing material is adjacent the back side of the substrate material. A laser is applied through the multi-layered assembly, by initially passing through the substrate material and being focused at and absorbed by the energy absorbing material, increasing a temperature of the back side of the substrate material to a greater extent than an increase in temperature of the front side of the substrate material. Continued application of such laser stimulus to the energy absorbing material may correspondingly heat and/or pressurize the back side of the substrate, which can initiate a drilling of or establishing a pore through the substrate. 
     It is therefore an object of at least one embodiment of the invention to utilize a laser to drill a pore in a substrate by indirectly heating the substrate by focusing the laser at or near an interface between the substrate and an energy absorbing material. This allows for laser micromachining of a material that might otherwise be transparent to light of a wavelength emitted by the laser. 
     The method can further include a step of producing a crater at the back side of the substrate material which, in some embodiments, can be produced by a shock wave that increases both a temperature and a pressure at an interface defined between the back side of the substrate material and the energy absorbing material. The crater formation can be an initiating step for drilling of the pore and the pore, in some embodiments, is drilled in a drilling direction that opposes a direction of laser transmission through the substrate. 
     It is thus an object of at least one embodiment of the invention to provide a method for forming craters in back sides of substrates and/or drilling pores from the crater toward the front side of the substrate. 
     The substrate material may be a single crystal quartz material and the laser can be a UV emitting excimer laser or other lasers, e.g., CO2 lasers, and/or others . . . . 
     It is therefore an object of at least one embodiment of the invention to generate pores and/or craters in a single crystal quartz wafer or chip and may be a further object of at least one embodiment of the invention to perform laser micromachining on a substrate that is substantially transparent to a wavelength of light that is emitted by the laser. 
     The energy absorbing material may be a liquid media, for example, an ultraviolet radiation absorbing organic liquid such as acetone and/or fluorescence immersion oil. Such liquid media can have a thermal expansion coefficient of at least about 700×10-6 K −1  and an ultraviolet absorption coefficient of at least about 1.46 cm −1 , for example, an ultraviolet absorption coefficient of between about 1.46 cm −1  to 1.65 cm −1 . 
     It is thus an object of at least one embodiment of the invention to provide a liquid medium for use as an energy absorbing material that can be placed under the substrate for performing a laser induce backside etching of the back side of the substrate. The backside etching may be performed on a quartz chip that has been pre-etched or thinned from an initially thick substrate. The initially thick substrate may be about 100-500 microns thick and can be either laser or wet etched, for example, with a buffered oxide etchant (BOE) or hydrofluoric (HF) etchant, in a pre-thinning procedure, down to about 20-50 microns in thickness before the nanopore laser drilling is initiated, which may improve suction and reduce series resistance. 
     The pore can have a smaller diameter than the crater which it intersects, and the pore diameter can be generally constant along its length or a major portion thereof, and a crater sidewall can include an undercut or groove extending thereinto. 
     It is thus an object of at least one embodiment of the invention to provide a patch clamp chip that has a smooth pore that opens into a relatively larger diameter crater. This configuration can provide suitable structure to which a cell can be mounted. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a schematic diagram of an apparatus used for producing a planar patch clamp chip or wafer per from a multi-layered assembly one embodiment of the present invention; 
         FIG. 2  is a pictorial view of the multi-layered assembly used in the machine of  FIG. 1 ; 
         FIG. 3  is a cross-section along line  3 - 3  of  FIG. 2  showing a spacing of a substrate from a backer plate by a gap filled with an energy absorbing material; 
         FIG. 4  is a figure similar to that of  FIG. 3  showing an initial stage of laser micromachining in which a laser beam is focused toward an interface between the substrate and energy absorbing material; 
         FIG. 5  is a figure similar to that of  FIG. 3  showing transmission of energy through the substrate into the energy absorbing material after a crater has been formed in a back side of the substrate; 
         FIG. 6  is a figure similar to that of  FIG. 5  showing an initiation of a pore drilling procedure; 
         FIG. 7  is a figure similar to that of  FIG. 5  showing the pore after completion of the drilling procedure; 
         FIG. 8  is an enlarged scanning electron microscope image of a substrate after focused ion beam milling to show a cross-section of a first embodiment of a substrate that was laser micromachined according to methods of the invention; 
         FIG. 9  is an enlarged scanning electron microscope image of a pictorial view of a variant of the substrate of  FIG. 8 ; 
         FIG. 10  is an enlarged scanning electron microscope image of a pictorial view of another variant of the substrate of  FIG. 8 ; 
         FIG. 11  is an enlarged scanning electron microscope image of a pictorial view of a second embodiment of a substrate that was laser micromachined according to methods of the invention; 
         FIG. 12  is a simplified representation of the use of the substrate of  FIG. 7  in a patch clamp application; and 
         FIG. 13  is a simplified representation of another use of the substrate of  FIG. 7  in a patch clamp application that incorporates piezoelectric actuation controls. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     Referring now to  FIG. 1 , the present invention may use an ArF excimer laser  10  having collimating and focusing optics  11  to direct a narrow collimated beam  12  of light along an axis  15  toward a back surface of a sandwich-like, multi-layered assembly  14  that is held on a mechanical stage  16 . The laser may, for example, have a wavelength range of about 155 nm to about 195 nm and is preferably operated to emit a beam  12  of light having a wavelength of about 193 nm. In other embodiments, laser  10  may be yet other types of lasers, such as CO2 lasers, and/or others, depending on, for example, a desired wavelength that is selected based on particular characteristics of components within the multi-layered assembly  14 , in other words, how the multi-layered assembly  14  reacts to exposure to light having such wavelength(s), and/or other factors. 
     The laser  10  may include a variable attenuating mirror for controlling how much of the laser beam is to be transmitted, and a stencil metal mask with an adjustable aperture that allows production of different laser beam shapes and sizes. Laser  10  may further include a stage  16  that can be controlled by an automated controller  18  of the type well known in the art for providing control signals  22  to the laser  10 , controlling its output power in a series of pulses as will be described and providing control signals  22  to actuator motors  24  providing x-y control of the stage  16 . 
     Referring now to  FIGS. 2 and 3 , the multi-layered assembly  14  may include a substrate  26 , an underlying energy absorbing material  34 , and a backer plate  32  that supports the energy absorbing material  34  and substrate  26 . The energy absorbing material  34  cooperates with the substrate  26  to perform a one-step micromachining process in which the laser  10  drills through the substrate  26 , forming small diameter smooth holes therethrough, while causing a nominal amount of surface roughness to the substrate  26 . For example, a synergistic relationship between the energy absorbing material  34  and the substrate  26  allow for using the laser  10  to perform a one-step micromachining process to create holes or pores that have diameters as small as 200 nm, while resulting in an end-of-procedure surface roughness of merely tens of nanometers as measured from the respective surface(s) of the substrate  26 . Furthermore, submicrometer holes, bores, or pores may be formed in the substrate  26  along with crater-shaped depressions that are formed in a surface of the substrate that faces a direction of back from the substrate. Such relationships between the components of multi-layered assembly  14 , and techniques for forming such hole, pores, and craters in the one-step micromachining procedure, are discussed in greater detail elsewhere herein. 
     Still referring to  FIGS. 2 and 3 , within the multi-layered assembly  14 , substrate  26  can be, for example, a clear solid material that is transparent to the UV light emitted by laser  10  ( FIG. 1 ), preferably a single crystal quartz chip or wafer. A suitable such single crystal quartz wafer for use as substrate  26  is the 350 μm thick quartz wafer that is readily available from the University Wafer Company of South Boston, Mass. 
     A front surface of the upper substrate  26  may have a series of depressions or wells  28  formed at regular x-y grid locations  29 . The wells  28  provide a thinned portion  30  at the locations  29  measured along axis  15  having a thickness of 100 to 1000μ and may be molded, ground, or etched in the substrate  26 . The diameter of the wells  28  may be relatively large, for example, 5.0 mm, and serve simply to permit a generally thicker substrate  26  in regions outside of the locations  29  for structural convenience. Referring specifically to  FIG. 2 , the substrate  26  can define a single, unitary structure with multiple wells  28  formed therein. However, it is also contemplated that substrate  26  can be an assembly of multiple, relatively smaller, individual quartz wafers or chips, each having only a single well  28  formed therein. 
     Referring yet further to  FIGS. 2 and 3 , in some embodiments, backer plate  32  is positioned as a lowermost component of the multi-layered assembly  14  and can serve as, for example, a structural base or part of a structural frame thereto. Backer plate  32  may be a supportive slide that is incorporated into the multi-layered assembly  14 , underlying and being spaced vertically below the substrate  26 . Optional spacer  31  is one suitable structure that can be used to establish such vertical spacing between the substrate  26  and backer plate  32 . The spacer  31  can be formed out of, for example, polydimethylsiloxane (PDMS). The PDMS may be cast on the rear surface of the substrate  26  through a mold produced using integrated circuit techniques to provide precisely controlled spacer thickness or may be spun-coated and selectively removed except at the edges of the substrate  26 . Regardless of the particular manner in which the PDMS is incorporated into the multi-layered assembly  14 , it is preferably configured to form a chamber defined by the PDMS at its outer perimeter and defining a space between the substrate  26  and backer plate  32 . 
     The space between the substrate  26  and the backer plate  32  is filled with energy absorbing material  34  which can be a liquid media and, in some embodiments, a UV absorbing organic liquid. The energy absorbing material  34  has thermal expansion and/or UV absorption coefficients that are large enough to heat and/or squeeze the substrate  26  to an extent that facilitates, catalyzes, or initiates laser drilling of the substrate  26 . The energy absorbing material  34  can have a thermal expansion coefficient of at least about 700×10 −6  K −1  and a UV absorption coefficient of at least about 1.46 cm −1 , preferably being within a range of between about 1.46 cm −1  to 1.65 cm −1 . Suitable UV absorbing organic liquids for use as energy absorbing material  34  include, for example, acetone and fluorescence immersion oil, along with other suitable materials that may increase temperature when exposed to UV radiation to an extent that may correspondingly heat the substrate material, interfacing therewith, so as to melt the substrate material. Examples of other suitable energy absorbing materials  34  include, but are not limited to, pyrene, naphthalene, and toluene, and/or other materials. 
     Still referring to  FIGS. 2 and 3 , the particular amount of volume of energy absorbing material  34  is selected based on the intended configuration of multi-layered assembly  14 , and laser drilling techniques that are implemented, and intended end-use configuration and characteristics of substrate  26  for its use in a patch clamping setup. In some embodiments, a relatively small amount of energy absorbing material  34  is used by sandwiching a thin layer of the energy absorbing material  34  between backer plate  32  and substrate  26 , without incorporating spacers  31 . In other embodiments, such as the one illustrated in  FIG. 2 , a relatively greater volume of energy absorbing material  34  is provided, with the particular volume being a function of the space between the backer plate  32  and substrate  26  as dictated by spacer  31 . 
     Regardless of whether the energy absorbing material  34  is implemented as a thin layer that is tightly sandwiched between the substrate  26  and a backer plate  32  substantially adjacent and below the substrate  26 , or implemented as a thicker layer that fills a larger space between the substrate  26  and backer plate  32  as dictated by spacer  31 , the energy absorbing material  34  and substrate  26  are preferably in a face-to-face or an abutting relationship with respect to each other. In such configuration, the substrate  26  and energy absorbing material  34  define an interface  35  therebetween. Interface  35  allows the energy absorbing material  34  to transmit heat and/or pressure to the substrate  26  with relatively little energy loss in so doing. 
     Referring now to  FIGS. 1 and 4 , the laser  10  may be positioned above a first location  29  and pulsed by the controller  18  to produce a series of controlled light pulses  40  of laser beam  12  that is focused at the interface  35  or substantially adjacent or proximate thereto. The light pulses  40  are directed toward a front side  46 , passing through an outer surface  47  thereof. Since substrate  26  is substantially transparent to UV light, the light pulses  40  pass through the entire thickness of the substrate  26 , leaving the substrate  26  through an outer surface  49  of a back side  48  of the substrate  26 . At this point, the substrate  26  is substantially unheated or otherwise affected by the light pulses  40 , at least compared to the temperature increase, thermal expansion, and/or other responses of the energy absorbing material to the stimulus of the light pulses  40 . 
     Still referring to  FIGS. 1 and 4 , the laser  10  indirectly heats the substrate  26  by primarily heating the energy absorbing material  34  which, in turn, secondarily heats the substrate  26  at its back side  48  or from below. In particular, the light pulses  40  that are focused at the interface  35 , heat the energy absorbing material  34  and therefore also the outer surface  49  of the back side  48  of substrate  26  to establish rapid increases temperature and pressure at the interface  35 . This correspondingly leads to rapid thermal expansion of the energy absorbing material  34  and/or exit side  48  of substrate  26  that can lead to an ablation of material from the exit side  48  or, in some embodiments, create a shock wave  52  at the interface  35 . In any event, a one-step emission of the laser  10  may be used for wholly fabricating a crater  53  and/or pore  54  in the substrate  26 , explained in greater detail elsewhere herein. The particular configuration and characteristics of the crater  53  and/or pore  54  are influenced by, amongst other things, the particular setup of the laser  10  and its output qualities, described in greater detail elsewhere herein, whereby the craters  53  and/or pores  54  are give desired sizes, shapes, and/or other characteristics by selecting a corresponding setup and output qualities of the laser  10 . 
     Referring now to  FIGS. 5-8 , shows an embodiment of crater  53  that can be a disc-shaped depression extending into the back side  48  of the substrate. Crater  53  has a relatively flat bottom wall  102  that can have a circular perimeter shape and extend generally parallel to the outer surface  49  of back side  48 . Seen best in the SEM (scanning electron microscope) image of  FIG. 8 , crater  53  of this embodiment includes a sidewall  105  that may taper slightly inwardly toward the bottom wall  102 . An upper portion of sidewall  105 , in its orientation of  FIG. 8 , transitions into the outer surface  49  of back side  58  by way of an arcuate surface that may have been at least partially smoothed during its formation. The smoothing is analogous to the smoothing achieved on an exponentially larger scale with open flame fire polishing, for example, while fire polishing a pipette or some other structure that is exponentially larger than the crater  53  and/or pore  54 . 
     Intuitively, overall dimensions of the crater  53  are functions of dimensions and characteristics of the sidewall  105 . Stated another way, crater  53  defines a crater depth which corresponds to a height dimension of the sidewall  105 . A crater width or diameter is defined by a (greatest) distance measured between facing surfaces of the sidewall  105 . As can be extrapolated from the size scale provided in  FIG. 8 , crater  53  may have a crater diameter of about 40 μm and a crater depth of about 10 μm. 
     In some embodiments, along at least part of the perimeter of crater  53 , an undercut  110  extends radially into the sidewall  53 , between the sidewall and the bottom wall  102 . Such undercut  110  may define a groove as an interlocking structure into which portions of a cell can flow under certain conditions, allowing parts of the cell&#39;s membrane to engage against, for example, a projecting shoulder defined between the undercut  110  and sidewall  105 . 
     Referring now to  FIGS. 7 and 8 , after completion of the laser micromachining of substrate  26 , a smoothed annular edge or shoulder can define an opening between the crater  53  and a pore  54 , such opening being labeled as a pore-crater opening  153 . Like various portions of the crater  53 , the pore-crater opening  153  can be fire polished and smoothed so as to eliminate substantially all surface irregularities. Seen best in  FIG. 8 , the pore-crater opening  153  maybe located in the middle of bottom wall  102  of the crater. 
     The pore  54  extends between the pore-crater opening  153  and a pore-surface opening  147  that opens into the pore  54  from the outer surface  47  of the front side  46  of substrate  26 . Accordingly, in embodiments of substrate  26  that include a crater  53  formed thereinto, pore  54  extends between the crater  53  and the front side  46 . In embodiments of substrate that do not include a crater  53 , the pore  54  extends between the front and exit sides  46 ,  48 , in other words, through the entire thickness dimension of the substrate  26 . Regardless of the particular end-use configuration of substrate  26 , pore  54  has a substantially constant diameter along at least a major portion of its length. Accordingly, the pore-surface and pore-crater openings  147 ,  153  may define opening diameters that are about the same size, for example with the larger of the two openings being no more than about 50% greater than the smaller of the two openings. As another example, the pore  54  can define minimum diameter and maximum diameter segments along its length, with the maximum diameter segment being no more than about 50% greater in magnitude than the smaller diameter segment. 
     Referring to  FIGS. 4-8 , a crater  53  and/or pore  54  may be formed into substrate  26  of the multi-layered stack  14  in the following way. The multi-layered stack  14  is assembled and the laser  10  is set up to based on intended characteristics of the crater  53  and/or  54 , such as pore diameter and/or others. As discussed above, light pulses  40  are focused away from the front side  46  of the substrate  26  and toward its interface  35  with the energy absorbing material  34 . Doing so causes a thermal expansion and also pressure increase of the affected material(s) within the multi-layered stack  14 , which may be largely the energy absorbing material  34  at this early stage. Correspondingly, the interface  35  and the back side  48  of the substrate  26  may be secondarily affected by the changes occurring within the energy absorbing material  34 . Such secondary affects may be an indirect heating characteristic of laser  10 , by way of the substrates&#39;  26  intimate interaction with the energy absorbing material  34 . 
     For example, by focusing the light pulses proximate the interface  35 , increasing temperature and pressure of the energy absorbing material  34  can be transmitted to the substrate  26 , establishing a localized zone of increasing temperature and pressure of the back side  48  nearest the point of focus of the light pulses  40 . This may cause temperature and pressure differentials between the front and exit sides  46 ,  48  of the substrate  26  but in any event will increase temperature and pressure at the back side  48 . When such values increase enough, a crater  53  and/or pore  54  can be established by way of this a one-step micromachining procedure. 
     Referring now to  FIGS. 5-7 , although the Applicant does not wish to be bound by a particular theory, it is contemplated that the formation of crater  53  may be an initiator of the drilling of pore  54 . In such embodiments, once crater  53  is formed, the near molten material of bottom wall  102  is more receptive to accepting energy from or, in other words, is less transparent to the UV from laser  10  than material in a room temperature at-rest substrate  26 . Accordingly, due to the preheating of bottom wall  102  during establishment of the crater  53 , the light pulses  40  are able to further ablate material at their point of interaction with the preheated bottom wall  102 , allowing the light pulses  40  to pierce therethrough and begin formation of the columnar pore  54 . 
     Referring now to  FIGS. 6 and 7 , while the emission of light pulses  40  continues, so does the ablation or melting away of more material at the particular location which the light pulses  40  pass through the material of the substrate ( FIG. 6 ). In this regard, the pore  54  may propagate upwardly toward the front side  46  of the substrate so as to define a drilling direction “D” that opposes the direction of the light pulses  40  passing through the substrate  26 . In yet other embodiments, the pore  54  is not formed in the drilling direction “D” but instead, the drilling direction can extend in the same direction as the light pulses  40  passing through the substrate  26 , whereby the pore  54  drilling may originate at the outer surface  47  of the front side  46 . Although the terms “front” and “back” in describing various portions of the substrate  26  have been implemented in a convenient sense, it is fully contemplated that the by, for example, inverting the arrangement of the components of stack  14 , or positioning the laser  10  on the opposing side of the stack  14 , such terms might then assume generally opposite means. In other words, regardless of the particular orientation of the stack  14  and/or relative positions between the stack  14  and laser  10 , in preferred embodiments, the drilling direction “D” extends from the interface  35  between the energy absorbing and substrate materials  34 ,  26 , toward the side or outer surface of the substrate  26  that opposes the energy absorbing material. That is, the drilling direction “D” typically extends away from the location where the shock wave  52  occurred, whether such direction is the same as or opposite to the direction of laser  10  emission. 
     Regardless of how the pore  54  is created in a particular embodiment, the pore characteristics such as, for example, pore diameter, may be controlled or manipulated at least to some extent by adjusting the set up and controls of the laser  10 . For some crater  53  and/or pore  54  formation procedures, the laser output power may be fixed at 5 W and a variable attenuator of the laser  10  can be set to allow about 70%, for example, 73% of total beam transmission, and operated for 2500 pulses at a 100 Hz repetition rate for the light pulses  40 . Yet other set ups are contemplated, again, based on the intended characteristics of the crater  53  and/or pore  54  that are being formed. 
     For example, Table 1 below illustrates how pore diameter can be influenced by changing transmission rate or repetition rate of the emitted light pulses  40 , from 50 Hz pulses to 100 Hz pulses during their emission from the laser  10 . To establish such data of Table 1, laser energy was fixed at 50 mJ and six drilling schemes (Recipes) were tested to show various combinations of the two transmission rates, and the corresponding influence on pore diameter. 
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE I 
               
             
             
               
                   
               
               
                 Drilling Recipes for Two Different Transmission Rates and Their Results 
               
             
          
           
               
                   
                   
                   
                 Pore Diameter 
                 Pore Diameter 
               
               
                   
                 50 Hz 
                 100 Hz 
                 (μm) at 83% 
                 (μm) at 87% 
               
               
                 Recipe 
                 Pulses 
                 Pulses 
                 Transmission Rate 
                 Transmission Rate 
               
               
                   
               
             
          
           
               
                 1 
                 3000 
                 0 
                 0.216 a   
                 3.841 
               
               
                 2 
                 500 
                 2000 
                 0.621 
                 4.168 
               
               
                 3 
                 1000 
                 4000 
                 0.342 
                 4.455 
               
               
                 4 
                 1500 
                 3000 
                 0.395 
                 4.511 
               
               
                 5 
                 2000 
                 2500 
                 1.594 
                 5.270 
               
               
                 6 
                 2000 
                 4000 
                 3.210 
                 5.421 
               
               
                   
               
               
                   a Note: This approaches the laser wavelength limit of 193 nm. 
               
             
          
         
       
     
     Referring generally to  FIGS. 8-11 , the different configurations of these different embodiments show that, similar to the data in Table 1, influencing output characteristics of laser  10  may be used to form craters  53  and/or pores  54  that have different features, configurations, and/or other characteristics, as desired. Namely, using (relatively) higher transmission rates for laser  10 , for example, transmission rates of greater than about 80%, produces craters  53  with flat bottom walls  102 , like those seen in  FIGS. 8-10 . Using (relatively) lower transmission rates for laser  10 , for example, transmission rates that are less than about 80%, produces craters  53  that have tapering sidewalls and substantially no discernible bottom wall, like that seen in  FIG. 11 . 
     Specifically regarding the embodiments of  FIGS. 8-10 , of the embodiments of craters  53  that have flat bottom walls  102  or semispherical configurations, crater depth and sidewall configuration can also be controlled by the transmission rate or power of laser  10 . Of the three different embodiments shown in  FIGS. 8-10 , the embodiment of crater  53  of  FIG. 10  has the most shallow crater depth and also has a semicircular or concave-up, arcuate transition between the bottom wall  102  and the sidewall  105 . The crater  53  of  FIG. 10  that was formed with a transmission rate of laser  10  of about 83%. 
     Again comparing the embodiments of  FIGS. 8-10 , the crater  53  of  FIG. 9  has a deeper crater depth than that of  FIG. 10  but is shallower than that of  FIG. 8 . The crater  53  of  FIG. 9  has a similar semicircular or concave-up, configuration to that seen in  FIG. 10 , with a somewhat flatter bottom wall  102  when compared thereto. The crater  53  of  FIG. 9  was formed with a transmission rate that is between 83% and 87% which was used in making craters  53  of  FIGS. 10 and 8 , respectively. The crater of  FIG. 8  has been previously discussed and, when compared to the craters  53  of  FIGS. 9 and 10 , has the deepest crater depth and the flattest bottom wall  102 , deviating the most from a semicircular cross-sectional configuration. As discussed elsewhere in greater detail, the crater  53  of  FIG. 8  includes an undercut  110  that is defined between the bottom and sidewalls  102  and  105 . 
     Referring now to  FIG. 11 , this embodiment of crater  53  is made by setting the laser  10  to a relatively low transmission rate, for example, a rate of about 74%. This sub-80% transmission rate forms a crater  53  that appears trumpet-like or arcuately tapering in cross-section, such that the sidewall  105  tapers conically down to where it connects to the pore  54 . The sidewall  105  can also, in some related embodiments, have scales or patterned discontinuities across surfaces thereof. 
     Referring now generally to  FIGS. 12-13 , regardless of the particular configuration of crater  53  and pore  54 , after the substrate  26  has been laser micromachined into a usable wafer or chip having a crater  53  and/or pore  54 , it can be implemented into a suitable investigative tool or instrument, depending on the particular intended end-use research or study that will be performed. It has already been shown that the craters  53  of the invention can provide gigaohm seals that are not only satisfactory in performance, but may be notably superior when compared to currently known techniques. For example, whereas known methods of glass chip production yield about 60% of produced units that can achieve 1.0-1.5 gigaohm seals, and a substantially lower percentage that can achieve about 5.0 gigaohm seals, preliminary mockup productions of glass chips using the inventive procedure(s) have already successfully produced a substantial percentage of produced units that have achieved 7.0 or greater gigaohm seals. In view of such promising preliminary results, it is fully contemplated and expected that the inventive methods disclosed herein are fully capable of producing chips that achieve or approach 15 gigaohm sealing capabilities. 
     Referring specifically now to  FIG. 12 , in some embodiments, the substrate  26  can be used in a planar patch clamp apparatus to investigate ion channel performance. As one example of such investigation, the substrate  26 , shown having the same orientation as seen in  FIG. 8  and therefore an inverted orientation with respect to that shown in  FIG. 7 , may receive a cell  60  within the crater  53  to expose a portion of the cell wall  62  to be accessible through the pore  54 . A light suction applied by a pump  67  from the front side  46  may adhere the cell wall  62  to the surface of crater  53  with a 5 to 30 gigaohm resistance between a solution  64  on the side of the substrate  26  holding the cell  60  and a solution  66  on the side of the substrate  26  opposite solution  64 . The application of suction may correspondingly also pull a portion of the cell wall  62  into the undercut  110  in a manner that enhances the seal of the cell  60  to the substrate  26  by way of the mechanical interlocking relationship therebetween. Although the cell  60  is shown in  FIG. 12  as having its membrane or cell wall  62  ruptured over the pore  54 , it is, of course, contemplated that the cell wall  62  remains intact for various other types of studies or investigations. 
     A sharp suction applied by a pump  67  at the outer surface  47  of the front side  46  or other means may be used to provide electrical connection to the interior of the cell  60  by a sensitive electrical detector  70  permitting measurement of electrical differences between the exterior and interior of the cell  60  through an electrode  72  communicating with the interior of the cell  60  referenced to solution  64  outside the cell  60 . 
     Referring now to  FIG. 13 , since preferred embodiments of substrate  26  are made from a single crystal quartz material, such embodiments may be incorporated into an apparatus to investigate mechanosensitive ion channel performance and function. The apparatus of  FIG. 13  is largely analogous to that of  FIG. 12 , only being configured to piezoelectrically actuate, stress, or otherwise stimulate the cell wall  62  so as to measure, by way of detector  70 , gating responses of the particular ion channel that cooperates with the pore  54 . 
     Still referring to  FIG. 13 , a barrier  200  can be provided that acts as an enclosure, retaining the solution  64  therein. Ends of the substrate  26  extend through opposing sides of the barrier  200 . A power source  210  provides electrical stimulus for stimulating the piezoelectric behaviors of the substrate. A controller  220  sends and controls an electrical signal to the substrate, through conductors  225  that lead to the substrate. The actual connection(s) of the conductors  225  to the substrate  26  can be accomplished with suitable terminals. For example, terminals  230  and  235  are attached to the opposing outer surfaces  46  and  47  at a first end of the substrate  26 , appearing as a left end in  FIG. 13 , and connected to the controller  220  by a first pair of conductors  225 . Terminals  240  and  245  are attached to the opposing outer surfaces  46  and  47  at a second end of the substrate  26 , appearing as a right end in  FIG. 13 , and connected to the controller  220  by a second pair of conductors  225 . 
     Referring yet further to  FIG. 13 , in such an embodiment, the controller  220  may place the cell wall  62  under compressive and/or tensile stresses along multiple axes of movement or actuation. Depending on the particular cut of the crystal, controller  220  may establish a voltage across the thickness of the substrate  26 , specifically by establishing a voltage between the upper terminals  235 ,  245  and the lower terminals  230 ,  240 . Depending on the polarity of the signal, doing so will cause the substrate  26  to compress or elongate with respect to its thickness dimension which correspondingly compresses or stretches the cell wall  62  in such direction. Here too, depending on the particular cut of the crystal, controller  220  may establish a voltage across the length of the substrate  26 , specifically by establishing a voltage between the left end terminals  230 ,  235  and the right end terminals  240 ,  245 . Again depending on the polarity of the signal, doing so will cause the substrate  26  to compress or elongate, only this time with respect to its length dimension, compressing or stretching the cell wall  62  in a corresponding manner. While doing so, the detector  70 , senses notable gating and/or other responses of the particular ion channel or other portions of the cell  60 , depending on the particular configuration of detector  70 . 
     Referring yet further to  FIG. 13 , regardless of the particular configuration of the cell membrane investigating apparatus and its setup and controls, the substrate  26  according to the invention provides an “on-chip” piezoelectric system that may be used as a suitable alternative for imposing mechanical deformations to a membrane which to date has been primarily studied with pipettes and conventional patch clamping. Substrate  26  can be configured and implemented specifically for ones of, e.g., (i) lower frequency actuation of the ion channels, (ii) higher frequency mechanical probing, as well as (iii) static stress modulation of the membrane, as desired based on the particular investigation being performed. 
     It is specifically intended that the present invention not be limited to the embodiments and illustrations contained herein, but include modified forms of those embodiments including portions of the embodiments and combinations of elements of different embodiments as come within the scope of the following claims.

Technology Classification (CPC): 1