as illustrated in the schematic of fig1 , an apparatus 10 ( as described below ) is illustrated schematically for intracellular electro - manipulation which includes a power supply 12 ( a high voltage power supply , e . g ., 20 kv or higher ) and a delivery system 14 may be adapted to direct the electric pulse , particularly a nanosecond pulsed electric field ( nspef ), output to a load 16 such as target cells positioned in proximity to the delivery system 14 . the pulse generator may include a pulse forming network 18 and a high voltage switch 20 , such as a spark gap . the pulse forming network 18 may be a high voltage cable , a strip - line , or a pulse forming network constructed of individual capacitors and inductors in a transmission line arrangement with a matching network 22 . the high voltage switch 20 can suitably be a gaseous , liquid or solid state switch , e . g ., a spark gap . the energy in the pulse forming network 18 may be stored capacitively , which utilizes a closing switch 20 to release a pulse , or inductively , which requires an opening switch to release a pulse . upon triggering of the switch 20 , an electrical pulse is delivered through the delivery system 14 and launched into the load 16 , e . g ., the target cells in suspension or tissue form . the switch 20 can be triggered by a variety of common methods , e . g ., optically or electrically . the power supply 12 may utilize a high voltage dc power supply ( e . g ., matsusada rb30 - 30p ) to charge up , e . g ., a coaxial cable ( such as through a current limiting resistor ), to a high voltage creating a capacitor on the coaxial cable between the inner conductor and its outer conductive shielding . when the inner conductor is rapidly brought to ground by a switch 20 , a corresponding pulse of high voltage with , e . g ., 5 - 15 ns rise and fall times , is generated across the load . the duration of this pulse may be determined by the length of cable used , and the amplitude may be determined by the voltage at which the coaxial cable was charged . thus , in one variation , a coaxial cable having a length of , e . g ., 20 meters , may be used to generate a pulse duration of , e . g ., 100 ns . by this method , it is possible to precisely control the number of pulses delivered as well as the frequency of those pulses simply by controlling the discharge switch for the coaxial cable &# 39 ; s inner conductor . the generator 10 itself ( including all components ) may be sufficiently portable to fit into a rolling suitcase . to control the system , a microcontroller such as a digital programmable logic device ( e . g ., microchip pic16f887 ) may be incorporated into the assembly to control the pulse delivery . the user may input information to the microcontroller - based system via , e . g ., a keypad , and a liquid crystal display ( lcd ) may be implemented to display information to the user . the circuit may be battery - powered . the “ load ” 16 , which includes the target cells in tissue or suspended in a medium , is placed between two or more electrodes . these electrodes may be solid material ( in any of a number of suitable shapes , e . g ., planar , cylindrical , spherical , etc ), wires or meshes or combinations thereof . one ( set of ) electrode ( s ) is connected to the high voltage connection of the pulse generator , and a second ( set of ) electrode ( s ) is connected to the ground connection of the pulse generator in a suitable manner , e . g ., via a second stripline or high voltage cable . the electrode material is a conductor , most commonly metal . if such a pulse - forming network is charged up to , e . g ., 18 kv , and then released , this charge can produce an almost rectangular ultra - short duration pulse which when applied to a load equal to twice the cable impedance can produce a maximum voltage of 18 kv . the corresponding electric field intensity between two electrodes separated by 1 . 0 mm is 180 kv / cm . the maximum electrical power , v 2 / r , which can be achieved with these conditions is 3 . 24 mw ( assuming r = 100ω ), while the energy ( power x pulse duration ) transferred into the load is only 0 . 32 joule if the pulse duration is 100 ns . for a 100 μl volume of cell suspension , this energy transfer results in a calculated maximum temperature increase of only about 1 k for a single pulse . as mentioned above , in applying a nanosecond pulsed electric fields ( nspef ) an electrode assembly may be utilized . an example of some electrode assemblies which may be utilized are shown and described in further detail in u . s . prov . pat . app . 60 / 916 , 898 filed may 9 , 2007 ( and in corresponding wo 2008 / 141221 a1 ), each of which are incorporated herein by reference in its entirety . in one variation of an electrode , a medical instrument for delivering electrotherapy illustratively comprises an outer support member having an open distal end and a base wall portion within the support member arranged to form a cup - like open volume in the distal end of the support member . at least one aperture is formed in the cup - like volume for applying a suction mechanism to the cup - like region . at least a first and a second electrode have at least a portion extending into the cup - like region . a system for delivering electrotherapy comprises a medical instrument having a suction mechanism for providing a source of suction within the cup - like volume to hold a tissue portion to be treated , and a power supply coupled to the first and second electrodes for applying electrical signals to provide electrotherapy to the tissue . when the tissue or tumor to be treated is adhered to the instrument by the suction mechanism , the tumor may be positioned in apposition to the electrode assembly for effectively delivering the pulses . a variety of pulse generators can be used . however , the pulse generator is desirably capable of delivering high voltage pulses ( e . g ., in the 1 - 900 ns range ) which are imposed across a pair of spaced apart electrodes , to generate electrical fields on the order of , e . g ., 20 to 100 kv / cm . the electrode assembly may be designed to draw tissue into the recessed cup - like volume in order to immobilize the tissue and position the electrodes firmly against or within the tissue . this allows for desirable targeted treatment of the tissue . for positioning electrodes against the tissue , flat ( planar ) electrodes can be positioned along the inner walls of the cup - like volume . for positioning electrodes within the tissue , needle - like electrodes protruding essentially perpendicular from the backside portion of the cup - like volume can be provided for penetrating a controllable constant distance into the tissue as it is drawn into the recessed cup - like volume . fig2 a to 2d show variations of electrode assemblies for delivering electrotherapy using suction electrodes . the electrode assemblies may utilize stainless steel electrodes which are electropolished to eliminate any sharp edges that can lead to corona formation at high voltages and are then embedded in a dielectric material , such as a plastic . very small holes 38 may be formed or drilled into the base of the recessed cavity 39 and during use a suction may be applied through the holes to draw the tissue to be treated into the recessed cavity 39 , e . g ., cup - like volume having an inner diameter of about 4 mm and a depth of about 2 mm , for electric field application . the dimensions of recessed cavity 39 are intended to be illustrative and not limiting . accordingly , recessed cavity 39 may be varied in its size to match a size of any particular tumor to be treated such that the treated tumor may be received within the cavity in close proximity or in direct contact against the electrodes . fig2 a shows an example of an electrode assembly comprising a support member 30 having a cylindrical cross section having , e . g ., six ( 6 ) spaced apart planar electrodes 32 , positioned circumferentially about the recessed cavity 39 . the electrodes 32 are electrically isolated from one another by a dielectric material 34 , such as plastic upon which the electrodes can be , e . g ., partially embedded . in the arrangements shown the electrodes 32 have one exposed side ( without plastic ) along the wall of the cavity 39 to allow direct contact to the skin . the opposite sides of the electrode 32 may be coated with plastic . in operation , electrodes in apposition from one another form bias pairs . a plurality of apertures 38 in the back plate 36 are shown for applying a suction force to immobilize a region of skin or epithelium therein . fig2 b shows another variation of the electrode assembly comprising support member 30 having a cylindrical cross section and having a pair of “ u ”- shaped planar electrodes 40 and 42 disposed on the periphery of the recessed cavity 39 . the electrodes 40 , 42 may be electrically isolated from one another by a dielectric material , such as plastic 44 upon which the electrodes can be embedded . fig2 c shows another variation comprising a support member 30 having a rectangular cross section and having a pair of spaced apart parallel plate electrodes 46 and 48 . the back plate 50 having a plurality of apertures 52 therein may be formed from a dielectric material . fig2 d shows another variation comprising a support member 30 having a plurality of needle electrodes 54 which are mounted at the base of the back plate 56 to control the penetration depth of the tissue as it is sucked into the cup - shaped volume . the distance between the center needle and each of the four outer needles may vary but is shown in this example as about 2 mm . fig2 e and 2f show perspective and end views , respectively , of yet another variation of an electrode assembly . in this example , support member 30 may also define the recessed cavity 39 and the plurality of apertures 38 defined over the back plate 36 . a plurality of needle electrodes 54 , e . g ., six ( 6 ) needle electrodes , may project into the cavity 39 from the back plate 36 with the needle electrodes 54 arranged in a parallel array . in this variation , a linear arrangement of three ( 3 ) needle electrodes 54 may be aligned in parallel with an adjacent linear arrangement of three ( 3 ) needle electrodes 54 , as shown , with the apertures 38 positioned about the electrode array . since each electrode is electrically isolated from one another , electrodes can be connected to separate electrically conductive ( e . g . copper ) wires , such as wires that end on a connector projecting out of the side of the plastic cylinder . this allows each electrode to be connected to a different pulse generator and biased differently for maximum versatility , if so desired . as previously mentioned , the back plate of each recessed cavity may have multiple apertures , such as on the order of 100 μm in diameter . an air pump , e . g ., an oscillating diaphragm air pump or other suction source , is then coupled to the support member on the side of the base wall support opposite the recessed cavity and is used to generate a mild suction that pulls the tissue to be treated into the cup - like volume . the electrodes may comprise an electrical conductor that is resistant to corrosion such as , for example , stainless steel . the electrodes portioned at the distal end are preferably electropolished or otherwise planarized . electropolishing removes corners and sharp edges to minimize undesirable corona discharge when large voltages associated with generating nspef are applied to the electrodes . in utilizing the generated nspef through any one of the electrode assemblies to treat tumors , such as melanoma tumors , the parameters in above - described u . s . pat . no . 6 , 326 , 177 are generally insufficient in effectively treating such tumors . the disclosed number of pulses , i . e ., 20 pulses , at 100 ns is insufficient ; rather , 1500 pulses at 100 ns instead would be optimal for treating such tumors . turning now to fig3 a , 4 a , and 5 a , side views of exemplary suction electrodes developed for use and experimentation are shown . fig3 b , 4 b , and 5 b show corresponding perspective views of the electrode treatment distal end effectors . each support member 60 may be seen having a recessed cavity 39 for contacting and receiving the tissue to be treated . fig3 b illustrates the recessed cavity 39 of support member 60 having a plurality of needle electrodes 62 , as previously described . fig4 b illustrates the recessed cavity 39 having at least two electrodes 64 positioned in apposition on either end of the cavity 39 for treating the tissue region positioned therebetween and fig5 b illustrates a variation having at least six electrodes 66 electrically isolated from one another and positioned about a circumference of recessed cavity 39 , as previously described . in use , fig6 a to 6d show side views of the support member 60 suctioning or drawing in tissue to be treated from various regions of the body to illustrate how the tissue may be drawn into the recessed cavity 39 into contact or proximity to the electrodes . drawing in the tissue may further facilitate tissue treatment by clearly defining the treatment area to be treated for the operator . as shown in fig6 a , a region of tissue 70 from a subject &# 39 ; s face may be readily drawn into recessed cavity 39 . fig6 b illustrates a region of tissue 72 from a subject &# 39 ; s arm , fig6 c illustrates a region of tissue 74 from a subject &# 39 ; s stomach , and fig6 d likewise illustrates a region of tissue 76 to be treated from the subject &# 39 ; s leg . because the components of the assembly are portable , the assembly may be stored or housed in a housing 80 for ready shipping or transportation , as shown in fig7 . an example is shown in the graph of fig8 of the applied pulse voltage 90 having a rise time of , e . g ., 15 ns in this particular example , and the resulting current waveform 92 delivered to the tissue region , e . g ., tumor , when utilizing an electrode treatment assembly having a six - electrode configuration , as previously described . when nspef is applied to a tissue region such as a tumor , if a large resistance between the electrode and the tumor restricts current flow ( such as the presence of the stratum corneum in skin ), the field may not pass into the tumor effectively . thus it may be desirable to apply , in one example , a minimum current of 20 a ( although lower currents may be applied if so desired ) that may pass through the tumor during nspef application to have a desired effect of triggering tumor apoptosis . in order to prevent damage to tissues surrounding the tumor , the nspef therapy may be applied at a pulse frequency that will not heat the tissue above , e . g ., 40 ° c . ( the minimum temperature for hyperthermia effects ). therapy with nspef treatment is thus able to initiate apoptosis within the tumor cells without raising the temperature more than a few degrees so as to prevent harm to surrounding tissues from heat transfer . in one example , if 100 ns pulses were applied , the frequency of the applied pulses is desirably 7 pulses per second ( hz ) or lower to prevent damage to surrounding tissues . with the electrode assemblies described herein , treatment of tissue regions such as skin tumors may be effected by applying nspef while specifying various parameters . for instance , one or all of the following parameters may be adjusted to provide optimal treatment of tissue to effect tumor apoptosis : ( 1 ) pulse amplitude ( kv / cm ); ( 2 ) pulse duration ( ns ); ( 3 ) pulse application frequency ( hz ); and / or ( 4 ) pulse number applied . because the value of these parameters may vary widely over a number of ranges , it has been determined that particular ranges may be applied for effecting optimal tissue treatment which may effect tumor apoptosis in as few as a single treatment . in varying pulse amplitude , an applied amplitude as low as , e . g ., 20 kv / cm , may be sufficient for initiating an apoptosis response in the treated tissue . the pulse amplitude may , of course , be increased from 20 kv / cm , e . g ., up to 40 kv / cm or greater . however , an applied amplitude of at least , e . g ., 30 kv / cm or greater , may be applied for optimal response in the treated tissue . in varying pulse duration , durations in the range of , e . g ., 50 - 900 ns , may be highly effective although shorter durations may be applied if the number of pulses is increased exponentially . in varying pulse application frequency , frequencies up to 7 hz may be applied with 100 ns pulses without heating surrounding tissues to hyperthermic levels . because tissue heating may be dependent on pulse width multiplied by the frequency of application , shorter pulses may be applied at proportionately higher frequencies with similar heat generation . in varying the number of pulses applied , the pulse number determines the total energy applied to the tissue region . generally , applying a minimum pulse number of 600 pulses may result in complete remission of tumors . in one example , nspef therapy having a pulse duration of 100 ns may be applied over a range of , e . g ., 1000 - 2000 pulses , to effectively treat the tissue region . given the range of parameters , a relationship between these parameters has been correlated to determine a minimum number of electrical pulses which may effectively treat a tissue region , e . g ., a tumor , with a single treatment of nspef therapy to cause complete apoptosis in the tumor tissue . generally , the number of electrical pulses increases exponentially as the pulse duration is shortened . data obtained and as shown in the following table 1 show the minimum number of pulses which may be applied for a given pulse width to completely eliminate , e . g ., a melanoma , with a single nspef treatment utilizing the devices described herein . the values of table 1 are plotted in the chart of fig9 and provides a correlation illustrated by curve 100 where for a given pulse duration or width , any number of pulses , n , at or above the curve 100 may result in complete tumor remission after a single treatment as described herein . the curve 100 may be described in the following equation ( 1 ): n = minimum number of pulses to cause tumor apoptosis with a single treatment this non - linear dependence of pulse number on pulse width suggests that the effectiveness of the nspef therapy described herein is not simply due to energy delivery to the tumor as that is linearly proportional to n times t given a constant voltage and current . in optimizing the device , multiple experiments have shown that tumors , such as melanoma tumors , may be eliminated utilizing nspef when exposed to 100 ns long pulses having a 15 ns rise time where the minimum number of pulses range from , e . g . 1500 to 2000 pulses , as illustrated in the chart of fig1 which shows the optimum pulse number where a majority of tumors were successfully treated after a single treatment when pulsed with at least 1500 to 2000 pulses . accordingly , as shown in the graph of fig1 , the percent efficacy after one treatment is shown to increase from 1500 pulses and higher . as also indicated in the chart of fig1 which illustrates optimum amplitude of the pulses , the number of tumors successfully treated after a single treatment begins to rise at higher amplitudes , e . g ., from 25 kv / cm . thus , the minimum pulse amplitude observed is 30 kv / cm in this example while the optimum pulse amplitude is 40 kv / cm or greater in this example for effectively treating tumors such as melanoma tumors . aside from pulse amplitude , another parameter is pulse frequency . it has been determined that the optimum pulse frequency is 7 hz as higher frequencies may result in excessive heat applied to the tissue , as previously described . thus , if a maximum frequency of 7 hz were utilized to deliver at least 2000 pulses , the treatment time to optimally treat a tumor would be at least 4 to 5 minutes at about , e . g ., 4 . 76 minutes . in utilizing the parameters described above for the nspef , a treated tumor may be effectively eliminated within a week or two following a single application of the nspef . additional treatments of the tumor or tumors may be effected if necessary or desired . in this particular example , murine b16 - f10 melanoma cells transfected with enhanced green fluorescent protein ( egfp ) were obtained and stored in liquid nitrogen until use . these cells were cultured and injected into 4 - 6 week old female nu / nu mice ( immunodeficient , hairless , albino ) using standard procedures at four injection sites each . tumors were detected visually by the bulges they produced and by gfp detection under fluorescent microscopy . various suction electrode assemblies , shown in fig1 , were used where electrode assemblies 110 , 112 , and 114 each had a recessed cavity with an inner diameter of about 4 mm and a depth of about 2 mm while the electrode assembly 116 utilized an array of needles positioned within the recessed cavity where a distance between the center needle and each of the outer needles was about 2 mm . in each of the assemblies , one or more electrodes 118 were used to discharge the energy into the treated tissue while the remaining electrodes functioned as return electrodes . a suction was drawn ( e . g ., 500 mm hg ) within the recessed cavity to pull the tumor therein and nspef therapy was applied with 100 ns pulse widths while either the pulse number , amplitude , or frequency was varied . a typical treatment applied 2700 pulses with a pulse width of 100 ns at 30 kv / cm and a frequency of 5 - 7 hz . the suction electrode assembly was rotated 45 ° every 500 pulses to ensure uniform field distribution across the tumor . gfp fluorescence changes following nspef application using the electrode pictured at the top of each column were noted at 0 min , 5 min , 1 hr , 2 hr , 3 hr , and 4 hr . gfp fluorescence changes , if any , were also noted for tumors which had only suction applied as well as suction plus heat applied ( 37 ° c . for 10 min ) but without nspef treatment . fig1 shows the resulting tumor activity for each of the electrode assemblies 110 , 112 , 114 , and 116 . tumors that showed signs of regrowth , generally indicated by renewed gfp production , were retreated using the same parameters as the first treatment . tumors were considered eliminated when no regrowth was detected within two weeks after the last treatment . fig1 also shows trans - illumination and reflected light images adjacent to one another of the treated tissues ( melanomas ) corresponding to the various electrode assemblies 110 , 112 , 114 , and 116 . the images were recorded on the days indicated in the upper left corner of each pair of figures , e . g ., 0 , 3 , 7 , etc ., where nspef treatment ( if applied ) occurred on day 0 . fig1 shows the measured change in gfp signal area normalized on a logarithmic scale over a period of time in hours following nspef treatment with the indicated suction electrode type , i . e ., the 6 - pole dual plot correlates to electrode assembly 110 , the 6 - pole plot correlates to electrode assembly 112 , the 2 - pole plot correlates to electrode assembly 114 , and the needle plot correlates to electrode assembly 116 . the indicated error bars represent the standard error of the mean ( sem ) with n = 10 - 12 . fig1 shows the measured change in tumor area normalized on a logarithmic scale over a period of days following nspef treatment with the indicated suction electrode type assembly . as above , the indicated error bars represent a standard error ( sem ) of n = 10 - 12 . as shown in fig1 , histological sections of the treated tumors and skin immediately adjacent to suction electrode location were taken at different times after treatment with 2000 pulses at 30 kv / cm and a frequency of 7 hz . images in the three left columns were taken from skin ( melanoma , epidermis , and blood cells ) with tumors before ( i . e ., 0 hr ) and several times after nspef treatment ( i . e ., at 1 hr , 6 hrs , 24 hrs , and 48 hrs ). images in the two right columns were taken from skin ( blood in adjacent skin and epidermis in adjacent skin ) immediately adjacent to the nspef - treated region and no morphological changes could be detected there indicating that the nspef treatment effects on skin and melanomas are highly localized to the region between the electrodes . thus , in varying the range of pulse numbers from 500 - 2700 while using the same amplitude ( e . g ., 30 kv / cm ), duration ( e . g ., 100 ns ), and frequency ( e . g ., 5 hz ), a minimum of 2000 pulses has shown desirable complete elimination of tumors , although fewer pulses may be applied if desired , as indicated in the chart of fig1 . in varying the range of pulse amplitude while using the same pulse duration ( e . g ., 100 ns ), pulse number ( e . g ., 2000 pulses ), and frequency ( e . g ., 7 hz ), a minimum amplitude of 30 kv / cm has shown desirable elimination of tumors , although the amplitude may be lowered or increased if so desired , as indicated in the chart of fig1 . in varying the range of pulse frequency , it is generally desirable to use the highest pulse application frequency possible without damaging the surrounding tissue given the temperature rise in the tissue , as indicated in the chart of fig2 . applying nspef at 1 hz at 30 kv / cm and 20 a to tissue may increase the tumor temperature by 2 ° c . whereas 5 hz increased the tumor temperature by 6 - 7 ° c . applying a frequency of 7 hz may limit the temperature rise of the treated tissue to 37 ° c . which is below the hyperthermia threshold of 41 ° c . for an amplitude of 30 kv / cm , applying pulses at 5 hz or higher was more effective than using lower frequencies . typical melanoma responses to nspef therapy in the 10 - 25 kv / cm range were recorded where four melanomas on one mouse were treated with either 10 , 15 , 20 or 25 kv / cm nspef ( 2000 pulses , 100 ns , 7 hz ). the gfp fluorescence at each respective pulse amplitude over a period of 0 , 1 , 6 , and 8 days were recorded , as shown in fig2 , as were the trans - illumination , as shown in fig2 , and reflected light images , as shown in fig2 . the temperature increase inside a tumor over a period of several minutes during nspef application were also recorded , as shown in fig2 . a pulse amplitude of 30 kv / cm with 100 ns long pulses were applied beginning at the indicated frequency 120 with frequency of 1 hz and 5 hz . pulsing was stopped at the indicated frequency 122 for 5 hz and at 124 for 1 hz . the 1 hz pulse application increased tumor temperature by 2 ° c . while the 5 hz pulse application increased the temperature by 7 ° c . the appearance of nspef - treated skin on the indicated day following nspef therapy is shown in fig2 in reflected light images of 20 different nspef - treated tumors . the electrode configuration 110 , 112 , 114 , and 116 used is shown with respect to each column . a number of tumors were treated with nspef therapy over a range of pulses . with a pulse duration of 25 ns at 30 kv / cm and with 20 - 25 hz pulse frequency , the number of tumors treated and the percentage of tumor regrowth were recorded over the range of pulses applied during a single treatment , as shown in the chart of fig2 . the chart illustrates how tumor elimination may be effected when at least 15 , 000 pulses were applied . although the percentage of tumors eliminated was less than 100 % at 15 , 000 pulses , complete tumor regrowth elimination may be potentially effected by treatment at a greater number of pulses when applied at these particular parameters . fig2 likewise illustrates the number of tumors treated and the percentage of tumor regrowth over the number of pulses applied . as shown , with a pulse duration of 50 ns at 30 kv / cm and a pulse frequency of 20 hz , the optimal pulse number applied for a single treatment may completely eliminate tumor regrowth at 8000 pulses . with these particular parameters , a number greater than 8000 pulses may also be potentially applied to effectuate complete tumor elimination . in one particular variation , an elongate instrument which may be delivered via or through an endoscopic device may utilize any one or more of the nspef parameters described herein for tumor treatment . the endoscopic device , particularly an eus device , may be used to image or locate a tissue region to be treated . ultrasound imaging may be particularly useful in locating one or more tumors for treatment although conventional endoscopic imaging may also be utilized . with the targeted tissue region located within the body , the nspef instrument 140 may be positioned or advanced within one or more working channels in the endoscope until a tapered piercing end of the nspef instrument is projected from a distal end of the endoscope to deliver a highly localized treatment . the nspef instrument 140 may be configured either as a bipolar or monopolar electrode configuration . generally , the nspef instrument 140 may be positioned upon the distal end of an elongate shaft 130 ( such as an insulated conducting cable disposed within a flexible stainless steel tubing ) which is sufficiently flexible for advancement through the endoscopic working lumen , as shown in the perspective view of fig2 . in its low - profile delivery configuration , the piercing tip 132 which may also function as a conducting electrode may project distally from the shaft 130 for piercing directly into the tissue to be treated or through a tissue region and / or for delivery and / or retraction through the endoscopic working lumen or patient body . because the nspef instrument 140 as well as shaft 130 is designed to be advanced through the working lumen of an endoscopic device , the nspef instrument 140 and / or shaft 130 may have a diameter of , e . g ., around 3 mm , for advancement through a working lumen having a diameter of e . g ., 3 . 7 mm , with a working length of , e . g ., about 125 cm . one or more reference electrodes 134 ( shown in this variation as elongate members ) may be positioned proximal to the piercing tip 132 in a low - profile configuration where the one or more reference electrodes 134 are placed against the conducting electrode . a proximal end of each reference electrode 134 may be pivotably connected to the elongate shaft 130 such that when the one or more electrodes 134 are actuated to reconfigure , each electrode 134 may rotate about its pivot to extend into a radial pattern . moreover , the piercing tip 132 and one or more reference electrodes 134 may be in connected in electrical communication with a pulse generator ( as described herein ) through corresponding conductors passed through elongate shaft 130 . the nspef therapy may be applied through nspef instrument 140 with the parameters as described herein . fig2 shows a perspective view of the one or more reference electrodes 134 being partially extended or reconfigured about its proximal pivot into its deployed configuration . the electrodes 134 may be actuated by the operator using a mechanism for deploying the electrodes 134 into their deployed configuration . in this variation , one or more corresponding cables or wires 136 attached to each electrode 134 may extend through openings 138 into and through the shaft 130 to an actuator positioned at or along a proximal end of shaft 130 . with the electrode assembly fully deployed , as shown in the perspective view of fig3 , the piercing tip 132 may be penetrated into or through the tissue to be treated ( such as the tumor ) while under the guidance of ultrasound imaging from the endoscope for desirably positioning the instrument . the one or more reference electrodes 134 are shown in their deployed configuration where each electrode 134 may extend radially from shaft 130 . although six electrodes 134 are illustrated circumferentially positioned and radially extending about shaft 130 in this embodiment , other variations may utilize different numbers of electrodes which are spread or configured into a variety of different shapes , e . g ., a hemi - circular plate configuration . a single treatment via nspef instrument 140 with the parameters described herein may stop blood flow to the tumor and initiate apoptosis and / or necrosis resulting in tumor shrinkage . as mentioned above , once the nspef instrument 140 and shaft 130 have been advanced through a working lumen 152 of endoscope 150 , the one or more reference electrodes 134 may be deployed , as shown in fig3 . the piercing tip 132 may be advanced into a tissue region to be treated , such as a tumor located on or within an organ 162 until the one or more reference electrodes 134 are positioned on or against a surface 164 of the organ or tumor . the endoscope 150 may be manipulated to position its distal end into proximity to the organ 162 and / or tumor while under guidance from an ultrasound transducer 154 positioned within the distal end of the endoscope 150 . the operator may utilize the captured ultrasound images from the transducer 154 to also guide the deployment of the nspef instrument 140 from endoscope 150 and insertion of the instrument into and / or against the organ 162 and into proximity to the tumor for nspef treatment , as shown in fig3 and as described above . moreover , to facilitate contact between the nspef instrument 140 and the tissue surface 164 of the targeted tumor , suction may be applied through , e . g ., an irrigation and / or suction lumen 156 either through the endoscope 150 , through one or more openings 166 defined along the nspef instrument 140 ( as shown in fig3 ), or both for drawing the tumor into apposition against the electrode assembly . other mechanisms such as tissue graspers 160 advanced through an adjacent working lumen 158 through endoscope 150 may also be used ( as shown in fig3 ) to draw the tumor or organ into apposition with the nspef instrument 140 . following nanosecond pulse application , the treatment instrument 140 may be withdrawn from the tumor and the outer reference electrodes 134 may be reconfigured back to their original low - profile configuration for retraction back into the working lumen 152 of endoscope 150 . alternatively , for particularly large tumors , nspef instrument 140 may be withdrawn from a first location within the organ and / or tumor and repositioned under the guidance of eus to a second location for additional treatment . this procedure may be repeated as many times as desired by relocating the nspef instrument 140 until the entire tumor is treated by nspef therapy . the nspef instrument 140 may penetrate through various tissue regions within the subject &# 39 ; s body , such as through a gastrointestinal wall for treating various tissue regions such as the pancreas , liver , lymph nodes , and other surrounding structures . one exemplary treatment which the nspef instrument 140 is suitable for treating may involve minimally invasive treatment of pancreatic carcinoma . because typical carcinoma sizes may range from 1 - 2 cm and are usually found within the pancreas 1 - 2 cm from the outer edge of the organ , the instrument 140 having a piercing tip with a length of about 1 - 2 cm may be particularly suitable for treating the tumor or tumors . in yet another variation , fig3 a and 34b show perspective views of a nspef instrument which may utilize any one or more of the nspef parameters described herein for tumor treatment . as previously described , because the nspef instrument as well as shaft 170 is designed to be advanced through the working lumen of an endoscopic device , the nspef instrument and / or shaft 170 may have a diameter of , e . g ., around 3 mm , for advancement through a working lumen having a diameter of , e . g ., 3 . 7 mm . a piercing tip 172 may project from shaft 170 and further define four openings ( in this particular example ) proximal to the piercing tip 172 through which a corresponding needle array may be deployed . as shown in fig3 a , the piercing tip 172 may be advanced from the endoscope lumen and / or pierced through a tissue wall ( such as a gastrointestinal wall ) or in proximity to an organ to be treated ( such as a pancreas ) into proximity with a tumor 176 to be treated . the ultrasound transducer 154 positioned within the distal end of the endoscope 150 may be utilized to locate and image a position of tumor 176 such that the nspef instrument may be guided into proximity to the tumor 176 , as shown . once in proximity to the tumor 176 , four conductive needles 178 , 180 , 182 , 184 circumferentially arranged about piercing tip 172 may be advanced distally , e . g ., 1 - 2 cm , from shaft 170 through their corresponding openings 174 such that the array of needles extend to span a distance of , e . g ., about 1 cm or more between the needles , sufficient to surround the tumor , as shown in fig3 b . the conductive needles may be optionally configured into bipolar needle electrode pairs for forming the pulsed electric field therebetween . with the needle array in position about the tumor 176 , a nspef having any of the parameters described herein may be applied through the needle array to generate an electric field pattern that may uniformly expose the carcinoma cells . fig3 c illustrates an example of the electric field distribution created between the needle array ( e . g ., a nanosecond pulsed electric field of 40 kv / cm ) for exposing and treating the tumor 176 between the needle array . another variation is shown in the perspective views of fig3 a and 35b which illustrate a nspef instrument similar to the previous variation but having a six - electrode array 190 , 192 , 194 , 196 , 198 , 200 positioned uniformly about piercing tip 172 and advanced through a corresponding opening 174 . as shown , each needle array may project distally and extend at an angle to allow the needle array to surround the tumor 176 , as above . with the tumor 176 desirably surrounded , a nspef treatment may be effected to treat the tissue enclosed within the array , as shown by the electric field distribution in fig3 c . this example illustrates the six - electrode array with a 40 kv / cm field applied although any of the nspef parameters described herein may be applied . fig3 a and 36b show perspective views of yet another variation which illustrates an eight - needle array . in this variation , the shaft 210 may comprise a piercing length 212 , e . g ., 1 - 2 cm or more , having a piercing tip 214 where the piercing length 212 is narrower in diameter relative to the shaft 210 . the piercing length 212 may be advanced from the endoscope lumen and pierced directly through the tumor 176 , as shown in fig3 b , while under guidance from the ultrasound transducer 154 positioned within the distal end of the endoscope 150 . once the tumor 176 has been impaled , one or more conductive needles may be advanced from the shaft 210 through openings 216 located proximally of tumor 176 as well as from piercing length 212 through openings 218 located distally of tumor 176 . the proximally located needle array 220 and distally located needle array 222 may extend radially from the instrument to completely surround the tumor 176 to be treated . although four radially extending needles are illustrated in the proximal needle array 220 and four radially extending needles are illustrated in the distal needle array 222 , the number of needles in either or both arrays may be varied . with the needle arrays suitably positioned , the nspef therapy may be applied to treat the tumor 176 located between the needle arrays , as shown by the electric field distribution in fig3 c , where 40 kv / cm is applied . the applications of the devices and methods discussed above are not limited to treatment of melanoma tumors but may include any number of further treatment applications . moreover , such devices and methods may be applied to other treatment sites within the body . modification of the above - described assemblies and methods for carrying out the invention , combinations between different variations as practicable , and variations of aspects of the invention that are obvious to those of skill in the art are intended to be within the scope of the claims .
systems and methods for treating tumors on or within internal organs of mammals that have been imaged with endoscopic ultrasound are described . the system uses an expandable bipolar electrode assembly that can be imaged by ultrasound and can penetrate , e . g ., the stomach , intestine or bowel wall , etc . and be positioned in or around the tumor on an internal organ while being guided by an operator who visualizes its position with ultrasound imaging . it utilizes an electrode assembly that extends down an internal cavity in the endoscope to allow the operator to spread the electrodes for pulse delivery of a nanosecond pulsed electric field to the tumor .