Scanning ion conductance microscope

Abstract

A scanning ion conductance microscope, SICM, which can image the topography of soft non-conducting surfaces covered with electrolytes by maintaining a micropipette probe at a constant conductance distance from the surface. It can also sample and image the local ion currents above the surfaces by scanning the micropipette probe in a plane located at a constant distance above the surface. Multiple micropipettes mounted in a multi-barrel head and containing various ion specific electrodes allow simultaneous scanning for different ion currents.

Wherefore, having thus described our invention, what is claimed is: 
     
       1. A scanning ion conductance microscope comprising: (a) a reservoir holding a sample to be scanned therein;   (b) a micropipette having an open tip communicating with a hollow shaft;   (c) an electrolyte solution disposed within said reservoir covering said sample and disposed within said tip .[.and shaft.]. of said micropipette;   (d) a first microelectrode .[.diposed.]. .Iadd.disposed .Iaddend.in said shaft in .[.electrical contact.]. .Iadd.ionic communication .Iaddend.with said electrolyte .[.therein.]. .Iadd.solution in said open tip.Iaddend., said first microelectrode being .[.spaced from inner sidewalls of said shaft to allow said electrolyte solution to pass between said first microelectrode and said inner sidewalls of said shaft.]. .Iadd.in ionic communication with electrolyte solution in said reservoir via said open tip by means of electrolyte solution within said tip.Iaddend.;   (e) a second microelectrode disposed in said reservoir in .[.electrical contact.]. .Iadd.ionic communication .Iaddend.with said electrolyte .[.therein.]. .Iadd.solution in said reservoir and forming a continuous ionic current path between said first and second microelectrodes via the electrolyte solution in said reservoir and in said open tip.Iaddend.;   (f) scanning means for scanning said tip of said micropipette over a top surface of said sample in a scanning pattern;   (g) voltage means for applying a voltage across said first and second microelectrodes;   (h) current means for measuring .[.the.]. .Iadd.an ionic .Iaddend.current flowing .Iadd.in the ionic current path .Iaddend.between said first and second microelectrodes through said open tip of said micropipette and for supplying an indication of said current at an output thereof; and,   (i) control logic means having an output connected to said scanning means and an input connected to said output of said current means for causing said scanning means to set the height of said tip at a desired distance above said top surface and for outputting data of interest related to said .[.top surface.]. .Iadd.sample .Iaddend.as it is scanned.   
     
     
       2. The scanning ion conductance microscope of claim 1 and additionally comprising: (a) feedback means connected between said scanning means and said control logic means for providing said control logic means with an indication of a z-directional component of the position of said tip of said micropipette; and wherein,   (b) said control logic means includes logic for causing said scanning means to position said tip of said micropipette at a distance above said top surface which will maintain the ion conductance between said first and second .[.electrodes.].electrodes .Iadd.microelectrodes .Iaddend.through said open tip of said micropipette at a constant value which will cause said tip to follow said top surface in close non-contacting proximity thereto whereby said data of interest output by said control logic means reflects the .[.topology.]. .Iadd.topography .Iaddend.of said top surface.   
     
     
       3. The scanning ion conductance microscope of claim 1 and additionally comprising: said control logic means includes logic for causing said scanning means to scan said tip of said micropipette in a plane parallel and close adjacent above said top surface whereby said data of interest output by said control logic means reflects the ion conductance of said .[.top surface.]. .Iadd.sample .Iaddend.at the positions of said tip.   
     
     
       4. The scanning ion conductance microscope of claim 1 and additionally comprising: (a) a plurality of said micropipettes disposed to form a multi-barrel scanning head; and,   (b) a plurality of said first microelectrodes disposed in respective ones of said micropipettes, each of said microelectrodes being specific to a different ion; and wherein,   (c) said control logic means includes logic for causing said scanning means to scan said tip of said micropipette in a plane parallel and close adjacent above said top surface whereby said data of interest output by said control logic means reflects the ion conductance of said .[.top surface.]. .Iadd.sample .Iaddend.at the positions of said tip of each of said micropipettes.   
     
     
       5. The scanning ion conductance microscope of claim 4 wherein: said second .[.electrode.]. .Iadd.microelectrode .Iaddend.is disposed within said shaft of one of said micropipettes.   
     
     
       6. A scanning ion conductance microscope capable of providing both topographic and ion conductance information about a sample comprising: (a) a reservoir holding a sample to be scanned therein;   (b) a micropipette having an open tip communicating with a hollow shaft;   (c) an electrolyte solution disposed within said reservoir covering said sample and disposed within said tip .[.and shaft.]. and shaft of said micropipette;   (d) a first microelectrode disposed in said shaft in .[.electrical contact.]. .Iadd.ionic communication .Iaddend.with said electrolyte .[.therein.]. .Iadd.solution in said tip.Iaddend., said first microelectrode being .[.spaced from inner sidewalls of said shaft to allow said electrolyte solution to pass between said first microelectrode and said inner sidewalls of said shaft.]. .Iadd.in ionic communication with electrolyte solution in said reservoir by means of the electrolyte solution within said open tip.Iaddend.;   (e) a second microelectrode disposed in said reservoir in .[.electrical contact.]. .Iadd.ionic communication .Iaddend.with said electrolyte .[.therein.]. .Iadd.solution in said reservoir and forming a continuous ionic current path between said first and second microelectrodes via the electrolyte solution in said reservoir and in said open tip.Iaddend.;   (f) scanning means for scanning said tip of said micropipette over a top surface of said sample in a scanning pattern;   (g) voltage means for applying a voltage across said first and second microelectrodes;   (h) current means for measuring .[.the.]. .Iadd.an ionic .Iaddend.current flowing .Iadd.in the ionic current path .Iaddend.between said first and second microelectrodes through said open tip of said micropipette and for supplying an indication of said current at an output thereof;   (i) control logic means having an output connected to said scanning means and an input connected to said output of said current means for causing said scanning means to set the height of said tip at a desired distance above said top surface and for outputting data of interest related to said .[.top surface.]. .Iadd.sample .Iaddend.as it is scanned;   (j) feedback means connected between said scanning means and said control logic means for providing said control logic means with an indication of a z-directional component of the position of said tip of said micropipette; and wherein,   (k) said control logic means includes first logic for causing said scanning means to position said tip of said micropipette at a distance above said top surface which will maintain the ion conductance between said first and second .[.electrodes.]. .Iadd.microelectrodes .Iaddend.through said open tip of said micropite at a constant value which will cause said tip to follow said top surface in close non-contacting proximity thereto whereby said data of interest output by said control logic means reflects the topology of said top surface; and,   (l) said control logic means includes second logic for causing said scanning means to scan said tip of said micropipette in a plane parallel and close adjacent above said top surface whereby said data of interest output by said control logic means reflects the ion conductance of said .[.top surface.]. .Iadd.sample .Iaddend.at the positions of said tip.   
     
     
       7. The scanning ion conductance microscope of claim 6 and additionally comprising: (a) a plurality of said micropipettes disposed to form a multi-barrel scanning head; and,   (b) a plurality of said first microelectrodes disposed in respective ones of said micropipettes, each of said microelectrodes being specific to a different ion whereby when said second logic of said control logic causes said scanning means to scan said tip of said micropipette in a plane parallel and close adjacent above said top surface said data of interest output by said control logic means reflects the ion conductance of said .[.top surface.]. .Iadd.sample .Iaddend.at the positions of said tip of each of said micropipettes.   
     
     
       8. The scanning ion conductance microscope of claim 7 wherein: said second .[.electrode.]. .Iadd.microelectrode.Iaddend.is disposed within said shaft of one of said micropipettes.   
     
     
       9. .[.The.]. .Iadd.A .Iaddend.method of .[.operating a scanning ion conductance microscope to provide both.]. .Iadd.providing .Iaddend.topographic and ion conductance information about a sample comprising the steps of: (a) disposing the sample to be scanned in a reservoir containing an electrolyte covering the sample;   (b) providing a micropipette having an open tip communicating with a hollow shaft;   (c) disposing an electrolyte within the tip .[.and shaft.]. of the micropipette;   (d) disposing a first microelectrode in the shaft in .[.electrical contact.]. .Iadd.ionic communication .Iaddend.with the electrolyte .[.therein in non-contacting relationship with inner sidewalls of the shaft and.]. .Iadd.in .Iaddend.the open tip;   (e) disposing a second microelectrode in the reservoir in .[.electrical contact.]. .Iadd.ionic communication .Iaddend.with the electrolyte .[.therein.]. .Iadd.in said reservoir and forming a continuous ionic current path between said first and second microelectrodes via the electrolyte solution in said reservoir and in said open tip.Iaddend.;   (f) applying a voltage across the first and second microelectrodes and measuring .[.the.]. .Iadd.an ionic .Iaddend.current flowing .Iadd.in the ionic current path .Iaddend.between the first and second microelectrodes through the open tip;   (g) scanning the tip of the micropipette over a top surface of the sample in a scanning pattern with the tip of the micropipette at a distance above the top surface which will maintain the ion conductance between the first and second electrodes through the open tip at a constant value which will cause the tip to follow the top surface in close noncontacting proximity thereto while providing a z-directional component of the position of the tip of the micropipette;   (h) outputting data of interest which reflects the topology of the top surface;   (i) scanning the tip of the micropipette over a top surface of the sample in a scanning pattern with the tip of the micropipette in a plane parallel and close adjacent above the top surface; and,   (j) outputting data of interest which reflects the ion conductance of the .[.top surface.]. .Iadd.sample .Iaddend.at the positions of the tip.   
     
     
       10. The method of claim 9 and additionally comprising the steps of: (a) providing a plurality of the micropipettes disposed to form a multi-barrel scanning head; and,   (b) disposing a plurality of the first microelectrodes in respective ones of the micropipettes with each of the microelectrodes being specific to a different ion whereby when the tip of the micropipette is scanned in a plane parallel and close adjacent above the top surface the data of interest output reflects the ion conductance of the .[.top surface.]. .Iadd.sample .Iaddend.at the positions of the tip of each of the micropipettes.   
     
     
       11. The method of claim 10 wherein said step of disposing a second microelectrode in the reservoir in electrical contact with the electrolyte therein comprises the step of: disposing the second microelectrode in the shaft of one of the plurality of micropipettes in electrical contact with the electrolyte therein. .Iadd.   
     
     
       12.  The method according to claim 9, comprising: measuring a time dependence of ion conductance of the sample at a selected location of said sample. .Iaddend. .Iadd.   
     
     
       13.  A method of providing topographic information about a sample, comprising the steps of: disposing the sample to be scanned in a reservoir containing an electrolyte covering the sample;   providing a micropipette having an open tip communicating with a hollow shaft and in which is disposed a first microelectrode spaced apart from the open tip within said micropipette;   disposing said microelectrode in said reservoir so that said electrolyte occupies said open tip and said first microelectrode in ionic communication with said electrolyte in said reservoir via said electrolyte in said open tip;   disposing a second microelectrode in the reservoir in ionic communication with the electrolyte in said reservoir and forming a continuous ionic current path between said first and second microelectrodes via the electrolyte solution in said reservoir and in said open tip;   applying a voltage across the first and second microelectrodes and measuring an ionic current following in the ionic current path between the first and second microelectrodes through the open tip;   scanning the tip of the micropipette over a top surface of the sample in a scanning pattern with the tip of the micropipette at a distance above the top surface which will maintain the ion conductance between the first and second microelectrodes through the open tip at a constant value which will cause the tip to follow the top surface in close non-contacting proximity thereto while providing a z-directional component of the position of the tip of the micropipette; and   outputting data of interest which reflects the topography of the top surface. .Iaddend. .Iadd.   
     
     
       14.  A method of measuring ion conductance of a sample, comprising: disposing the sample in an electrolyte solution;   providing a first microelectrode in a micropipette having an open tip filled with said electrolyte solution;   providing a second microelectrode;   disposing the micropipette with said first microelectrode and said second microelectrode in said electrolyte solution with said first and second microelectrodes each in ionic communication with said electrolyte solution to form a continuous ionic current path through said sample and between said first and second microelectrodes via the electrolyte solution;   positioning the first microelectrode over at least one selected location above the top surface of the sample;   applying a voltage across the first and second microelectrodes;   measuring an ionic current flowing in the ionic current path between said first and second microelectrodes and through said sample at the selected location; and   outputting data which reflects the ion conductance of said sample at said selected location based on the measured ionic current. .Iaddend. .Iadd.15. The method according to claim 14, further comprising:   scanning said micropipette with said first microelectrode over the top surface of the sample in a scanning pattern with the tip in a plane parallel above the top surface,   measuring the ionic current flowing in said ionic current path between said first and second microelectrodes and through the sample during scanning of the tip; and   outputting data which reflects the ion conductance of said sample as function of said scanning pattern and the measured ionic current.   
     
     
        .Iaddend. .Iadd.16.  The method of claim 15, further comprising: (a) providing a plurality of the first microelectrodes in respective of a plurality of micropipettes each having an open tip and disposed to form an multielectrode scanning head with each of the first microelectrodes being specific to a different ion; and,   (b) disposing said plurality of the micropipettes with said first microelectrodes in said electrolyte solution so that said first microelectrodes are located in close adjacent position above the top surface of the sample during scanning and the data of interest output reflects the respective ion conductances of the sample at the scanning   
     
     
        positions of the respective first microelectrodes. .Iaddend. .Iadd.17. The method according to claim 14, further comprising: measuring time dependance of ion conductance of said sample at said   
     
     
        selected location. .Iaddend. .Iadd.18.  The method of claim 14, further comprising: (a) providing a plurality of the first microelectrodes disposed in respective of a plurality of said micropipettes to form a multi-electrode scanning head with each of the microelectrodes being specific to a different ion; and,   (b) disposing said plurality of the micropipettes with said microelectrodes in said electrolyte solution so that said microelectrodes are located in close adjacent position above the top surface of the sample at said at least one selected position and the data of interest output reflects the ion conductance of the sample at the position of the tip of each of the microelectrodes. .Iaddend.