Patent Number: RE0347086
Section: description

DESCRIPTION OF THE PREFERRED EMBODIMENT The Scanning Ion Conductance Microscope (SICM) of the present invention is shown in simplified form in a preferred embodiment in FIG. 2 where it is generally indicated as 22. As depicted in FIG. 2, the SICM 22 is in the process of determining surface topography of the sample 14. The sample 14 is disposed in a reservoir 24 filled with an electrolyte 26. In the SICM 22, the scanning is accomplished by a micropipette 28 which is scanned by apparatus 30 according to techniques well known in the art as mentioned above. Again as in the prior art, the micropipette 28 could be stationary and the reservoir 24 holding the sample 14 moved by the scanning apparatus 30. The micropipette 28 (which is non-conductive and preferably of glass) has one electrode 32 disposed therein while a second electrode 32 is disposed in the electrolyte 26 within the reservoir 24. To complete the ion conductance path at the micropipette 28, the micropipette 28 is also filled with the electrolyte 26. A voltage source 34 is connected to the electrodes 32 and a current measuring transducer 36 is placed in series with the voltage source 34 to measure the current flowing and provide an indicative signal thereof to the control logic 38 on line 40. The control logic 38 performs two functions. First, it controls the scanning apparatus 30 over line 42. Second, by receiving a z-positional feedback signal from the scanning apparatus 30 it outputs the data on line 44 employed to visualize the scan results according to techniques well known in the art which, per se, form no part of the invention. In operation, the micropipette 28 is filled with electrolyte 26 and lowered through the reservoir 24 toward the surface 12 of the sample 14 while the conductance between the electrode 32 inside the micropipette 28 and the electrode 32 in the reservoir 24 is monitored. As the tip of the micropipette approaches the surface 12, the ion conductance decreases because the space through which ions can flow is decreased. The micropipette 28 is then scanned laterally over the surface 12 while the feedback system comprising the scanning apparatus 30 and the control logic 38 as described above raises and lowers it to keep the conductance constant. The path of the tip, as indicated by the dashed line in FIG. 2, follows the topography of the surface 12, therefore. As in prior art scanning microscopes, the z-directional signal developed in the process can be employed to display the surface topography in an manner desired as, for example, by displaying on a CRT (with or without color and/or other enhancements) or by plotting on a plotter, or the like. With respect to the micropipettes as employed by the inventors in tested embodiments to date, the early micropipettes were made from 1.5 mm outer diameter, 0.75 mm inner diameter Omega Dot capillary capillary tubing. Later micropipettes were made with similar tubing on a Brown-Flaming puller. The micropipette tip diameters were estimated using a non-destructive bubble pressure method which correlates the pipette's outer diameter to the internal pressure required for the pipette to produce a fine stream of bubbles in a liquid bath. The ratio of outer diameter to inner diameter has been found to be essentially constant along the entire length of the pipette. Inner diameters were thus estimated from the OD/ID ratio of the unpulled capillary tubing. Typically recently employed micropipettes have had tips with outer diameters of order 0.1 to 0.2 .mu.m and inner diameters of order 0.05 to 0.1 .mu.m. Samples were glued onto glass substrates or directly onto electrodes and then covered with a few drops of 0.1M NaCl. The micropipette tips were allowed to fill by capillary action and then their shafts were backfilled with a syringe. The 0.1M NaCl was also employed in the micropipettes to avoid concentration cell potentials and liquid junction potentials. Reversible Ag/AgCl microelectrode holders and bath electrodes provided the necessary stability for reliable current and topographic imaging. In their testing, the inventors herein applied DC voltages of 0.03 to 0.4 V and measured DC currents (typically 1 to 10 nA) to find the conductance: generally 10-8 to 10-7S. The microscope was operated with conductances 0.9 to 0.98 of the conductance when the tip was far from the surface. At smaller conductances, the inventors found that the micropipette tip was sometimes actually pressing into the sample surface. The inventors generated topographic images of their test samples by measuring the voltage that the feedback system applied to the z-axis of a single-tube x,y,z piezoelectric translator to keep the conductance constant. For ion current images, the local current was monitored as the micropipette 28 was scanned over the surface 12 at a constant height (i.e. at a constant z, being a plane parallel to the sample 14) as depicted in FIG. 3. A digital scanner supplied the x and y scan voltages for both topographic and ion current images. The z values (or ion currents) together with their x and y coordinates were recorded on a video cassette recorder via a digital data acquisition system. A program developed at the University of California Santa Barbara was used to filter the resulting image and added shading or color scales to allow surface features to be seen more easily. The inventors found that the method of statistical differences, which enhances features on their local background while suppressing noise, was especially useful for processing ion current images. The resolution of the SICM as a function of pipette diameter was measured with a large-scale model. A glass pipette, inner diameter 0.71 mm, outer diameter 1.00 mm, was scanned at a constant height over plastic blocks with regularly spaced grooves 0.71 mm deep. The height was set by lowering the pipette until the ion conductance went from 4.2.times.10-5S, its value far from the surface, down to 4.0.times.10-5S. These conductances were measured at a frequency of 10 KHz. This resolution test showed that it should be possible, in principle at least, to resolve features as small as the micropipette's inner diameter if the noise on the ion conductance signal could be reduced below 1%. So far, in practice the inventors have resolved features down to several times the micropipette's inner diameter of 0.05 to 0.2 .mu.m. There is a compromise between averaging the ion conductance signal from a long time to reduce noise and obtaining entire images in a reasonable time. The inventors have chosen to acquire their images in about five minutes and found that in so doing the smallest resolvable features are of order 0.2 .mu.m. The most promising application for the SICM is not simply imaging the topography of surfaces at submicron resolution. As mentioned above with respect to FIG. 3, the SICM 22' shown therein can image not only the topography but also the local ion currents coming out through pores in a surface. Comparison of topographic and ion current images can give a more detailed picture of the type of surface features that correlate with ion channels. This will be important in the evaluation of biological samples where not every hole is an ion channel. For images of the local ion currents, the micropipette 28 was scanned over the surface 12 at a preselected height, as indicated by the dashed line in FIG. 3, without moving up and down. It was also possible to hold the micropipette 28 over various locations on the imaged surface and measure local electrical properties. Thermal drift was small enough, approximately 0.004 .mu.m/minute, so that it was possible to look, for example, at the time dependence of the ion currents above a pore. While the current was constant for the model system employed, it would be more subtle for biological samples. As should now be appreciated from the foregoing description, the SICM of this invention is the first microscope that offers both high resolution topographic and ion current images of non-conductors. Much of the necessary apparatus employed in the SICM such as the micropipettes, microelectrodes, and current amplifiers, are already used routinely by electrophysiologists. Most of the rest of it is substantially the same as used in scanning tunneling microscopy and is readily available commercially. Because the SICM operates in a saline solution or other ionic solution, the microscope is well suited for bilogical applications. As also depicted in FIG. 3, an exciting extension of the basic SICM would be to use a scanning head 46 employing multiple barrel micropipettes 28 with ion-specific electrodes 32',32". The total current into all barrels (or the current into one barrel with a non-specific electrode provided for the purpose) could be used for feedback while the microscope could simultaneously measure and image the flow of different ions. It is anticipated by the inventors herein that such a technique will prove invaluable in the future to electrophysiologists to combine spatially resolved ion flow measurements and topological imaging of biological membranes. Another version of the SICM is depicted in FIG. 4 wherein it is labelled as 22". As with the embodiment of FIG. 4, there is a scanning head 46 employing multiple barrel micropipettes 28 with ion-specific electrodes 32', 32" and a non-specific electrode 32. The free electrode 32 (i.e. the electrode 32 in the reservoir 24) of the previous embodiments is replaced by the non-specific electrode 32 in one of the micropipettes 28 in the scanning head 46. All the active electrodes 32,32',32" . . . are, therefore, included in the scanning head 46. In this way, the scanning head 46 becomes self contained and only needs electrical connections thereto. This could be of particular interest in an arrangement where the scanning head 46 was fixed and the reservoir 24 containing the sample 14 is moved to create the scanning action of the micropipettes 28 over the surface 12 of the sample 14.