U.S. patent number RE34,708 [Application Number 07/875,441] was granted by the patent office on 1994-08-30 for scanning ion conductance microscope.
This patent grant is currently assigned to The Regents of the University of California. Invention is credited to Barney Drake, Paul K. Hansma.
United States Patent |
RE34,708 |
Hansma , et al. |
August 30, 1994 |
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.
Inventors: |
Hansma; Paul K. (Santa Barbara,
CA), Drake; Barney (Santa Barbara, CA) |
Assignee: |
The Regents of the University of
California (Berkeley, CA)
|
Family
ID: |
23180912 |
Appl.
No.: |
07/875,441 |
Filed: |
April 29, 1992 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
Reissue of: |
305465 |
Feb 1, 1989 |
04924091 |
May 8, 1990 |
|
|
Current U.S.
Class: |
250/306;
250/423F |
Current CPC
Class: |
G01Q
60/44 (20130101); Y10S 977/86 (20130101); Y10S
977/852 (20130101); G01Q 70/06 (20130101) |
Current International
Class: |
G01N
27/416 (20060101); G01N 27/49 (20060101); G12B
21/20 (20060101); G12B 21/00 (20060101); G12B
21/02 (20060101); G21K 005/08 () |
Field of
Search: |
;250/306,307,423F |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
"Scanning Tunneling Microscope for Electrochemistry-a New Concept
for the In Situ Scanning Tunneling Micro. in Electrolyte
Solutions", Itaya et al., Surface Sci. Letters, Jul. 88, 201 pp.
L507-L512. .
"Tunneling Microscopy in an Electrochemical Cell: Images of Ag
Plating" Sonnenfeld, et al., App. Phy. Lett. vol. 49, No. 18, Nov.
3, 1986, pp. 1172-1174. .
"The Scanning Ion-Conductance Microscope", Hansma et al., Science,
vol. 243, pp. 641-643, Feb. 3, 1989..
|
Primary Examiner: Anderson; Bruce C.
Attorney, Agent or Firm: Oblon, Spivak, McClelland, Maier
& Neustadt
Government Interests
This invention was made with Government support under Contract No.
N00014-86-K-2058 awarded by the Office of Naval Research. The
Government has certain rights in this invention.
Claims
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.
Description
BACKGROUND OF THE INVENTION
This invention relates to scanning microscopes used for imaging the
topography of surfaces and, more particularly, to a scanning ion
conductance microscope comprising, a reservoir holding a sample to
be scanned therein; a micropipette having a tip communicating with
a shaft; an electrolyte solution disposed within the reservoir
covering the sample and disposed within the tip and shaft of the
micropipette; a first microelectrode disposed in the shaft in
electrical contact with the electrolyte therein; a second
microelectrode disposed in the reservoir in electrical contact with
the electrolyte therein; scanning means for scanning the tip of the
micropipette over a top surface of the sample in a scanning
pattern; voltage means for applying a voltage across the first and
second microelectrodes; current means for measuring the current
flowing between the first and second microelectrodes and for
supplying an indication of the current at an output thereof; and,
control logic means having an output connected to the scanning
means and an input connected to the output of the current means for
causing the scanning means to set the height of the tip at a
desired distance above the top surface and for outputting data of
interest related to the top surface as it is scanned.
The family of scanning probe microscopes that have been introduced
to the scientific community of recent years is broadening the
frontiers of microscopy. As typified by the greatly simplified
general example of FIG. 1, these microscopes scan a sharp probe 10
over the surface 12 of a sample 14 to obtain surface contours, in
some cases actually down to the atomic sale. The probe 10 may be
affixed to a scanning mechanism and moved in a scan pattern over
the surface 12 or alternately (and equally effectively because of
the small sizes involved) the probe 10 may be stationary with the
sample 14 mounted on a scanning mechanism that moves the surface 12
across the probe 10 in a scanning pattern. The point 16 of the
probe 10 rides over the surface 12 as the probe is moved across it
as indicated by the arrow 18. As the point 16 follows the
topography of the surface 12, the probe 10 moves up and down as
indicated by the bi-directional arrow 20. This up and down movement
of the probe 10 is sensed to develop a signal which is indicative
of the z directional component of the 3-dimensional surface 12.
The use of a piezoelectrically driven tube to affect the x-, y-,
and z-directional movements employed in the scanning process is
generally accepted in such devices and such well known equipment is
preferred for use in the scanning aspects of this invention as
well. Various methods are employed to sense the vertical movement
of the probe 10. Where the sample 14 is of an electrically
conductive material and the scanning is conducted in a
non-conductive environment such as air, current flow between the
probe point 16 and the surface 12 can be employed to control the
vertical position of the probe 10. The vertical control signal then
supplies the z-directional component. For non-conducting materials
it is more common to measure the vertical deflection of the probe
10 directly in order to develop the z-directional component.
While the contact type of scanning probe microscope as described
above works well in certain environments, in other environments it
is virtually worthless. This is particularly true where the sample
is of a soft material which cannot be subjected to the contacting
probe described above. While the biasing force of the probe against
the surface of the sample in such prior art apparatus is
exceedingly small, it is still there and the probe itself is quite
sharp in order to follow the contours demanded of it. Accordingly
for example, if a soft membrane is contact scanned, it is torn by
the probe.
Additionally, despite their various attributes, such prior art
scanning microscopes can only supply a visualization of the surface
topography. They cannot show, for example, ion flow capabilities of
and through the surface under examination.
Wherefore, it is an object of the present invention to provide a
non-contacting scanning microscope which can be used to display an
indication of the surface topography of materials which cannot be
scanned with a contacting probe.
It is another object of the present invention to provide a scanning
microscope which can be used to display an indication of ion flow
capabilities of and through a surface under examination.
Other objects and benefits of this invention will become apparent
from the description which follows hereinafter when taken in
conjunction with the drawing figures which accompany it.
SUMMARY
The foregoing objects have been achieved in the scanning ion
conductance microscope capable of providing both topographic and
ion conductance information about a sample of the present invention
comprising, a reservoir holding a sample to be scanned therein; a
micropipette having a tip communicating with a shaft; an
electrolyte solution disposed within the reservoir covering the
sample and disposed within the tip and shaft of the micropipette; a
first microelectrode disposed in the shaft in electrical contact
with the electrolyte therein; a second microelectrode disposed in
the reservoir in electrical contact with the electrolyte therein;
scanning means for scanning the tip of the micropipette over a top
surface of the sample in a scanning pattern; voltage means for
applying a voltage across the first and second microelectrodes;
current means for measuring the current flowing between the first
and second microelectrodes and for supplying an indication of the
current at an output thereof; control logic means having an output
connected to the scanning means and an input connected to the
output of the current means for causing the scanning means to set
the height of the tip at a desired distance above the top surface
and for outputting data of interest related to the top surface as
it is scanned; feedback means connected between the scanning means
and the control logic means for providing the control logic means
with an indication of a z-directional component of the position of
the tip of the micropipette; and wherein, the control logic means
includes first logic for causing the scanning means to position the
tip of the micropipette at a distance above the top surface which
will maintain the ion conductance between the first and second
electrodes at a constant value which will cause the tip to follow
the top surface in close non-contacting proximity thereto whereby
the data of interest output by the control logic means reflects the
topology of the top surface; and, the control logic means includes
second logic for causing the scanning means to scan the tip of the
micropipette in a plane parallel and close adjacent above the top
surface whereby the data of interest output by the control logic
means reflects the ion conductance of the top surface at the
positions of the tip.
In an alternate embodiment, there are a plurality of the
micropipettes disposed to form a multi-barrel scanning head and a
plurality of the first microelectrodes are disposed in respective
ones of the micropipettes. Each of the microelectrodes is specific
to a different ion whereby when the second logic of the control
logic causes the scanning means to scan the tip of the micropipette
in a plane parallel and close adjacent above the top surface, the
data of interest output by the control logic means reflects the ion
conductance of the top surface at the positions of the tip of each
of the micropipettes.
DESCRIPTION OF THE DRAWINGS
FIG. 1 is a simplified drawing showing the operation of prior art
scanning microscopes.
FIG. 2 is a simplified drawing of the components of a scanning ion
conductance microscope according to the present invention when
operated to obtain surface topography.
FIG. 3 is a simplified drawing of the scanning ion conductance
microscope of FIG. 2 showing a modification thereof wherein
multiple scanning pipettes are employed and its manner of operation
to obtain the imaging of ion currents through channels in membranes
and the like.
FIG. 4 is a simplified drawing of the scanning ion conductance
microscope of FIGS. 2 and 3 showing a further modification thereof
wherein the free electrode is replaced by an electrode contained in
one of the multiple pipettes.
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.
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