Apparatus and method for a near field scanning optical microscope in aqueous solution

• US 6,621,079 B1
• Filed: 06/29/2000
• Issued: 09/16/2003
• Est. Priority Date: 07/02/1999
• Status: Expired due to Fees

• Current Assignee: University of Virginia Patent Foundation (University of Virginia)
• Original Assignee: University of Virginia Patent Foundation (University of Virginia)
• Inventors: Shao, Zhifeng, Szabo, Gabor, Mannelqvist, Anders
• Primary Examiner(s): Lee, John R.
• Assistant Examiner(s): Gill, Erin-Michael
• Application Number:
  US09/606,343
• Time in Patent Office: 1,174 Days
• Field of Search: 250/306, 250/234, 250/440.1, 250/440.2, 751/05, 356/375, 356/376, 731/05
• US Class Current: 250/306
• CPC Class Codes: G01Q 60/22 Probes, their manufacture, or their related instrumentation, e.g. holders
• CPC Class Codes: G01Q 60/44 SICM [Scanning Ion-Conductance Microscopy] or apparatus therefor, e.g. SICM probes

Abstract

The present invention near field scanning optical microscope NSOM, and related method thereof, provides a high resolution image of a sample in aqueous solution without damaging the sample. This attribute will greatly expand the applications and utility of a NSOM in biomedicine, among other fields. Moreover, the NSOM can be further extended to include signals other than light. In operation, a pipette is filled with an electrolyte solution (aqueous solution) and lowered through the reservoir toward the surface of the sample while the current between the electrode inside the pipette and the electrode in the reservoir is monitored. As the tip of the pipette approaches the surface, the ion current decreases because the space through which ions can flow is reduced. The pipette is then scanned laterally over the surface and the path of the tip pipette follows the topography of the surface. An optical image is simultaneously provided as the light source is incident on the sample and the emitted light reaches the image acquisition device after being transmitted through the sample. The acquisition device converts the light energy into electrical signals where they are processed for displaying the acquired images on the monitor or printer.

Claims

1. A near field scanning optical microscope (NSOM) comprising:

a reservoir holding a sample to be scanned therein;

a pipette having an open tip communicating with a hollow shaft;

an electrolyte solution disposed within the reservoir covering the sample and disposed within the tip of said pipette;

a first electrode disposed in said shaft in ionic communication with said electrolyte solution in said open tip, said first electrode being in ionic communication with electrolyte solution in said reservoir via said open tip by means of electrolyte solution within said tip;

a second electrode disposed in said reservoir in ionic communication with said electrolyte solution in said reservoir and forming a continuous ionic current path between said first and second electrodes via the electrolyte solution in said reservoir and in said open tip;

scanning means for scanning said tip of said pipette over a top surface of said sample in a scanning pattern;

voltage means for applying a voltage across said first and second electrodes;

current means for measuring a current flowing in the ionic current path between said first and second electrodes through said open tip of said pipette and for supplying an indication of said current at an output thereof; and

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;

wherein the improvement comprises;

a light source disposed on said microscope for emitting light through said shaft of said pipette and onto the sample;

said hollow shaft being opaque to substantially prevent light emitted from said light source from being transmitted through the walls of said shaft; and

an image acquisition means in optical communication with said light source so that the sample is in optical communication between said light source and said image acquisition means, whereby said image acquisition means for acquiring an image of the sample.

2. The near field scanning optical microscope (NSOM) of claim 1 wherein:

said image acquisition means converts the acquired light into electrical signals for purpose of providing a high resolution image of the sample.

3. The near field scanning optical microscope (NSOM) of claim 2 wherein:

said control logic means includes logic for causing said scanning means to position said tip of said pipette at a distance above said top surface which will maintain the current between said first and second electrodes through said open tip of said pipette at a constant value which will cause said tip to follow said top surface in close non-contacting proximity thereto.

4. The near field scanning optical microscope (NSOM) of claim 3 wherein:

said control logic means includes logic for causing said scanning means to scan said tip of said pipette in a plane parallel and close adjacent above said top surface.

5. The near field scanning optical microscope (NSOM) of claim 4 wherein:

said control means for outputting data of interest related to said sample as it is scanned.

6. The near field scanning optical microscope (NSOM) of claim 5 further comprising:

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 pipette.

7. The near field scanning optical microscope (NSOM) of claim 6 wherein:

said data of interest output by said control logic means reflects the topography of said top surface.

8. The near field scanning optical microscope (NSOM) of claim 2 further comprising:

a plurality of said pipettes disposed to form a multi-bore pipette having respective tip openings;

a plurality of said first electrodes disposed in respective ones of said plurality of pipettes, each of said plurality pipettes being specific for a different function;

a first of said plurality of pipettes for optically imaging the sample;

a second of said plurality of pipettes for collecting secondary information; and

said control logic means includes logic for causing said scanning means to position said tips of said multi-bore pipettes at a distance above said top surface which will maintain the current between said first electrode disposed in said optical imaging pipette and said second electrode through said open tip of said optical imaging pipette at a constant value which will cause said tips of said multi-bore pipettes to follow said top surface in close non-contacting proximity thereto.

9. The near field scanning optical microscope (NSOM) of claim 8 further comprising:

a flow inhibitor means is disposed at said tip opening of said secondary information pipette, wherein said flow inhibitor means effects the ion current flow of ion current through the tip opening of said secondary information pipette.

10. The near field scanning optical microscope (NSOM) of claim 9 wherein:

said flow inhibitor means is selected from the group consisting of a vesicle and membrane.

14. The near field scanning optical microscope (NSOM) of claim 2 further comprising:

a flow inhibitor means is disposed at said tip opening of said pipette, wherein said flow inhibitor means effects the ion current flow of ion current through the tip opening of said pipette.

15. The near field scanning optical microscope (NSOM) of claim 14 wherein:

said flow inhibitor means is selected from the group consisting of a vesicle and membrane.

11. A method for optically imaging a sample comprising the steps of:

disposing the sample to be scanned in a reservoir containing an electrolyte covering the sample;

providing a pipette having an open tip communicating with a hollow shaft, wherein said shaft is at least partially opaque;

disposing an electrolyte within the tip of the pipette;

disposing a first electrode in the shaft in ionic communication with the electrolyte in the open tip;

disposing a second electrode in the reservoir in ionic communication with the electrolyte in said reservoir and forming a continuous ionic current path between said first and second electrodes via the electrolyte solution in said reservoir and in said open tip;

applying a voltage across the first and second electrodes and measuring an ionic current flowing in the ionic current path between the first and second electrodes through the open tip;

scanning the tip of the pipette over a top surface of the sample in a scanning pattern with the tip of the pipette at a desired distance above the top surface which will maintain the current flow 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 non-contacting proximity thereto so as to provide a z-directional component of the position of the tip of the pipette;

scanning the tip of the pipette over a top surface of the sample in a scanning pattern with the tip of the pipette in a plane parallel and close adjacent above the top surface;

emitting light through said shaft of said pipette onto the sample; and

acquiring light having been transmitted through the sample for acquiring an image of the sample and outputting a corresponding acquisition signal.

12. The method of optically imaging the sample of claim 11, further compressing:

outputting data of interest which reflects the topography of the top surface.

13. The method of claim 11 further comprising the steps of:

a plurality of said pipettes disposed to form a multi-bore pipette having respective tip openings;

providing a plurality of said first electrodes disposed in respective ones of said plurality of pipettes, each of said plurality pipettes being specific for a different function, whereby a first of said plurality of pipettes for optically imaging the sample and whereby a second of said plurality of pipettes for collecting secondary information; and

scanning said tips of said multi-bore pipettes at a distance above said top surface which will maintain the current between said first electrode disposed in said optical imaging pipette and said second electrode through said open tip of said optical imaging pipette at a constant value which will cause said tips of said multi-bore pipettes to follow said top surface in close non-contacting proximity thereto.