WO2008075842A1 - Microscopie à conductance ionique comprenant une multipipette - Google Patents

Microscopie à conductance ionique comprenant une multipipette Download PDF

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Publication number
WO2008075842A1
WO2008075842A1 PCT/KR2007/006244 KR2007006244W WO2008075842A1 WO 2008075842 A1 WO2008075842 A1 WO 2008075842A1 KR 2007006244 W KR2007006244 W KR 2007006244W WO 2008075842 A1 WO2008075842 A1 WO 2008075842A1
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WO
WIPO (PCT)
Prior art keywords
pipette
multipipette
ion
conductance microscope
noise
Prior art date
Application number
PCT/KR2007/006244
Other languages
English (en)
Inventor
Dal-Hyun Kim
Byong-Chon Park
Sang-Jung Ahn
Ja-Yong Koo
Dae-Won Moon
Original Assignee
Korea Research Institute Of Standards And Science
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Korea Research Institute Of Standards And Science filed Critical Korea Research Institute Of Standards And Science
Publication of WO2008075842A1 publication Critical patent/WO2008075842A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01Q—SCANNING-PROBE TECHNIQUES OR APPARATUS; APPLICATIONS OF SCANNING-PROBE TECHNIQUES, e.g. SCANNING PROBE MICROSCOPY [SPM]
    • G01Q60/00—Particular types of SPM [Scanning Probe Microscopy] or microscopes; Essential components thereof
    • G01Q60/44—SICM [Scanning Ion-Conductance Microscopy] or apparatus therefor, e.g. SICM probes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01Q—SCANNING-PROBE TECHNIQUES OR APPARATUS; APPLICATIONS OF SCANNING-PROBE TECHNIQUES, e.g. SCANNING PROBE MICROSCOPY [SPM]
    • G01Q70/00—General aspects of SPM probes, their manufacture or their related instrumentation, insofar as they are not specially adapted to a single SPM technique covered by group G01Q60/00
    • G01Q70/06—Probe tip arrays

Definitions

  • the present invention relates to a scanning ion conductance microscope having a pipette, and more particularly to a scanning ion conductance microscope having a multipipette which includes multiple pipettes to measure ion currents so that it is less affected by noise, thereby achieving a more accurate image .
  • a scanning ion conductance microscope generally includes a pipette 100 for detecting an ion current, a current amplifier 300 for amplifying and transferring the current detected by the pipette to an analysis computer, a scanner 200 for horizontally moving glass with a sample placed on it, and a scanner controller 400 for processing an ion current signal and driving the scanner.
  • the scanning ion conductance microscope is a type of scanning probe microscope that uses, as a feedback signal, an ion current flowing through a hole of about lOOnm in a single pipette 100. As the pipette hole approaches a sample at a distance, which corresponds to about the size of the sample hole, the ion current decreases in proportion to the distance. The curvature of the surface of the sample can be imaged by performing scanning while keeping the ion current at a constant level .
  • the conventional scanning ion conductance microscope has a low signal-to-noise ratio (SNR) and thus has a low resolution since the ion current measured by the microscope not only depends on the distance but also varies with changes in the ion concentration of a buffer solution and noise such as electrode noise and external noise.
  • SNR signal-to-noise ratio
  • This scanning ion conductance microscope which uses AC ion currents changing while a pipette vibrates vertically, has been developed recently to overcome these problems.
  • This scanning ion conductance microscope includes vertically vibrating means 120 for vibrating a pipette 100 vertically as shown in FIG. 4.
  • the distance-modulation (or distance-changing-mode) scanning ion conductance microscope having the vertically vibrating means that vibrates the pipette vertically has attracted attention since has high resolution and high sensitivity.
  • this microscope requires the vertically vibrating means to alternately increase and decrease the distance and needs to measure the AC ion current using a lock- in amplifier and thus has a limited imaging speed and is not suitable for high-speed imaging.
  • This scanning ion conductance microscope has been used for limited imaging applications on the cellular level since it is very difficult for the microscope to perform the high-speed ion conductance imaging with high sensitivity and high resolution.
  • the present invention has been made in view of the above problems, and it is an object of the present invention to provide a scanning ion conductance microscope having a multipipette which can be used for in-vivo measurement of biological phenomena, which occur in cellular or living tissues, such as measurement of cellular dynamics.
  • a multipipette-equipped scanning ion conductance microscope comprising a pipette having a pipette electrode for measuring a change in the level of an ion current flowing through a pipette hole of the pipette and then imaging the measurement, wherein the pipette is a multipipette having a group of multiple pipettes, each including a pipette electrode, and a sample is measured by comparing ion currents flowing through pipette holes of barrels of the multiple pipettes.
  • FIG. 1 illustrates an example of a scanning ion conductance microscope having a multipipette according to the invention
  • FIGS. 2A to 2C are perspective views showing different examples of the multipipette included in the scanning ion conductance microscope according to the invention.
  • FIG. 3 is a cross-sectional view showing how an ion current flows in one direction in the scanning ion conductance microscope having a multipipette according to the invention
  • FIG. 4 is a cross-sectional view showing how an ion current flows in another direction in the scanning ion conductance microscope having a multipipette according to the invention
  • FIG. 5 illustrates how ion currents flow between pipette holes in various directions in the scanning ion conductance microscope having a multipipette according to the invention.
  • FIG. 6 illustrates the configuration of an example of a conventional scanning ion conductance microscope having a single pipette.
  • FIG. 1 illustrates an example of a scanning ion conductance microscope having a multipipette according to the invention
  • FIGS. 2A to 2C are perspective views showing different examples of the multipipette included in the scanning ion conductance microscope according to the invention
  • FIG. 3 is a cross-sectional view showing how an ion current flows in one direction in the scanning ion conductance microscope having a multipipette according to the invention
  • FIG. 4 is a cross-sectional view showing how an ion current flows in another direction in the scanning ion conductance microscope having a multipipette according to the invention
  • FIG. 5 illustrates how ion currents flow between pipette holes in various directions in the scanning ion conductance microscope having a multipipette according to the invention.
  • the scanning ion conductance microscope according to the invention includes a multipipette 1.
  • the multipipette 1 has multiple pipettes, each including a pipette electrode 12 inside a pipette hole 11.
  • the multipipette 1 may be constructed by combining a plurality of individually manufactured pipettes as shown.
  • the multipipette 1 may also be constructed by mounting a pipette electrode 12 in each of a number of pipette holes 11 arranged longitudinally in a vertical direction. That is, the multipipette 1 may be constructed by mounting pipette electrodes 12 in a glass capillary having multiple holes arranged in a longitudinal direction of the glass capillary.
  • the multipipette 1 can be constructed of any number of pipettes such as 2, 3, or 7 pipettes as shown in FIG. 2.
  • An ion current detected by the pipette electrode 12 may be an ion current that flows through the pipette hole 11 by a voltage difference between the pipette electrode 12 and a reference electrode 13 immersed in a buffer solution or may be an ion current that flows by a voltage difference between two adjacent pipette electrodes 12.
  • the scanning ion conductance microscope having the multipipette 1 may be a general scanning ion conductance microscope or a distance-modulation scanning ion conductance microscope.
  • the scanning ion conductance microscope according to the invention may further include a noise reference pipette 10 provided at a flat portion of the sample to measure an ion current at the flat portion and to detect noise, thereby extracting noise included in ion currents measured by the multipipette 1.
  • a noise reference pipette 10 provided at a flat portion of the sample to measure an ion current at the flat portion and to detect noise, thereby extracting noise included in ion currents measured by the multipipette 1.
  • the scanning ion conductance microscope according to the invention constructed as described above compares ion currents flowing through the holes of the multipipette as described above to remove noise not associated with the surface curvature, thereby achieving both high resolution and high signal-to-noise ratio (SNR) characteristics.
  • the multipipette may not only be manufactured using a generally used multi-barrel glass capillary but also be manufactured using a semiconductor Micro-Electro-Mechanical Systems (MEMS) process.
  • MEMS Micro-Electro-Mechanical Systems
  • Ion current flows in the buffer solution containing a large number of ions through the pipette hole when there is a voltage difference between the pipette electrode and the reference electrode placed in the buffer solution.
  • the buffer solution may be any solution containing ions.
  • the multipipette scanning ion conductance microscope mode method is basically based on experimental discoveries through experimental comparison of the general scanning ion conductance microscope and the distance-modulation scanning ion conductance microscope.
  • the conventional scanning ion conductance microscope has been limited to measurement of a large structure such as a large hole of lOOnm. That is, the conventional scanning ion conductance microscope has been limited to use for processes such as measurement of a large structure of about lOOnm having a large curvature since the conventional scanning ion conductance microscope has a low SNR ratio and a low resolution.
  • the scanning ion conductance microscope having a multipipette combines the advantages of both the well-known general scanning ion conductance microscope and the high-resolution, high SNR distance- modulation scanning ion conductance microscope that has been developed recently.
  • Ion current flows by a voltage difference between a reference electrode 13 immersed in a buffer solution and pipette electrodes 12 provided in pipette holes 11 formed in a pipette 1 as shown in FIG. 3.
  • an ion current flows to the reference electrode 13 through the two adjacent pipette holes 11 of the pipette electrodes 12.
  • the level of the ion current flowing through each of the small pipette holes 11 of the multipipette 1 is proportional to the distance between the pipette hole 11 and the surface of the sample.
  • ion currents flowing through the pipette holes are compared with each other to extract a noise-related ion current not associated with the distance, thereby removing a noise-related measured level.
  • Pure ion currents depending on the distance are used alone to form an image, thereby increasing the SNR and achieving high-sensitivity, high-speed measurement .
  • FIG. 5 illustrates measurement of a sample using an ion current flowing from one pipette hole 11 of the multipipette 1 to an adjacent pipette hole 11. As shown, the ion current flows from one pipette hole 11 of the multipipette 1 to an adjacent pipette hole 11 by a voltage difference between two pipette electrodes 12 of the two pipette holes 11.
  • the respective flow directions of the ion currents are denoted by arrows in FIG. 5. If the sample and the multipipette come close to each other, the ion current is reduced since the flow of the current is impeded. This method can be used to image the surface curvature .
  • the multipipette When the multipipette has three or more pipettes, it is possible to remove noise, increase the signal-to-noise ratio, and achieve high-sensitivity, high-speed imaging by comparing ion currents of their pipette electrodes 12 and therefore there is no need to separately install the reference electrode 13, resulting in that the multipipette can be used for in-vivo cell imaging.
  • a voltage difference between the two pipette electrodes 12 in the two adjacent pipette holes 11 of the multipipette 1 causes an ion current to flow from one of the two pipette holes 11 to the other.
  • the multipipette 1 includes three pipettes, one of the pipettes is set as a reference Ia such that ion currents flow from a reference pipette hole 11 of the reference Ia to pipette holes 11 of the two adjacent pipette Ib.
  • the multipipette 1 includes seven pipettes, ion currents flow from a pipette electrode 12 of one of the pipettes Ia, which is set as a reference, to pipette electrodes 12 of the other pipettes Ib.
  • the pipette electrode 12 of the central pipette Ia is set as a reference electrode such that currents flow to the pipette electrodes 12 of the other pipettes Ib that are radially arranged.
  • the central pipette Ia is used as a reference, it is possible to more accurately detect noise since currents are measured with reference to the pipette Ia located at the center of the other pipettes Ib.
  • ion currents are measured at multiple pipette electrodes 12 at once to obtain the same number of images as the number of the pipettes of the multipipette 1, thereby increasing the imaging speed and efficiency as high as the number of the pipettes of the multipipette 1. Measuring ion currents over a wide area at once in this manner can more quickly measure the sample.
  • a measurement signal output by the noise reference pipette 10 also includes a noise component due to external noise caused by a change in the buffer solution or the like although the measurement signal of the noise reference pipette 10 should be constant since it measures an ion current at a constant height.
  • the noise component is equally applied to the multipipette 1. Accordingly, it is possible to detect the noise using an ion current measured by the noise reference pipette 10 and, by removing the measured noise from ion currents measured by the pipettes of the multipipette, it is possible to obtain correct versions, from which the noise has been removed, of the signals measured by the pipettes of the multipipette.
  • the noise reference pipette it can be combined with a conventional single-barrel pipette to remove noise, thereby achieving high-speed multipipette imaging.
  • the present invention provides a high-speed, high SNR scanning ion conductance microscope mode using multipipette ion currents, wherein ion currents flowing through holes of a multipipette are compared to remove noise not associated with the surface curvature, thereby achieving both the high speed characteristics of the general scanning ion conductance microscope and the high-resolution, high-SNR characteristics of the distance-modulation scanning ion conductance microscope, and also provides a microscope using such a mode.

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  • Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Electrochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Radiology & Medical Imaging (AREA)
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  • Investigating Or Analyzing Materials By The Use Of Electric Means (AREA)

Abstract

La présente invention concerne un microscope à conductance ionique comprenant une multipipette qui comprend plusieurs pipettes servant à mesurer les courants ioniques de sorte qu'il soit moins affecté par le bruit, ce qui permet d'obtenir une image plus précise. La multipipette mesure les changements des niveaux des courants ioniques s'écoulant dans les trous de pipette des multiples pipettes, chaque pipette comportant une électrode, puis la multipipette produit une image de la mesure. Un échantillon est mesuré par comparaison des courants ioniques s'écoulant dans les trous de pipette de la multipipette.
PCT/KR2007/006244 2006-12-19 2007-12-04 Microscopie à conductance ionique comprenant une multipipette WO2008075842A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2006-0130405 2006-12-19
KR1020060130405A KR100816088B1 (ko) 2006-12-19 2006-12-19 멀티 피펫을 구비한 이온전도현미경

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130140191A1 (en) * 2010-08-11 2013-06-06 Patrick Unwin Pipets containing electrolyte and electrodes
US9598281B2 (en) 2011-03-03 2017-03-21 The Regents Of The University Of California Nanopipette apparatus for manipulating cells
CN109142797A (zh) * 2018-09-05 2019-01-04 西安交通大学 一种液滴型扫描离子电导显微镜及其探针和扫描方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4868396A (en) * 1987-10-13 1989-09-19 Arizona Board Of Regents, Arizona State University Cell and substrate for electrochemical STM studies
US4924091A (en) * 1989-02-01 1990-05-08 The Regents Of The University Of California Scanning ion conductance microscope
US5202004A (en) * 1989-12-20 1993-04-13 Digital Instruments, Inc. Scanning electrochemical microscopy

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4868396A (en) * 1987-10-13 1989-09-19 Arizona Board Of Regents, Arizona State University Cell and substrate for electrochemical STM studies
US4924091A (en) * 1989-02-01 1990-05-08 The Regents Of The University Of California Scanning ion conductance microscope
US5202004A (en) * 1989-12-20 1993-04-13 Digital Instruments, Inc. Scanning electrochemical microscopy

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130140191A1 (en) * 2010-08-11 2013-06-06 Patrick Unwin Pipets containing electrolyte and electrodes
JP2013533497A (ja) * 2010-08-11 2013-08-22 ザ ユニヴァーシティ オブ ウォーリック 電解質および電極を含むピペット
US9316613B2 (en) * 2010-08-11 2016-04-19 The University Of Warwick Pipets containing electrolyte and electrodes
EP2603801B1 (fr) * 2010-08-11 2019-07-31 The University Of Warwick Dispositifs de microscopie électrochimique à balayage comprenant pipettes qui contiennent un électrolyte et des electrodes et méthode de microscopie électrochimique à balayage
US9598281B2 (en) 2011-03-03 2017-03-21 The Regents Of The University Of California Nanopipette apparatus for manipulating cells
US10513434B2 (en) 2011-03-03 2019-12-24 The Regents Of The University Of California Nanopipette apparatus for manipulating cells
CN109142797A (zh) * 2018-09-05 2019-01-04 西安交通大学 一种液滴型扫描离子电导显微镜及其探针和扫描方法
CN109142797B (zh) * 2018-09-05 2020-03-17 西安交通大学 一种液滴型扫描离子电导显微镜及其探针和扫描方法

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