Scanning ion-conductance microscopy (SICM) is a scanning probe microscopy technique that uses an electrode as the probe tip. [1] SICM allows for the determination of the surface topography of micrometer and even nanometer-range [2] structures in aqueous media conducting electrolytes. The samples can be hard or soft, are generally non-conducting, and the non-destructive nature of the measurement allows for the observation of living tissues and cells, and biological samples in general.
It is able to detect steep profile changes in samples [3] and can be used to map a living cell's stiffness [4] in tandem with its detailed topography, or to determine the mobility of cells during their migrations. [5]
Scanning ion conductance microscopy is a technique using the increase of access resistance in a micro-pipette in an electrolyte-containing aqueous medium when it approaches a poorly conducting surface. It monitors the ionic current flowing in and out of the micro/nano-pipette, which is hindered if the tip is very close to the sample surface since the gap through which ions can flow is reduced in size.
The SICM setup is generally as follows: A voltage is applied between the two Ag/AgCl electrodes, one of which is in the glass micro-pipette, and the other in the bulk solution. The voltage will generate an ionic current between the two electrodes, flowing in and out of the micro-pipette. The conductance between the two electrodes is measured, and depends on the flux of ions.
Movements of the pipette are regulated through piezoelectrics.
The micro-pipette is lowered closer and closer to the sample until the ionic flux starts to be restricted. The conductance of the system will then decrease (and the resistance will increase). When this resistance reaches a certain threshold the tip is stopped and the position recorded. The tip is then moved (in different ways depending on the mode used, see below) and another measurement is made in a different location, and so on. In the end, comparing the positions of all the measurements provides a detailed height profile of the sample.
It is important to note that the tip is stopped before contacting the sample, thus it does not bend nor damage the surface observed, which is one of the major advantages of SICM.
The total resistance of the setup (Rtot) is the sum of the three resistances: Rb, Rm, and Rt. Rb the resistance of the electrolyte solution between the tip of the micro-pipette and the electrode in the bulk of the solution. Rm is the resistance of the electrolyte solution between the electrode in the micro-pipette and the tip. Rt is the resistance of the current flowing through the tip
Rb and Rm depend on the electrolyte conductivity, and the position and shape of the Ag/AgCl electrodes. Rt depends on the size and shape of the aperture, and on the distance between the tip and the sample.
All the parameters except the distance between tip and sample are constant within a given SICM setup, thus it is the variation of Rt with the distance to the sample that will be used to determine the topography of the sample.
Usual approximations are: 1) the voltage drop at the surfaces of the Ag/AgCl electrodes is neglected, it is assumed that it is negligible compared to the voltage drop at the tip, and constant, 2) the fact that the bulk resistance is a function of d is neglected since it depends on the distance between the tip and the electrode in the bulk.
SICM has a worse resolution than AFM or STM, which can routinely reach resolutions of about 0.1 nm. The resolution of SICM measurement is limited to 1.5 times the diameter of the tip opening [7] in theory, but measurements taken with a 13 nm opening-diameter managed a resolution of around 3–6 nm. [2]
SICM can be used to image poorly or non-conducting surfaces, [6] which is impossible with STM.
In SICM measurements, the tip of the micro-pipette does not touch the surface of the sample; which allows the imaging of soft samples (cells, biological samples, cell villi) [8] [9] [10] without deformation.
SICM is used in an electrolyte-containing solution, so can be used in physiological media and image living cells and tissues, and monitor biological processes while they are taking place. [10]
In hopping mode, it is able to correctly determine profiles with steep slopes and grooves.
There are four main imaging modes in SICM: constant-z mode, Direct current (constant distance) mode, alternating current mode, and hopping/backstep/standing approach mode.
In constant-z mode, the micro-pipette is maintained at a constant z (height) while it is moved laterally and the resistance is monitored, its variations allowing for the reconstitution of the topography of the sample. This mode is fast but is barely used since it only works on very flat samples. If the sample has rugged surfaces, the pipette will either crash into it, or be too far for imaging most of the sample.
In direct current (DC) mode (constant distance mode), the micro-pipette is lowered toward the sample until a predefined resistance is reached. The pipette is then moved laterally and a feedback loop maintains the distance to the sample (through the resistance value). The z-position of the pipette determines the topography of the sample. This mode does not detect steep slopes in sample, may contact the sample in such cases and is prone to electrode drift.
In alternating current (AC) mode, the micro-pipette oscillates vertically in addition to its usual movement. While the pipette is still far from the surface the ionic current, and the resistance is steady, so the pipette is lowered. Once the resistance starts oscillating, the amplitude serves as feedback to modulate the position until a predefined amplitude is reached. [8] [9]
The response of the AC component increases much steeper than the DC, and allows for the recording of more complex samples.
In hopping (/backstep/standing approach) mode, the micro-pipette is lowered to the sample until a given resistance is reached, and the height is recorded. Then the pipette is dragged back, laterally moved and another measurement is made, and the process repeats. The topography of the sample can then be reconstituted.
Hopping mode is slower than the others, but is able to image complex topography and even entire cells, without distorting the sample surface. [11] [12]
SICM was used to image a living neural cell from rat brain, [5] determine the life cycle of microvilli, [8] observe the movement of protein complexes in spermatozoa. [2]
SICM has been combined with fluorescence microscopy [2] and förster resonance energy transfer. [13]
SICM has been used in a "smart patch-clamp" technique, clamping the pipette by suction to the surface of a cell and then monitoring the activity of the sodium channels in the cell membrane. [14]
A combination of AFM and SICM was able to obtain high resolution images of synthetic membranes in ionic solutions. [15]
Scanning near-field optical microscopy has been used with SICM; the SICM measurement allowed for the tip of the pipette to be placed very close to the surface of the sample. Fluorescent particles, coming from the inside of the micro-pipette, provide a light source for the SNOM that is being continuously renewed and prevent photobleaching. [16] [17]
FSICM [18] (Fast SICM), improving notably the speed of hopping mode has recently been developed.
A scanning tunneling microscope (STM) is a type of scanning probe microscope used for imaging surfaces at the atomic level. Its development in 1981 earned its inventors, Gerd Binnig and Heinrich Rohrer, then at IBM Zürich, the Nobel Prize in Physics in 1986. STM senses the surface by using an extremely sharp conducting tip that can distinguish features smaller than 0.1 nm with a 0.01 nm (10 pm) depth resolution. This means that individual atoms can routinely be imaged and manipulated. Most scanning tunneling microscopes are built for use in ultra-high vacuum at temperatures approaching absolute zero, but variants exist for studies in air, water and other environments, and for temperatures over 1000 °C.
A scanning electron microscope (SEM) is a type of electron microscope that produces images of a sample by scanning the surface with a focused beam of electrons. The electrons interact with atoms in the sample, producing various signals that contain information about the surface topography and composition of the sample. The electron beam is scanned in a raster scan pattern, and the position of the beam is combined with the intensity of the detected signal to produce an image. In the most common SEM mode, secondary electrons emitted by atoms excited by the electron beam are detected using a secondary electron detector. The number of secondary electrons that can be detected, and thus the signal intensity, depends, among other things, on specimen topography. Some SEMs can achieve resolutions better than 1 nanometer.
Electrophysiology is the branch of physiology that studies the electrical properties of biological cells and tissues. It involves measurements of voltage changes or electric current or manipulations on a wide variety of scales from single ion channel proteins to whole organs like the heart. In neuroscience, it includes measurements of the electrical activity of neurons, and, in particular, action potential activity. Recordings of large-scale electric signals from the nervous system, such as electroencephalography, may also be referred to as electrophysiological recordings. They are useful for electrodiagnosis and monitoring.
Atomic force microscopy (AFM) or scanning force microscopy (SFM) is a very-high-resolution type of scanning probe microscopy (SPM), with demonstrated resolution on the order of fractions of a nanometer, more than 1000 times better than the optical diffraction limit.
The patch clamp technique is a laboratory technique in electrophysiology used to study ionic currents in individual isolated living cells, tissue sections, or patches of cell membrane. The technique is especially useful in the study of excitable cells such as neurons, cardiomyocytes, muscle fibers, and pancreatic beta cells, and can also be applied to the study of bacterial ion channels in specially prepared giant spheroplasts.
Scanning probe microscopy (SPM) is a branch of microscopy that forms images of surfaces using a physical probe that scans the specimen. SPM was founded in 1981, with the invention of the scanning tunneling microscope, an instrument for imaging surfaces at the atomic level. The first successful scanning tunneling microscope experiment was done by Gerd Binnig and Heinrich Rohrer. The key to their success was using a feedback loop to regulate gap distance between the sample and the probe.
Kelvin probe force microscopy (KPFM), also known as surface potential microscopy, is a noncontact variant of atomic force microscopy (AFM). By raster scanning in the x,y plane the work function of the sample can be locally mapped for correlation with sample features. When there is little or no magnification, this approach can be described as using a scanning Kelvin probe (SKP). These techniques are predominantly used to measure corrosion and coatings.
Nanoelectrochemistry is a branch of electrochemistry that investigates the electrical and electrochemical properties of materials at the nanometer size regime. Nanoelectrochemistry plays significant role in the fabrication of various sensors, and devices for detecting molecules at very low concentrations.
Photothermal microspectroscopy (PTMS), alternatively known as photothermal temperature fluctuation (PTTF), is derived from two parent instrumental techniques: infrared spectroscopy and atomic force microscopy (AFM). In one particular type of AFM, known as scanning thermal microscopy (SThM), the imaging probe is a sub-miniature temperature sensor, which may be a thermocouple or a resistance thermometer. This same type of detector is employed in a PTMS instrument, enabling it to provide AFM/SThM images: However, the chief additional use of PTMS is to yield infrared spectra from sample regions below a micrometer, as outlined below.
Scanning thermal microscopy (SThM) is a type of scanning probe microscopy that maps the local temperature and thermal conductivity of an interface. The probe in a scanning thermal microscope is sensitive to local temperatures – providing a nano-scale thermometer. Thermal measurements at the nanometer scale are of both scientific and industrial interest. The technique was invented by Clayton C. Williams and H. Kumar Wickramasinghe in 1986.
In microscopy, conductive atomic force microscopy (C-AFM) or current sensing atomic force microscopy (CS-AFM) is a mode in atomic force microscopy (AFM) that simultaneously measures the topography of a material and the electric current flow at the contact point of the tip with the surface of the sample. The topography is measured by detecting the deflection of the cantilever using an optical system, while the current is detected using a current-to-voltage preamplifier. The fact that the CAFM uses two different detection systems is a strong advantage compared to scanning tunneling microscopy (STM). Basically, in STM the topography picture is constructed based on the current flowing between the tip and the sample. Therefore, when a portion of a sample is scanned with an STM, it is not possible to discern if the current fluctuations are related to a change in the topography or to a change in the sample conductivity.
Photoconductive atomic force microscopy (PC-AFM) is a variant of atomic force microscopy that measures photoconductivity in addition to surface forces.
Scanning electrochemical microscopy (SECM) is a technique within the broader class of scanning probe microscopy (SPM) that is used to measure the local electrochemical behavior of liquid/solid, liquid/gas and liquid/liquid interfaces. Initial characterization of the technique was credited to University of Texas electrochemist, Allen J. Bard, in 1989. Since then, the theoretical underpinnings have matured to allow widespread use of the technique in chemistry, biology and materials science. Spatially resolved electrochemical signals can be acquired by measuring the current at an ultramicroelectrode (UME) tip as a function of precise tip position over a substrate region of interest. Interpretation of the SECM signal is based on the concept of diffusion-limited current. Two-dimensional raster scan information can be compiled to generate images of surface reactivity and chemical kinetics.
Automated patch clamping is beginning to replace manual patch clamping as a method to measure the electrical activity of individual cells. Different techniques are used to automate patch clamp recordings from cells in cell culture and in vivo. This work has been ongoing since the late 1990s by research labs and companies trying to reduce its complexity and cost of patch clamping manually. Patch clamping for a long time was considered an art form and is still very time consuming and tedious, especially in vivo. The automation techniques try to reduce user error and variability in obtaining quality electrophysiology recordings from single cells.
Non-contact atomic force microscopy (nc-AFM), also known as dynamic force microscopy (DFM), is a mode of atomic force microscopy, which itself is a type of scanning probe microscopy. In nc-AFM a sharp probe is moved close to the surface under study, the probe is then raster scanned across the surface, the image is then constructed from the force interactions during the scan. The probe is connected to a resonator, usually a silicon cantilever or a quartz crystal resonator. During measurements the sensor is driven so that it oscillates. The force interactions are measured either by measuring the change in amplitude of the oscillation at a constant frequency just off resonance or by measuring the change in resonant frequency directly using a feedback circuit to always drive the sensor on resonance.
Sir David Klenerman is a British biophysical chemist and a professor of biophysical chemistry at the Department of Chemistry at the University of Cambridge and a Fellow of Christ's College, Cambridge.
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Electrochemical AFM (EC-AFM) is a particular type of Scanning probe microscopy (SPM), which combines the classical Atomic force microscopy (AFM) together with electrochemical measurements. EC-AFM allows to perform in-situ AFM measurements in an electrochemical cell, in order to investigate the actual changes in the electrode surface morphology during electrochemical reactions. The solid-liquid interface is thus investigated. This technique was developed for the first time in 1996 by Kouzeki et al., who studied amorphous and polycrystalline thin films of Naphthalocyanine on Indium tin oxide in a solution of 0.1 M Potassium chloride (KCl). Unlike the Electrochemical scanning tunneling microscope, previously developed by Itaya and Tomita in 1988, the tip is non-conductive and it is easily steered in a liquid environment.
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