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3.3G: Scanned-Probe Microscopy - Biology

3.3G: Scanned-Probe Microscopy - Biology


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Learning Objectives

  • Describe the different types of scanning probe techniques and their advantages over other types of microscopy

3-D Images

Scanned-probe microscopy (SPM) produces highly magnified and three-dimensional-shaped images of specimens in real time. SPM employs a delicate probe to scan the surface of the specimen, eliminating the limitations that are found in electron and light microscopy. SPM covers several related technologies for imaging and measuring surfaces on a fine scale, down to the level of molecules and groups of atoms.

A scan may cover a distance of over 100 micrometers in the x and y directions and 4 micrometers in the z direction. SPM technologies share the concept of scanning a sharp probe tip with a small radius of curvature across the object surface. The tip is mounted on a flexible cantilever, allowing the tip to follow the surface profile. When the tip moves in proximity to the investigated object, forces of interaction between the tip and the surface influence the movement of the cantilever. Selective sensors detect these movements. Various interactions can be studied depending on the mechanics of the probe.

There are three common scanning probe techniques: atomic force microscopy (AFM) measures the interaction force between the tip and surface. The tip may be dragged across the surface, or may vibrate as it moves. The interaction force will depend on the nature of the sample, the probe tip and the distance between them. Scanning tunneling microscopy (STM) measures a weak electrical current flowing between tip and sample as they are held apart. Near-field scanning optical microscopy (NSOM) scans a very small light source very close to the sample. Detection of this light energy forms the image.

Key Points

  • Scanned-probe microscopy has enabled researchers to create images of surfaces at the nanometer scale with a probe.
  • The probe has an extremely sharp tip that interacts with the surface of the specimen.
  • There are several variations of scanned-probe microscopy of which atomic force microscopy, scanning tunneling microscopy, and near-field scanning optical microscopy are most commonly used.

Key Terms

  • micrometer: An SI/MKS unit of measure, the length of one one-millionth of a meter. Symbols: µm, um, rm

3.3G: Scanned-Probe Microscopy - Biology

The early pioneers of microscopy opened a window into the invisible world of microorganisms. But microscopy continued to advance in the centuries that followed. In 1830, Joseph Jackson Lister created an essentially modern light microscope. The twentieth century saw the development of microscopes that leveraged nonvisible light, such as fluorescence microscopy, which uses an ultraviolet light source, and electron microscopy, which uses short-wavelength electron beams. These advances led to major improvements in magnification, resolution, and contrast. By comparison, the relatively rudimentary microscopes of van Leeuwenhoek and his contemporaries were far less powerful than even the most basic microscopes in use today. In this section, we will survey the broad range of modern microscopic technology and common applications for each type of microscope.


Scanning Probe Microscope (SPM)

The scanning probe microscope gives researchers imaging tools for the future as these specialized microscopes provide high image magnification for observation of three-dimensional-shaped specimens.

This renders not only enhanced images but specimen properties, response and reaction or non-action when specimens are stimulated or touched.

History

Working in an IBM research laboratory in Zurich, Switzerland Dr. Gerd K. Binning and Dr. Heinrich Rohrer conducted the first successful scanning tunneling microscopic observation at the atomic level.

Rohrer and Binning were awarded the Nobel Prize in Physics in 1986 for their work in bringing scanning probe microscope technology from the drawing board to the laboratory.

Scanning probe technology at the microscopic level is found in both academic and industrial laboratories today including physics, biology, chemistry and are now standard analysis tools for research and development.

SPM Technology

The ability to observe a specimen in three dimensions, in real time plus manipulating specimens through the application of an electrical current with a physical interaction using the tip of the probe has incredible potential for research.

Viewing a specimen in a variety of environments is why scanning probe microscopes, SPMs, are so widely used.

Specimens can now be viewed at the nanometer level and instead of light waves or electrons, SPMs use a delicate probe to scan a specimen’s surface eliminating many of the restrictions that light waves or electron imaging has.

How Does a Scanning Probe Microscope work?

Tracing the surface of a specimen is done through the use of a sharp, electrically charged probe, much in the way an old record player created sound through a needle following the grooves on an LP.

Unlike a record player needle, the SPM probe does not touch the surface but traces the specimen nanometers above the surface.

Plus, the probe can be used to interact with a specimen allowing researchers to observe how a substance attracts or detracts, responds to electrical currents. Since SPM technology can operate in a wide variety of environments even non-conductive specimens can be manipulated and observed.

Development of scanning probe microscopes has allowed specialized microscopes to be created including:

Scanning Tunneling Microscopes

The scanning tunneling microscopes use a piezo-electrically charged wire, a very small space between the charged wire and the surface and the specimen to produce enhanced images of the specimen.

The charged wire forces energy across the small space and onto the specimen where the current meets with the specimens surface and decays.

This decay is measured and a high resolution image is produced from the information collected.

Tunneling microscopy allows imaging at the atomic level to be produced plus different types of information can be obtained by altering the environment that the specimen is observed in such as a gaseous environment, vacuum, or a liquid environment.

Atomic Force Microscopy

Atomic force microscopy uses a cantilever with a sharp probe that scans the surface of the specimen allowing for a resolution that you can measure in fractions of a nanometer in other words "feeling" the surface of an object in order to produce a visual image.

The flexibility of these types of microscopes are allowing for additional specialized instruments including the near field scanning optical microscope that utilizes optical fibers to stimulate specimens.

Advantages of SPM Technology

Scanning Probe Microscopy provides researchers with a larger variety of specimen observation environments using the same microscope and specimen reducing the time required to prepare and study specimens.

Specialized probes, improvements and modifications to scanning probe instruments continues to provide faster, more efficient and revealing specimen images with minor effort and modification.

Disadvantages of SPM Technology

Unfortunately, one of the downsides of scanning probe microscopes is that images are produced in black and white or grayscale which can in some circumstances exaggerate a specimens actual shape or size.

Computers are used to compensate for these exaggerations and produce real time color images that provide researchers with real time information including interactions within cellular structures, harmonic responses and magnetic energy.

SPM Technological Evolution

As researchers continue to improve and expand the abilities of scanning probe microscopes the technological evolution will include better observation equipment, improved data analysis, and processing equipment.

Additionally, micro-manipulation of molecules, DNA, biological and organic specimens using these precision instruments will produce a greater understanding of and new methods for:

Scanning probe microscopes have improved microscopy research in many ways like the invention of the microscope improved the world.

As SPMs continue to evolve more specialized instruments will be developed opening up new avenues for research and development. Thus, the field of Nanotechnology will become all the more fascinating.

For further information, please follow the links below.

Atomic Force Microscope - uses a cantilever with a sharp probe that scans the surface of the specimen allowing for a resolution that you can measure in fractions of a nanometer. That is serious resolution!

Scanning Tunneling Microscope - is commonly used in fundamental and industrial research offering a three dimensional profile of a surface looking at microscopic characteristics to your astonishment.

Nanonics Optometronic 4000 - Companies such as Nanonics have lead the way in SPM technologies, and continue to provide researchers systems with previously unimaginable potential. Check out this systems meld of the most powerful and versatile devices available.


Abstract submissions

The organizers welcome the submission of technical abstracts for oral or poster presentations at the 2021 i(SPM) 3 . Please use the attached template to prepare your 1 page abstracts:

Abstract submission for ISPM3 2021 is open!

Abstracts due on April 9th, 2021

Submissions covering all forms of SPM techniques, instrumentation and application are welcome. Specific target areas include, but are not limited to:


Scanned Probe Microscopies in Chemistry

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3.3G: Scanned-Probe Microscopy - Biology

Nanonics produces unique cantilevered Pt thermal resistive probes. This probe has all the capabilities of the AFM probe with abilities to image variation of the temperature and thermal conductivity on the sample surface. Nanomeric tip size and fast response time enable to get high resolution AFM/thermal mapping.

Click here for more information on probes and how to order

There are two main modes of thermal resistive probe operation:
  • Temperature Contrast Mode: In this mode, the resistive element of Nanonics thermal resistive probe is used as a Pt resistance thermometer. The temperature of the thermal probe changes as the tip scans the surface according to the surface temperature. Change of the Pt wire temperature leads to change of its resistance. The current passed through the probe in temperature contrast mode is set to be small enough that no self-heating of the probe occurs.
  • Thermal Conductivity Contrast Mode: In Thermal Conductivity Contrast Mode the resistive element of the Nanonics Thermal Resistive Probe is used as a resistive heater. The constant current passed through the Pt wire is significant and thus the probe temperature is much higher than a sample temperature. When the probe contacts with the sample, heat flows from the probe to the sample which results in the cooling of the probe.

Probe Design

A double-wire cantilevered glass probe has been produced for scanned probe microthermal, resistivity, and topographic measurements. The structure has many potentially unique properties for scanned probe microscopy and other nanotechnological measurements. Double Pt wire probe was fused at the tip and applied to thermal resistive measurements. The probe operation is based on the linear dependence of Pt resistance on temperature.

Key Features

HIGH SENSITIVITY

Thermal resistive probe has a high sensitivity and microseconds response time due to the nano metric size of the Pt thermal junction.

EXPOSED THERMAL TIP FOR OPTICAL INTEGRATION

Provides complete optical access from above and below for full integration with optical microscopes, Raman spectrometers and thus enables combining thermal characterization with optical and spectral mapping.

MULTIPROBE CAPABILITES

Cantilevered shape and exposed tip enable to bring the tips of the thermal resistive probe and probe for other SPM methods (AFM, NSOM, Conductive, and Nanoheater) within nanometric distance one to another. The multiprobe capabilities allow to study heat transport properties as function of optical, electrical and other action on the nanoscale level.


Nanonics congratulates 2014 Chemistry Nobel laureates!

The entire Nanonics team wishes a heartfelt congratulations to Dr. Eric Betzig and his co-recipients Professor Stefan Hell and Professor William Moerner, upon receiving the 2014 Nobel Prize in Chemistry for the development of super-resolution far-field fluorescence microscopy. Dr. Betzig was introduced to the quest of breaking the Abbe diffraction limit of resolution in optical imaging in his graduate studies in the 1980s at Cornell University under Professor Aaron Lewis, the founder of Nanonics who had started the field of near-field optics. Near-field optics at Cornell, definitively broke this limit of optical resolution through the doctoral work of Alec Harootunian and Eric Betzig in what was at the time a new area of imaging science.

When Eric Betzig joined the laboratory, Prof. Lewis had shown that light was transmitted with great efficiency through apertures as small as 30nm, beyond what was classically predicted by electromagnetic field calculations [A. Lewis, M. Isaacson, A. Harootunian and A. Murray, "Development of a 500ֵ Angstrom Spatial Resolution Light Microscope: Light is Efficiently Transmitted through lambda/16 Diameter Apertures," Ultramicroscopy 13, 227 (1984)]. These apertures, which even passed fluorescent light, had been produced and characterized in a verifiable way by the emerging techniques of electron beam lithography being developed at the time at Cornell by Professor Michael Isaacson.

During this period Alec Harootunian in the Lewis laboratory also developed the thermal pulling of glass for near-field optical probes that today is widely used and together with Eric Betzig demonstrated the breaking of the diffraction limit even for fluorescence imaging [A. Harootunian, E. Betzig, M. S. Isaacson and A. Lewis, "Superresolution Fluorescence Near-Field Scanning Optical Microscopy (NSOM)," Appl. Phys. Lett. 49, 674 (1986)] achieving a resolution on the order of 100nm.

Eric Betzig (left), Alec Hartoonian (center) and Prof. Aaron Lewis (right) in the Lewis lab at Cornell University, 1983.

Along with the above Eric Betzig perfomed the first theoretical simulation of what would be expected in the near-field of a near-field optical microscope [E. Betzig, A Harootunian, A. Lewis, and M. Isaacson, "Near-Field Diffraction from a Slit: Implications for Superresolution Microscopy," Applied Optics 25, 1890 (1986)] and was the first author in the paper in which the term Near-field Scanning Optical Microscopy (NSOM) was coined [E. Betzig, A. Lewis, A. Harootunian, M. Isaacson and E. Kratschmer,"Near-Field Scanning Optical Microscopy (NSOM): Development and Biophysical Applications," Biophys. J. 49, 269 (1986)]. He was also a part of the commercial results of this research and is listed as a co-author in one of the first near-field patents [Aaron Lewis, Michael Isaacson, Eric Betzig, and Alec Harootunian &ldquoNear-field scanning optical microscopy&rdquo (US 4917462A, 1990)].

Eric Betzig was an important member of the team in the Lewis laboratory&rsquos seminal role in near-field optics which has broken the diffraction limit in X, Y and Z in all modes of light interaction with matter be it fluorescence, absorption, collection or illumination. Furthermore, near-field optics remains the only optical technique today that not only breaks the Abbe diffraction limit in 3 dimensions but also can provide in parallel the phase of the optical wave while providing full correlation of the optical properties of a material with its 3D structure. It also critically is the only technique capable of imaging near-field evanescent waves which are lost in any far-field image.

Nanonics&rsquo continues in this quest of fully addressing the super-resolution and phase aspects of imaging in all regimes of the electromagnetic spectrum. This is certainly the case in the near-field but, as has been recently shown, by effectively designing atomic force/near-field optical systems for transparent optical integration Nanonics systems allow for pushing the envelope of far-field imaging science. An example of this is the confluence of far-field phase interference with the light being emitted by a near-field source placed with AFM precision at one position on a sample. This advance has now allowed for solving, in a parallel fashion, the 3D far-field phase of an object using a CCD camera with parallel fast data acquisition [Danielle R. Honigstein, Jacques Weinroth, Michael Werman, and Aaron Lewis, &ldquoNoniterative Exact Solution to the Phase Problem in Optical Imaging Implemented with Scanning Probe Microscopy,&rdquo ACSNano doi.org/10.1021/nn203427z (2011)]. The technique also has super-resolution potential in the far-field.

We at Nanonics are very proud of our continuing work originally initiated by Lewis first at Cornell, then at the Hebrew University of Jerusalem and of course also at Nanonics where state of the art Scanned Probe Microscopes have been developed for transparent NSOM including pioneering multiprobe AFM/NSOM technology. Through this commitment to super-resolution optical and structural imaging hundreds of laboratories throughout the world are able to obtain near-field and far-field structurally correlated information in areas such as silicon photonics, photonic band gap materials, plasmonics, organic and inorganic material science, and more recently in biological materials [V. Dalal, M. Bhattacharya, D. Narang, P.K. Sharma & S. Mukhopadhyay "Nanoscale Fluorescence Imaging of Single Amyloid Fibrils". J. Phys. Chem. Lett. 3, 1783 (2012)]

Our dedicated and experienced staff continues to push the envelope to bring to market the most accurate, sensitive, and highest resolution instruments. The importance of this effort is underscored by this year&rsquos awarding of the Nobel Prize in Chemistry to Dr. Betzig, a physicist who made enormous contributions in this area and for whom the seeds for such investigations were sown by his graduate experience at Cornell under Prof. Lewis, the founder of Nanonics Imaging.


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3.3G: Scanned-Probe Microscopy - Biology

National Biomedical Center
for Advanced Electron Spin Resonance Technology

Our research is supported by a grant from the National Institute of General Medical Sciences (NIGMS), part of the National Institutes of Health.


Scanned-probe detection of electron spin resonance from a nitroxide spin probe

We have developed an approach that extends the applicability of ultrasensitive force gradient detection of magnetic resonance to samples with spin-lattice relaxation times (T1) as short as a single cantilever period. To demonstrate the generality of the approach, which relies on detecting either cantilever frequency or phase, we used it to detect electron spin resonance from a T1 = 1 ms nitroxide spin probe (cf. Figure) in a thin film at 4.2 K and 0.6 T. Using a custom fabricated cantilever with a 4 &mum diameter nickel tip, we achieve a magnetic resonance sensitivity of 410 Bohr magnetons in a 1 Hz bandwidth. Our theory for this quantitatively predicts both the lineshape and the magnitude of the observed cantilever frequency shift as a function of field and cantilever-sample separation. Good agreement was found between nitroxide T1's measured mechanically and inductively, indicating that the cantilever magnet is not an appreciable source of spin-lattice relaxation here. We suggest that the new approach has a number of advantages that make it well suited to push magnetic resonance detection and imaging of nitroxide spin labels in an individual macromolecule to single-spin sensitivity. In the figure below, we show the schematic of the scanned-probe electron spin resonance experiment. A microstripline half-wave resonator delivers a transverse magnetic field, B1, oscillating at 17.7 GHz. In the center of the resonator, the microwave field oscillates along the x direction. A longitudinal Zeeman field of magnitude B0

0:6 T is applied along the z axis. The high-compliance cantilever has its long axis along y and oscillates in the x direction. The cantilever's 4 &mum-diameter nickel tip was affixed by hand. The sample is a 230 nm-thick film of 40 mM TEMPAMINE in perdeuterated polystyrene, coated with 20 nm of gold. The sample film was spin-coated onto a 250 &mum-thick quartz wafer. For clarity, sample and substrate are not drawn to scale.


An all-digital cantilever controller for MRFM and scanned probe microscopy using a combined DSP/FPGA design

SC Solutions, Sunnyvale, CA, Cornell University, Ithaca, NY and the U.S. Army Research Laboratory, Adelphi, MD, worked jointly to develop an all-digital cantilever controller for magnetic resonance force microscopy (MRFM) using a combined digital signal processor (DSP)/ field-programmable gate array (FPGA) design. One of the significant advantages of the all-digital cantilever controller was its absence of thermal drift and as effective tuning flexibility. The controller comprised a FPGA connected via a low-latency interface to an analog input and an analog output and a DSP with additional analog outputs. The performance of the controller was demonstrated in investigations employing ultrasensitive silicon microcantilevers fabricated at Cornell University's Nanoscale Science and Technology Facility. The results revealed that the digital MRFM controller performed without the need to know the exact cantilever resonance frequency.

Original languageEnglish (US)
Pages12-16
Number of pages5
Volume40
No8
Specialist publication American Laboratory
State Published - Apr 1 2008
Externally publishedYes



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