Editorial Feature

Nanoscale Imaging Techniques: An Overview

There are a wide range of nanoscale imaging tools that include microscopic methods such as scanning probe microscopy (SPM), electron microscopy (EM), high resolution optical microscopy as well as different types of spectroscopy; for instance, based on auger electrons (AES) or photoelectrons (XPS/UPS); or even secondary-ion mass spectrometry (SIMS).

The advances in nanoscale materials such as graphene, carbon nanotubes, and nanoformulated versions of all kinds of materials have led to advances in the tools and techniques required to examine and manipulate them - these advanced tools in turn allow greater understanding and control of nanomaterials, making nanoscale imaging instruments a crucial part of the world of nanotechnology.

Limits of Optical Microscopy

Optical microscopes are not well suited for observing materials with dimensions below 100 nm, due to the limitation imposed by the wavelength of light - it is definitely not possible to obtain an accurate image with a probe that is almost three times the size of the object!

The images or nanostructures obtained using optical techniques are usually blurred and out-of-focus, leading to the need for higher-resolution instruments.

Electron Microscopy and Scanning Electron Microscopy

The electron microscope is a microscope that uses an electron beam instead of light to form the image of a specimen. Compared to a light microscope, much higher magnifications and higher resolutions are possible. These microscopes are costly, large, and are typically kept in a room specifically designed for the equipment, and need trained personnel to operate them.

SEM image of E. coli cells.

SEM image of E. coli cells. Image Credits: Biodefense Image Library

In order to control the electron path, electron microscopes use electrostatic or electromagnetic lenses. The electromagnetic lens includes a solenoid through which a current is passed, thus introducing an electromagnetic field. The electron beam moves through the solenoid down the electron microscope column towards the sample. Electrons are highly sensitive to magnetic fields and can be controlled by a change in current through the lenses.

The two types of electron microscopes are:

  • Transmission electron microscope (TEM)
  • Scanning electron microscope (SEM)

In a TEM, a cathode emits a high voltage electron beam, which is focused by magnetic lenses. The electron beam is transmitted partially through the very thin specimen and contains data about the specimen structure. A series of magnetic lenses magnifies the spatial variation in the data till it is recorded by striking a fluorescent screen, or electronic detector. The detected image can be seen in real-time on a computer or monitor. Two-dimensional, black and white images are produced using transmission electron microscopes.

In a TEM, the electrons in the primary beam are made to move through the sample, but in an SEM, images are produced by detecting secondary electrons that are emitted from the surface because of excitation by the primary electron beam. The electron beam is scanned in a raster pattern across the sample surface and the detected signals based on beam position are mapped by detectors, forming an image. The TEM has a better resolution than the SEM; however the SEM can perform bulk imaging and has a greater depth of view.

The disadvantages of electron microscopes are:

  • These are quite expensive both in terms of purchase and maintenance
  • They require very stable high-voltage supplies
  • They require highly stable currents to each electromagnetic lens, cooling water supply circulation and constantly pumped ultra-high vacuum systems through the pumps and lenses.
  • Since the microscopes are highly sensitive to external magnetic fields and vibrations, these need to be housed in specially designed facilities.
  • Only trained personnel can operate the microscope.
  • Preparation of the sample is tedious as they have to be viewed in a vacuum.

SEM image of the mineral hexahydrite.

SEM image of the mineral hexahydrite. Image Credits: Research.gov

Scanning Tunneling Microscopy and Atomic Force Microscopy

Scanning Tunneling Microscope

The scanning tunneling microscope (STM) can be used to obtain 3D images of a sample. In an STM, a stylus analyzes the surface structure of the sample by scanning the surface from a specified distance.

A very fine conducting probe is held close to the sample. Electrons jump from the stylus to the sample surface due to the effect of quantum tunnelling, producing an electrical signal. The stylus tip, sharpened down to a single atom, scans across the surface at a distance of around 1 Angstrom (the diameter of an atom). The shape of the surface is determined in one of two ways - either the stylus is raised and lowered so that the signal stays constant and the displacement of the stylus is measured, or the stylus is fixed, and the variations is current are measured.

Using this technique, even minute details can be observed. A surface profile is created and a computer- generated contour map is produced.

The STM is highly suitable for conducting materials, and organic molecules can also be fixed on a surface, and their structure studied. This technique is used to study DNA molecules.

Atomic Force Microscope

The atomic force microscope was designed to enhance the capabilities of the STM. STMs are capable of imaging only conducting or semiconducting surfaces. However, the AFM can image almost any surface type including ceramics, polymers, biological samples and glass.

The AFM was invented in 1985 by Quate, Binnig and Gerber. The original AFM comprised a diamond shard attached to a gold foil strip. The sample surface is in direct contact with the diamond tip, and the interaction mechanism is provided by the interatomic van der Waals forces. The cantilever’s vertical movement is detected by a second tip that is an STM above the cantilever.

In most modern AFMs, a laser deflection system is used wherein a laser is reflected from the back of the AFM cantilever and onto a position-sensitive detector. Silicon or silicon nitride (Si3N4) are used in the micro-fabrication of AFM tips and cantilevers. The beam position is determined using a beam deflection system comprising a photodetector and a laser.

AFMs operate in the following modes:

  • Contact mode
  • Lateral force microscopy
  • Non-contact mode
  • Dynamic force / Intermittant-contact / “tapping mode” AFM
  • Force modulation
  • Phase imaging

AFM image of collagen fibres.

AFM image of collagen fibres. Image Credits: NIST

Nanoscale Spectroscopy

While AFM and other probe-based techniques offer mechanical, topographic, electromagnetic, thermal and near-field optical properties with molecular resolution, confocal Raman spectroscopy and imaging helps obtain specific chemical information about nano-materials, with sub-micron spatial resolution. Nanomaterials often have a strong and specific chemical signature, which can be easily identified using ultra-fast Raman mapping, after which mechanical, thermal, electrical or topographic analysis is done at the location of interest.

Using nanoscale spectroscopy it is possible to:

  • Confirm specific characteristics of a material
  • Obtain chemical information on nano-structures of interest

Hybrid Techniques

Tip-enhanced Raman scattering (TERS) uses a phenomenon where pressure on the sample surface from an AFM tip can increase the intensity of Raman-scattered signals. This can greatly increase the sensitivity of the Raman spectroscopy on the sample, increasing the resolution of the combined Raman-AFM image. This technique can be used on a range of samples, from nanotubes to DNA.

Single molecules can be studied using a combination of atomic force microscopy and confocal fluorescence microscopy. Both these techniques have individual features that enable the study of several aspects, such as dynamics using fluorescence spectroscopy, or topography using AFM. Combining these two techniques into one offers improved methodologies and opens up new investigation schemes such as the following:

  • Investigation, prediction and control of photophysical processes on the nanometer-scale
  • High-resolution imaging
  • Investigation of inter- and intramolecular interactions
  • Nanomanipulation: assembly and fabrication of nanomaterials

The nature of atomic force microscopy makes it highly adaptable to use in conjunction with other analytical techniques. Hybrid techniques such as these have had a great deal of research attention in recent years, and new ways of using AFM are being developed all the time.

Sources and Further Reading

Will Soutter

Written by

Will Soutter

Will has a B.Sc. in Chemistry from the University of Durham, and a M.Sc. in Green Chemistry from the University of York. Naturally, Will is our resident Chemistry expert but, a love of science and the internet makes Will the all-rounder of the team. In his spare time Will likes to play the drums, cook and brew cider.


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