1.
Superposition Method
The first reconstruction procedure involves superposing all the images of the
same object. When identical images are placed over one another, the true
parts of the images, known as the signal, become reinforced. At the same
time, the background noise tends to cancel out. As a result, the final image
becomes clearer.
The selection of identical parts of the images for superposition can be
accomplished visually. However, a more rigorous method involves calculating
a superposition function in two dimensions. This function is known as the
cross-correlation function. The cross-correlation function attains a maximum
value when one image is rotated and translated so that it optimally overlaps
with another image.
This optimisation can be performed using a computer if the images are
digitised and supplied to it for analysis.
Diffraction-Based Reconstruction
A more accurate method of obtaining a clear image from several indistinct
images is possible when the images are arranged in a regular repeating two-
dimensional pattern. In such cases, the image can be treated as an imperfect
diffraction grating.
When this image is placed in the path of a light source, a diffraction pattern
is formed. The diffraction pattern is then filtered so that only those parts
corresponding to the periodic lattice are allowed to pass through. The filtered
diffraction pattern is subsequently used as a grating and placed again in the
path of a light beam. The image formed in this way corresponds to a much
clearer version of the original object.
Mathematical Basis of Image Reconstruction
Mathematically, the procedure of image reconstruction is equivalent to
performing a Fourier transform of the image. The Fourier coefficients
obtained are then cleaned by filtering. After filtering, a reverse Fourier
transform is performed. This process results in a clearer and more accurate
image.
Applications of Image Reconstruction
The method of image reconstruction has been widely used to study
crystalline materials. It has also been applied to biological structures such as
virus structures including the stem of the T7 phage, membranes, and two-
dimensional arrays of ribosomes.
The method can be extended to three dimensions by taking several two-
dimensional images obtained by tilting the specimen at different angles.
Even a single micrograph of a suspension of particles contains images of the
particle in various three-dimensional orientations. Image reconstruction
techniques can be used to combine these images to form a three-
dimensional picture of the particle.
[Link] Diffraction
Electron diffraction is a technique in which the electron microscope is slightly
modified to obtain diffraction patterns from two-dimensional crystals. These
diffraction patterns are analysed using Fourier theory to convert them into a
three-dimensional image of the particle.
The wavelength of electrons is very small (about 0.03 Å), which is much
smaller than X-rays. Because of this, very high resolution is expected.
However, in practice, some problems reduce the resolution.
Limitations of Electron Diffraction
Electrons are easily absorbed by matter, so only very thin samples can be
used.
It is mainly useful when three-dimensional crystals do not form easily, but
two-dimensional arrays are formed, such as in membrane proteins.
In thin two-dimensional crystals, the diffraction pattern lies mostly in one
plane. Therefore, complete three-dimensional information is not available.
There is loss of phase information, which makes Fourier analysis difficult. This
problem is similar to X-ray diffraction.
Solution to Phase Problem
The phase problem can be partially solved by using an electron micrograph
to estimate approximate phase information. These estimated phases are
combined with the measured diffraction intensities to perform Fourier
transform and obtain the image.
Even with these difficulties, electron diffraction is widely used to obtain low-
resolution images of large particles that do not form suitable crystals for X-
ray [Link] Method
Sample preparation is similar to that used for electron microscopy.
The electron beam can focus on very small areas, so crystal ordering only
needs to extend a few microns.
Diffraction spot intensities are measured using densitometry.
Measured intensities and estimated phases are combined to perform Fourier
transform.
This produces electron density images of the particles.
Three-Dimensional Images
Diffraction patterns can also be obtained from tilted samples. By analysing
patterns taken at different angles, three-dimensional images can be
calculated. This improves the quality of the final result.
[Link] Tunnelling Electron Microscope
The tunnelling electron microscope has become very important in
laboratories, especially for studying surfaces. Its use in biological imaging is
less common, but in recent years it has been used to image DNA molecules,
proteins, and large macromolecular assemblies. It has helped in accurately
measuring the pitch of the DNA double helix.
Basic Principle (Electron Tunnelling)
The working of this microscope is based on the quantum mechanical
principle of electron tunnelling.
According to quantum mechanics, there is a small but finite probability that
an electron can move from one region of low potential energy to another,
even if it does not have enough energy to cross the barrier normally.
This can be compared to a football moving from one well to another nearby
well without coming to the surface or breaking the wall between them.
Working of the Microscope
A very sharp needle (tip) is brought extremely close to the surface to be
studied.
The distance between the tip and surface is only a few angstroms.
At this very small distance, electrons tunnel from the surface to the needle.
This creates a small measurable tunnelling current.
The tunnelling current depends on the distance between the tip and the
surface. Therefore, it acts as a very precise indicator of surface position.
The needle is moved across the surface in a raster pattern. At each point, the
height of the needle is adjusted so that the tunnelling current remains
constant.
The vertical movement of the needle at each position gives information
about the surface shape. This information is then converted into an image
showing the surface topography.
Special Features
The tip of the needle can be made extremely sharp, sometimes ending in a
single atom.
Modern instruments can measure very small tunnelling currents (even nano-
amperes).
Because of these technological improvements, it is possible to visualize
individual atoms on material surfaces.
Use in Biological Samples
The use of this microscope in biology is still limited.
Main reasons:
Biological materials are usually non-conducting or semi-conducting.
They must be stained with heavy metals to make them conductive.
Staining can reduce resolution.
The preparation and positioning of the needle tip must be extremely precise
for biological samples.
[Link] Force Microscope (AFM)
The Atomic Force Microscope is a modified form of the tunnelling microscope.
In this microscope, instead of keeping the needle tip slightly away from the
surface, the tip is brought into direct contact with the surface.
The force acting on the tip is kept constant while the tip is scanned across
the surface. The movement of the tip over the surface is similar to a
phonograph needle moving in the groove of a record.
Working Principle
The needle tip is pushed against the surface.
The force on the tip is maintained constant.
The tip scans across the surface.
The vertical movement of the tip is recorded.
To measure the movement of the tip:
A laser beam is reflected from a mirror fixed to the top of the needle.
An interferometer is used to accurately measure the distance travelled by
the laser beam.
This measurement gives the exact position of the tip.
The recorded vertical motion is converted into an image of the surface.
Advantages of AFM
It does not depend on electric current.
It can be used to study both conducting and non-conducting materials.
This makes it more versatile than the tunnelling microscope.
Limitations in Biological Samples
Although AFM can be used on many materials, studying biological samples
such as cells and proteins is still difficult. The main problem is that the tip
may distort or damage soft biological materials during scanning.
Other Tip-Scanning Microscopes
Several other microscopes have been developed based on the same principle
of a scanning tip. In each type, a different kind of force is measured.
Examples include:
Friction Force Microscope – measures frictional forces.
Magnetic Force Microscope – measures magnetic forces.
Electrostatic Force Microscope – measures electrostatic forces.
Scanning Thermal Microscope – measures temperature.
The scanning thermal microscope can be considered the world’s smallest
thermometer. A thermocouple attached to the needle tip measures
temperature at each point on the surface.
Since heat is related to motion at infrared wavelengths, it may become
possible in the future to measure the infrared spectrum of a single molecule.