DNA Under The Microscope
Electron & Atomic Force Microscopy
DNA (Deoxyribonucleic acid) is the molecule that
contains within it all the instructions and information about an organism. This
is to say that DNA contains information regarding how the organism will
develop, how it lives and reproduces etc. Therefore, the DNA may be described
as the blueprint of a living organism.
Given that DNA molecules are found
inside the cells, they are too small to be seen with the naked eye. For this
reason, a microscope is needed. While it is possible to see the nucleus
(containing DNA) using a light microscope, DNA strands/threads can only be
viewed using microscopes that allow for higher resolution.
To view the DNA as well as a variety of other
protein molecules, an electron microscope is used. Whereas the typical light
microscope is only limited to a resolution of about 0.25um, the electron
microscope is capable of resolutions of about 0.2 nanometers, which makes it
possible to view smaller molecules. This is achieved because electron
microscopes use electron beams rather than the visible light used for light
DNA Electron Microscopy
- Electron microscope
- Heavy metal salts (Lead
perchlorate, uranyl acetate, lanthanum nitrate)
- DNA sample (nucleic acids)
For this procedure, the steps involved:
- Spraying DNA onto a grid
with a glass nebulizer (using freshly cleaved mica is advised)
- Add latex spheres - Latex
spheres or ferritin used here serve as reference particles
- Spread the DNA
solution/preparation and protein (1:10 to 1:100) on water surface in a Langmuir
trough - This is known as the Kleinschmidt's technique. It is used for
spreading nucleic acid in protein to form a film of protein that retains DNA on
Once the preparation is ready, it is ready for
- The nucleic acid is applied
to the grids and immersed in the staining solution for between 10 minutes and 4
- Rinse the grids using
redistilled water at a pH of 6.0 three times and view under the microscope
- In the event that the
stained grid will not be viewed immediately, then they can be stored in an
evacuated desiccators over phosphate P205
Using the heavy salts allow for higher contrast
that makes it possible to view single molecules of the DNA
STEM microscopy has been shown to operate in a
dark-field mode thus providing high contrast of biological molecules. Because
of its dark-field images, this technique has also been shown to have a great
advantage in that it allow for direct visualization of unstained strands of
DNA. Through the high contrast
provided, the technique also makes it possible for researchers to be able to identify
any problems with the sample. The procedure for this technique is a lot similar
to typical electron microscopy for DNA. However, through STEM, researchers
obtain both mass and structural information of single-stranded DNA.
Cryo-electron microscopy is one of the
techniques that have been shown to be particularly successful in revealing the
structure of DNA.
Unlike the transmission electron microscope, Cryo-EM uses
frozen samples and electron beams that are gentler to view the sample. This
allows researchers to view biological molecules without causing any damage to
them in the process.
For this technique, a small amount of the sample
in solution is first applied to an EM grid similar to the process used to view
DNA strand under electron microscope (EM). The grid with the thin layer is then
immersed in liquid ethane (at -180 degrees c) to trap the molecules in water
crystal/ice. This ensures that the sample remains is not destroyed when being
viewed under the microscope.
Here, it is worth noting that samples prepared for
this technique (Cryo-EM samples) tend to be highly sensitive to electron
damage. For this reason, low electron doses of about 10–20 e−/Å2 are used to
ensure that the sample is not damaged.
While the sample is exposed to low doses
of the electrons, the layer of ice around the sample also helps protect the
sample during the process.
Cryo-Electron Tomography (CET)
Through recent advancements in this technique,
researchers were able to develop an improved technique of Cryo-EM known as Cryo-electron
tomography (CET). Using this technique, it has become possible for researchers
to develop 3D structures of various proteins and DNA strands.
process involves the capture of many images of the sample from various angles
and using the images to build a 3D structure. Using this technique, researchers
have managed to develop and present a variety of 3D images of DNA strands
showing the structure of DNA from different angles.
Apart from the electron microscope techniques
used to study DNA, methods such as Atomic Force Microscopy (AFM) are also being
used for the same purpose. Using this technique, it has become possible for researchers to measure the length of these strands.
Requirements (for AFM)
For this technique, some of the materials
- DNA samples (such as X174
- AFM microscope
- Freshly-cleaved ruby mica
- Magnesium acetate
- AC glow-discharge
- For single stranded DNA,
the procedure involves the following steps:
- Soak freshly-cleaved ruby
mica circles in 33 mM of magnesium acetate for between 4 and 24 hours
- Sonicate in Millipore water
for about 5 minutes to remove any excess magnesium acetate
- Use compressed air for
- Expose to AC glow-discharge
for about 20 seconds in 100 militorr air
- Almost immediately invert
the circles in about 7 micro liter drops of the DNA strands (singles stranded)
in 0.5 percent formaldehyde and 15 mM ammonium acetate - This is deposited to a
- Allow to stand for about 4
minutes and rinse the mica using about 3 drops of water
- Dry using compressed air
- If it is not to be used
immediately, store the preparation in a desiccator on drierite
For Double stranded DNA, the same procedure is used but
formaldehyde and ammonium acetate is not used.
- During imaging
(AFM-imaging) the process was carried out under 100 percent propanol using
either Nanoscope II or III AFM. Here, an O-ring was used to apply the propanol
by laying it on the sample in the device. The process involved:
- Placing a drop of alcohol
on to the cantilever in the fluid cell
- Positioning the cantilever
over the O-ring and firmly clamped
- Ensuring that the amount of
fluid was sufficient (this was determined by ensuring that no bubbles remained
on the light path)
During imaging, the minimum force was used. This ensured that the
cantilever did not lift the sample.
To measure the length of DNA strands using this
technique, the images are first enlarged and a fine chain laid along the DNA
contours. However, a better way of making the measurements has been shown to
involve direct measuring of the top-view of the DNA images in the nanoscope.
This method simply involves summing up given points in the image.
The STM can also be used to view DNA molecules.
It is capable of imaging objects at atomic levels, which makes it a good tool
for viewing DNA molecules.
For this technique, several methods may be used
to prepare the sample for imaging, these include:
- Place a droplet of the DNA
solution on the substrate
- Allow it to dry in air (air
- Contain the solution in
10mM potassium chloride to stabilize the DNA
- Sonicate the solution using
- Biosonik IV probe sonicator
for about30 minutes at 160 W
- Imaging - Imaging using STM
can be directly carried out on the layer
Method #2 - This method is similar to the first
method, but involves vacuum drying the solution and containing it in 10mM
ammonium acetate. Sonication was also skipped.
- Place 10 micro liters of
distilled water on a piece of parafilm - Water causes the phage to burst releasing
Add 10 micro liters droplet
of lysed DNA particles (lysed T7 bacteriophage)
- Briefly place a substrate
on the droplet to pick up the lysed particles
Following imaging, it is possible to identify
the DNA through their heights and width. Here, DNA strands can be clearly seen
arranged parallel to one another.
One of the most recent methods slightly deviates
from the others and involves dissolving DNA in an aqueous solution and placing
it in a layer of highly oriented graphite before allowing it to air dry. Here,
tungsten-wire tips are used while scanning is carried out under atmospheric
conditions. Scanning produces high resolution images of the double helix DNA.
Wolfgang Schonert, original author at GSI.de
As mentioned, DNA (deoxyribonucleic acid)
carries within it genetic, hereditary material and resides in the cell nucleus
of every organism. Essentially, the strands of DNA are composed of repeated patterns
of six molecules which include; deoxyribose (a five carbon sugar) a phosphate
group as well as four nitrogenous bases (cytosine (C), thymine (T), adenine (A)
and guanine (G)).
The linear order of the bases represents one of the most
important features of DNA given that their paring allows for important encoding
of information required for development and life of the organism. The structure
of DNA (double-helical) is of great significance given that it serves to
protect the base atoms.
* Nucleotides are the basic units of DNA. Each
nucleotide is composed of a single molecule of sugar, a single phosphate
molecule and one nitrogenous base.
- Viruses under the Microscope
- Atom under the Microscope
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Walther Stoeckenius. Electron Microscopy of DNA
Molecules "Stained" With Heavy Metal Salts .
Hele G.Hansma Rober L.Sinsheimer Min-Qia Li and
Pau K.Hansma (1992) Atomic force microscopy of single and double-stranded DNA.