Studying Cells
Recall the things you saw on the way coming back home? There were
trees, grass, people, and perhaps squirrels and birds. Although all of
these things are composed of cells, you hardly recall seeing a
cell. That's because cells are very tiny entities. An average animal
cell is about 10 to 20 µm in diameter (about five times smaller
than the smallest visible particle).
The small size has made the study of cells difficult until the
invention of light microscope. However, animal cells are not only tiny
but also translucent and colorless. Techniques for understanding the
finer structure of cells weren't developed until the latter part of
the19th century, when stains that can provide sufficient contrast
became available. However, the magnification provided by light
microscopes still couldn't resolve the finest details of the cell. It
wasn't until the 1940s, when the electron microscope was developed,
that scientists were able to visualize the inside of a cell with
sufficient detail to understand the internal complexity of the
cell. To elucidate even smaller structures inside of the cell such as
proteins, X-ray crystallography is used.
Light Microscope
The light microscope can provide enough magnification to visualize
the cell. However, because it uses light to resolve objects, the light
microscope cannot be used to probe structural details below 500nm. For
the most part, scientists using light microscope can see the major
organelles of cells such as the mitochondria and nucleus. To help
visualize the internal structure of the cell, different organelles
could also be selectively stained with different organic dyes such as
Malachite green, Sudan black, and Commassie blue.
Different variations of the light microscope have also been
created. Currently, the fluorescent light microscope has been widely
used to visualize cells stained with different fluorescent
dyes. Proteins could also be tagged with fluorescent proteins and
visualized under a fluorescent microscope.
Electron Microscope
To increase the resolution of images, the electron microscope is
developed to use electrons instead of light as the resolving
agent. Electrons have a much shorter wavelength than light and can be
used to resolve objects on the scale of 0.1nm. However, in order to
use the transmission electron microscope, specimens must undergo
special preparation. Because electron doesn't have very high
penetrating power, all samples must be sliced into 50 to 100nm thin
sections.
Besides having high resolution than the light microscope, scanning electron microscopes
(SEM) can be used to construct three-dimensional images. Here the electron beam is reflected from the surface of the specimen and an object is scanned as a series of layers. The data from the layers are then overlaid on top of each other to create a 3D image. The resolution of most SEM is about 10nm, so they are mostly used for intact cells and small organisms.
Schematic diagram demonstrating the principal features of a light
microscope, a transmission electron microscope, and a scanning electron
microscope.
X-Ray Crystallography
X-ray Crystallography is developed to reveal the three-dimensional arrangement of atoms in a molecule. Using x-ray beams with 0.1nm wavelength, a diffraction pattern can be generated by shooting X-ray beams through crystals. The diffraction data can then be used to construct a 3D model of the object. This technique is widely used today to elucidate protein structures.
Reconstructing the image from the X-ray diffraction data is a tedious and difficult task. Oftentimes, resolution of the structure of one protein requires months of automated data collection and numerous hours of data processing. Sometimes, the slowest step in X-ray crystallography is generation of crystals suitable for producing diffraction patterns. |
Human cells expressing a fluorescent protein
A Fluorescent Microscope
Rosalind Franklin's X-ray Diffraction Photograph of DNA,
1953
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