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