HOW ARE CELLS STUDIED?
MICROSCOPY & CELL
FRACTIONATION
MICROSCOPY
Magnification
Resolution
Contrast
LIGHT MICROSCOPES - LM
COMPOUND MICROSCOPE - 2 lenses: eyepiece and objective
Image is produced by passing light through the specimen
Living cells may be studied
Contrast can be improved by stains and manipulating light
Cells are visible but very few organelles are visible
In use since 1590s
LIGHT MICROSCOPES
Resolution lost when two points are closer than 0.2 micrometers
This corresponds to the smallest wavelength of light we can see
The best LM magnify specimens about 1000X; further
magnification possible but resolution decreases – image gets
blurry
ELECTRON MICROSCOPES - EM
Permit greater magnification with good resolution
Use electron beam not visible light
Resolution is inversely related to wavelength of the radiation the
microscope uses: wavelength of electron beam is much shorter
than visible light.
THINK: Can you use glass lenses to focus electron beams?
Would the image produced by EM be directly visible to you?
What does “inversely related” mean?
ELECTRON MICROSCOPES - EM
Introduced early in 1950’s; Rapid advance in cytology esp. when
used with cell fractionation
Revealed ultrastructure of cells for the first time
Living cells cannot be viewed due to preparation of specimen before
viewing
Electrons focused by magnetic field
Specimen is stained – ex. heavy metals
Image is visible by looking at screen or photograph
Image is shade of gray – color may be added to image later
TRANSMISSION ELECTRON MICROSCOPES - TEM
Specimen is sliced VERY thin
Electron beam passes through the specimen (transmitted)
To understand 3-D shape of structures you need to examine
multiple slices
SCANNING ELECTRON MICROSCOPES - SEM
Electron beam passes back and forth over the surface of
specimen
Image has great depth of field
Creates 3-dimensional image
BUT WHAT DID ALL OF THOSE STRUCTURES DO?
How can you divide the cell into individual “fractions” so you can
study each part individually?*
Are these structures real or simply “artifacts” of the process used
to prepare the specimens?
*Does the technique illustrate the concept of emergent properties, reductionism, or
systems biology?
CELL FRACTIONATION
A technique for isolating individual cell parts for further study
Study of each fraction’s biochemistry reveal function while study
of electron micrographs reveal structure
CELL FRACTIONATION
STEP 1: Break open the cell (homogenize) – there are a variety of
techniques
Example: Blender
Resulting mixture is called
homogenate
CELL FRACTIONATION
STEP 2: Separate the mixture into its different components using a
centrifuge (ultracentrifuge – most powerful)
Basically a test tube holder mounted on a
motor!
An ultracentrifuge spinning at
130,000 rpm applies of a force
>1 million times the force of gravity!
This is a desk to model for
small samples - microfuge
CELL FRACTIONATION
Centrifugation separates the particles by spinning them; larger particle settle to the
bottom, lighter ones to the top
By varying the speed
and duration, various
cell components can be
separated from each
other.
CELL FRACTIONATION
STEP 2:
CELL FRACTIONATION
What is the point?
Individual cell parts can be isolated in sufficient
quantities to carry additional study to determine their
function.
LINING OF THE TRACHEA
Light microscope
TECHNOLOGY CONTINUES TO ADVANCE
You might enjoy reading about the scanning
tunneling microscope
SEM MICROGRAPHS
SO COOL