MICROSCOPY

The Instruments

a.) Light Microscopy

a. Bright-field microscopes
i. Shows colored or darkened organisms or specimens (achieved
through staining) against a light background
ii. Has several objective lenses (parfocal microscopes remain in
focus when objectives are changed)
iii. Light reflected off the specimen does not enter the
objective lens
iv. The simplest and most common
v. Uses white light to illuminate specimen
vi. Disadvantages:
1.

Limited resolving power

2. Not ideal for observing colourless organisms (unless

staining will be performed)
vii. Two types
1. Simple
a. Contain a single magnifying lens
b. Similar to magnifying glass
c. Leeuwenhoek used simple microscope to
observe microorganisms
2. Compound
a. Precision instrument composed of a series of
lenses for magnification
b. Uses light (natural & artificial) as source of
illumination
c. Light passes through specimen into objective
lens
d. Oil immersion lens increases resolution
e. Have one or two ocular lenses

f. Total magnification = magnification of objective

lens X magnification of ocular lens
g. Most have condenser lens (direct light through
specimen)
b. Dark-field microscopes
i. Limits the entry of light creating a dark background against
the illuminated specimen
ii. Improves the contrast of unstained, transparent specimens
iii. Excludes the unscattered beam from the image
iv. Best for observing pale objects
v. Only light rays scattered by specimen enter objective lens
vi. Increases contrast and enables observation of more details
vii. Disadvantage: Sample must be very strongly illuminated,
which can cause damage to the sample
c. Phase microscopes
i. Used to examine living organisms or specimens that would be
damaged/altered by attaching them to slides or staining
ii. Light rays in phase produce brighter image, while light rays
out of phase produce darker image
iii. Contrast is created because light waves are out of phase
iv. Two types
1. Phase-contrast microscope
a. Employs special condensers that allow light to
pass through the specimen at different speeds
allowing for an effective contrast between the
different structures in the organism
b. Accentuates diffraction of the light that passes
through a specimen
c. Difference in densities and composition within
the imaged objects however often give rise to
changes in the phase of light passing through
them, hence they are sometimes called "phase
objects".
d. Makes cell structures visible

e. Allows their study with the specimen still alive,

thus an excellent way to observe living cells
f. Enhances the contrast between intracellular
structures having slight differences in refractive
index
2. Differential interference contrast microscope
a. Accentuates diffraction of the light that passes
through a specimen; uses two beams of light
b. Creates image by detecting differences in
refractive indices and thickness of different
parts of specimen
c. Excellent way to observe living cells
d. Fluorescent microscopes
i. Direct UV light source at specimen
ii. Makes use of fluorescent dyes
- May have affinity to different molecules
Cellular
Immunological
Molecular
iii. Makes use of high energy light (eg. UV)
- Fluorochromes will emit lower frequency lights
iv. Specimen radiates energy back as a longer, visible wavelength
v. UV light increases resolution and contrast
vi. Some cells are naturally fluorescent; others must be stained
vii. Specimens usually stained with fluorochromes
viii. Shows a bright image of the object resulting from the
fluorescent light emitted by the specimen
ix. Used in immunofluorescence to identify pathogens and to
make visible a variety of proteins
e. Confocal microscopes
i. Use fluorescent dyes
ii. Use UV lasers to illuminate fluorescent chemicals in a single
plane

iii. Resolution increased because emitted light passes through

pinhole aperture
iv. Computer constructs 3-D image from digitized images
b.) Electron Microscopy
a. Light microscopes cannot resolve structures closer than 200 nm
b. Beams of electrons are used to produce images instead of light
c. Wavelength of electron beam is much shorter than light, resulting
in much higher resolution
d. Electron microscopes have greater resolving power and
magnification making very small structures and details visible
e. Magnifies objects 10,000X to 100,000X
f. Detailed views of bacteria, viruses, internal cellular structures,
molecules, and large atoms
g. Needs preparation of samples
h. Two types
i. Transmission electron microscopes
1. Internal structure Need slicing
2. Uses electrons instead of light
3. The shorter wavelength of electrons gives greater
resolution
4. Ultrathin sections of specimens.
5. Beam passes through specimen, then an
electromagnetic lens, to a screen or film.
6. Specimens may be stained with heavy metal salts.
7. 10,000-100,000x; resolution 2.5 nm
ii. Scanning electron microscopes
1. Surface of the specimen
2. An electron gun produces a beam of electrons that
scans the surface of a whole specimen
3. Secondary electrons emitted from the specimen
produce the image
4. 1000-10,000x; resolution 20 nm

c.) Probe Microscopy

a. Magnifies more than 100,000,000 times
b. Two types
i. Scanning tunneling microscopes
1. Scanning tunneling microscopy uses a metal probe to
scan a specimen
2. Resolution 1/100 of an atom
ii. Atomic force microscopes
1. Atomic force microscopy uses a metal and diamond
probe inserted into the specimen
2. Produces 3-D images
3. Metal coat not needed