Microscopy
Outline
 Using the metric system to express the sizes of
microbes
 Microscopes
 Simple microscopes

Compound microscopes

Electron microscopes

Atomic force microscopes
Using the Metric System
• Metric units are used to express the sizes of
microbes.
• The basic unit of length in the metric system is the
meter (m); it is equivalent to 39.4 inches.
• The sizes of bacteria and protozoa are usually
expressed in terms of micrometers (µm). A
micrometer is one millionth of a meter.
Using the Metric System
Using the Metric System
• A typical spherical
bacterium (coccus) is
approximately 1 µm in
diameter.
• A typical rod-shaped
bacterium (bacillus) is
approximately 1 µm wide
by ~3 µm long.
Using the Metric System
• The sizes of viruses are expressed in terms of
nanometers (nm). A nanometer is equal to one
billionth of a meter.
• Most of the viruses that cause human diseases
range in size from 10 nm to 300 nm.
• One exception is Ebola virus, a cause of viral
hemorrhagic fever. Ebola viruses can be as long
as 1,000 nm (1 µm).
Measuring Microbes
When using a microscope, the sizes of
microorganisms are measured using an
ocular micrometer.
Fundamentals of Microscopy
• A microscope is an optical instrument that is used
to observe tiny objects; objects so small that they
cannot be seen with the unaided human eye.
• the resolving power or resolution of a
microscope is the limit as to what can be seen using
that instrument.
• The resolving power of the unaided human eye is
approximately 0.2 mm.
Simple Microscopes
• A simple microscope is one that contains
only one magnifying lens.
• A magnifying glass could be considered a
simple microscope; when using a magnifying glass,
images appear 3-20 times larger than the object’s actual
size.
• Leeuwenhoek’s simple microscopes had a
maximum magnifying power of about X300
(about 300 times).
Compound Microscopes
• A compound microscope contains more than one
magnifying lens.
• Because visible light is the source of illumination, a
compound microscope is also referred to as a compound
light microscope.
• Compound light microscopes usually magnify objects
about 1000 times.
• The resolving power of a compound light microscope is
approximately 0.2 µm (about 1,000 times better than the
resolving power of the unaided human eye).
Compound Microscopes
• It is the wavelength of visible light (~0.45 µm) that
limits the size of objects that can be seen.
• Objects cannot be seen if they are smaller than half
of the wavelength of visible light.
• Today’s laboratory microscope contains two
magnifying lens systems:
–
–
The eyepiece or ocular lens (usually X10)
The objective lens (X4, X10, X40, and X100 are the four
most commonly used objective lenses)
Compound Microscopes
• Total magnification is calculated by multiplying the
magnifying power of the ocular lens by the
magnifying power of the objective lens being used.
–
X10 ocular x X4 objective = X40 total mag.
–
X10 ocular x X10 objective = X100 total mag.
–
X10 ocular x X40 objective = X400 total mag.
–
X10 ocular x X100 objective = X1000 total mag.
Compound Microscopes
Photographs taken through the lens system of the
compound light microscope are called
photomicrographs.
Anatomy of a Compound Microscope
Compound Microscopes
• Because objects are observed against a bright
background or “bright field,” the compound light
microscope is sometimes referred to as a brightfield
microscope.
Compound Microscopes
• If the condenser is replaced with what is known as a
darkfield condenser, illuminated objects are seen
against a dark background or “dark field;” the
microscope is now called a darkfield microscope.
Darkfield Microscopy of
Treponema pallidum (the
bacterium that causes syphilis)
Compound Microscopes
• Other types of compound microscopes include:
–
Phase contrast microscopes
–
Fluorescence microscopes
Phase Contrast Microscopes
• Phase contrast microscopes are used to observe
unstained living microorganisms.
–
Organisms are more easily seen because the light refracted
by living cells is different from the light refracted by the
surrounding medium.
Fluorescent Microscopes
• Fluorescent microscope contains a built-in
ultraviolet (UV) light source.
–
When UV light strikes certain dyes and pigments these
substances emit a longer wavelength light causing them to
glow against a dark background
Fluorescent Microscopy
Electron Microscopes
• Electron microscopes enable us to see extremely small
microbes such as rabies and smallpox viruses.
• Living organisms cannot be observed using an electron
microscope – the processing procedures kill the
organisms.
• An electron beam is used as the source of illumination
and magnets are used to focus the beam.
• Electron microscopes have a much higher resolving
power than compound light microscopes.
• There are 2 types of electron microscopes - transmission
and scanning.
Scanning Electron Microscopes
 A scanning electron
microscope (SEM) produces
images of a sample by
scanning it with a focused
beam of electrons.

The electrons interact with
electrons in the sample, producing
signals that can be detected and
contain information about the
sample's surface topography and
composition.
Scanning Electron
Micrographs
Transmission Electron Microscopes
• Uses an electron gun to fire a beam of electrons
through an extremely thin specimen (<1 µm thick).
• An image of the specimen is produced on a phosphor-
coated screen.
• Magnification is approx. 1000 times greater than the
compound light microscope.
• Resolving power is approx. 0.2 nm.
S. aureus in the
process of binary fission
Transmission Electron
Micrographs
Atomic Force Microscopes
• Enable scientists to observe
living cells at extremely high
magnification and
resolution under
physiological conditions.
• Can observe single live cells
in aqueous solutions.
• Provides a true three-
dimensional surface profile.