Microscopy - MATCOnline
Download
Report
Transcript Microscopy - MATCOnline
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.