Download Microscopy Professor Spencer

Microscopy
CHE 113 Forensic Science
James T. Spencer, Copyright 2004-2008
Microscopy and Stains
Micro means small
Skopein means to
see
Microscopes
Preparation of
specimens for light
microscopy
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Sizes
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The Instruments
Brightfield Microscope
Compound
Comparison
Stereoscopic
Polarizing
Microspectrophotometer
Darkfield Microscope
Phase-contrast Microscope
Fluorescence Microscope
Electron Microscope
Transmission
Scanning
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How Microscopes Work
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Microscopy History
•Dutch spectacle-makers (1590), Janssens,
discovered that nearby objects appeared
greatly enlarged with lenses.
•Galileo (late 1600s), based on the Janssens
experiments, worked out a
much better instrument
with a focusing device.
Janssen
Galileo
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Microscopy History
Late 1600’s
•Other Early
Microscopes
Leeuwenhoek Microscope
Hooke Microscope
1670
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Microscopy History
•Later Microscopes
Olympus
(modern)
Pacino, 1870
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Microscopy History
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Compound Light Microscope
The most common microscope compound light microscope (LM). Two
sets of lenses: ocular and objective.
Brightfield Mic.
The total magnification: multiply
magnification of the objective lens with
the ocular lens.
e.g., ocular is 10x and the objectives is
100, total mag. will be 1000x.
Optical system comprised of
condenser, objective lens, eyepiece
lens and illuminator.
The compound light microscope uses
visible light. ( = 400 - 700).
Virtual image is any specimen viewed
through a lens.
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Electromagnetic Radiation
Magnetic and Electronic Parts
mutually perpendicular
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Electromagnetic Spectrum
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Darkfield Microscopy
The darkfield microscope shows a light silhouette of an
organism against a dark background. The light reaches
the specimen from an angle with the help of an opaque
disk.
The specimen then appears "lit up" against the dark
background.
Most useful for extremely small living organisms that are
invisible in the light microscopes. An example would
Treponema pallidum, the causative agent for syphilis.
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Darkfield Microscopy
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Brightfield
Darkfield
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Comparison Microscope
Important tool for
firearms examiner
Two compound
microscopes combined
into one unit
When viewer looks
through the eyepiece, a
field divided into two
equal parts is
observed
Bullet comparisonsrequires reflective light
• Hair & Fiber
comparisons
• Questioned documents16
Comparison Microscope
Split-image
comparison of firing
pin
imprints in coaxial
incident light
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Comparison Microscope
Split-image comparison of banknotes:
on the left the original, on the right the forgery
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Phase-Contrast Microscope
A phase-contrast microscope
splits a beam of light into 2 types
of light, direct and refracted
(reflected) and brings them
together to form an image of the
specimen.
Where they are "in-phase" the
image is brighter, where they are
"out of phase" the image is
darker, and by amplifying these
differences in the light, it
enhances contrast
It allows the detailed observation
of living organisms, especially
the internal structures.
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Polarizing Microscope
Based upon the rotation of polarized
light.
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Polarizing Microscope
Light confined to a
single plane of
oscillation is said to
be polarized.
Many crystals are
birefringent (light is
refracted into 2
separate rays).
Reduces glare by
transmitting light in a
vertical plane only.
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Why do molecules rotate
polarized light?
A molecule possessing nonsupeposable mirror
images is termed chiral.
Naturally occurring substances are often found
as just one mirror image (enantiomer).
Enantiomers are identical in physical properties
and identical chemical properties when they
react with nonchiral reagents; only in a chiral
environment will the differences show.
Enantiomers rotate of polarized light in different
directions.
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Optical Activity
Non-superimposable mirror images
Mirror
Left Hand
Right Hand
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Optical Isomers
Amino Acids
are Chiral
H2N
O
H
C
alanine
C OH
CH 3
Mirror Plane
HO2C
H2N
H
H
C
C
CH3
H3C
Enantiomers
CO2H
NH2
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Optical Isomerism
Enantiomers have the same physical
properties (i.e., b.p., m.p., viscosity, etc...).
Enantiomers react identically toward other
non-chiral molecules
Enantiomers frequently react very differently
toward chiral reagents (v. impt. biologically)
Frequently, one enantiomer will be biol.
active while the other is biol. inactive.
Interaction of Chiral
Reagents
(L and R hands)
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Fluorescence Microscope
In fluorescence microscopy,
specimens are first stained with
fluorochromes and then viewed
through a compound microscope
by using an ultraviolet (or nearultraviolet) light source.
The microorganisms appear as
bright objects against a dark
background.
Fluorescence microscopy is used
primarily in a diagnostic
procedure called fluorescentantibody (FA) technique, or
immunofluorescence.
This technique is especially
useful in diagnosing syphilis &
rabies.
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Hand section
of sugarcane
vascular
bundle viewed
with
fluorescence
microscope
Sugarcane
vascular bundle
viewed with
traditional
staining and
transmitted
light (Bright
Field)
microscopy
Hand-section of
Sugarcane stem
with a vascular
bundle Stained
with Toluidine Blue
& Viewed with
Bright Field
microscopy
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An unfertilized egg stained
with a dye that appears red
in a fluorescence
microscope. The line
indicates size (10
micrometers = 1/100 of a
millimeter). The egg is about
100 micrometers in diameter,
so about ten eggs would fit
side by side on a pencil line.
An egg that was
stained with a dye that
appears yellow in a
fluorescence
microscope.
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Stereomicroscope
Also called the dissecting microscope
Working distance below objective lens &
specimen. Image is 3D.
Doesn’t reverse or invert the image, so
examiner can manipulate the specimen
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Electron Microscope
A beam of electrons, instead of
light, is used with an electron
microscope.
Electron microscopes can
magnify greater because the
wavelengths of electrons are
much smaller than those of
visible light = 0.005nm as
opposed to 500nm (one hundred
thousand times smaller)
The best compound light
microscopes can magnify 2000x,
electron microscopes can
magnify up to 100,000x
2 types: TEM & SEM
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Transmission Elec. Microsc.
Transmission electron microscopes (TEM): Thin
sections (or layers) (2-D) can be seen in a
transmission electron micrograph.
Electrons pass directly through the specimen.
Magnification: 10,000-100,000x Resolving power:
2.5 nm.
The specimens must be fixed, dehydrated and
viewed under a high vacuum. These treatments
not only kill the specimens but may cause
shrinkage and distortion.
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Transmission Elec. Microsc.
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TEM Pictures
Silver Nanoprisms
Gold Nanoparticle
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Scanning Elec. Microscopy
Scanning electron microscopes (SEM): 3-D
views of the surfaces by aiming a beam of
electrons onto the specimen.
Electrons are bounced off the surface of the
specimen and form a 3D image that is
stereoscopic in appearance.
Magnification: 1000-10,000x and Depth of Field
very high.
Can be used to identify the elements present in
the specimen under examination.
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Scanning Elec. Micros.
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SEM Images
Human Hair (1100X)
Diatom
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SEM Images
Semiconductor Chip (600X)
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SEM Images
Bread Mold (200X)
Ni Boride
(SU Chem)
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Stains (Microscopy)
Definitions
Types
Simple
Differential
Special
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Staining Samples
Staining simply means coloring the
sample with a dye that emphasizes
certain structures or components.
“Fixing”- Before a sample can be
stained, it must be attached or fixed, to
the slide, otherwise the stain might
wash it from the slide.
“Smearing” - When a specimen is fixed,
a thin film of material containing the
sample is spread over the surface of the
slide. This film (smear) is allowed to air
dry or by flame.
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Stains
Stains are salts composed of a positive
and a negative ion, one of which is
colored and is known as the
chromophore. The color of “basic dyes”
originates in the positive ion. In “acidic
dyes” it originates in the negative ion.
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Dyes: Acids and Bases
Basic dyes (cation) include crystal violet,
methylene blue and safranin are more
commonly used for bacterial cells.
Acidic dyes (anion) are not attracted to most
bacterial cells - the stain colors the background.
This preparation of colorless bacteria against the
colored background is called negative staining.
It’s valuable for cell shapes, sizes & capsules.
Distortions are kept to a minimum because heat
fixing in not necessary. Examples of acidic dyes
are eosin, nigrosin & India ink.
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Simple Stains
Aqueous or alcohol solution of a
single basic dye. Used to highlight
microorganisms to determine cellular
shapes and arrangements. Examples
include, methylene blue,
carbolfuchsin, crystal violet, &
safranin.
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Differential Stains
Differential Stains React differently with different
kinds of bacteria in order to distinguish them.
Gram Stain Divides bacteria into two large
groups, gram-positive & gram-negative.
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Special Staining
Negative Staining for Capsules. Since most
capsules do not accept stains, the capsules
appear as halos around bacterial cells & stand out
against a dark background.
Endospore (Spore) Staining. Used to detect the
presence of endospores in bacteria. When stain
(malachite green) is applied to a heat-fixed smear
of bacterial cells, the stain penetrates the
endospores and stains them green.
Flagella Staining Used to demonstrate the
presence of flagella.
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End Chapter 7
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