Download Chapter 4-Microscopes

Microscopy
Chapter 4
CHE 113 Forensic Science
Microscopy and Stains
Micro=“small”
Skopein= “to see”
Microscopes
Preparation of
specimens for light
microscopy
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Microscope Types
• Brightfield Microscope
– Compound
– Comparison
– Stereoscopic
– Polarizing
– Microspectrophotometer
• Darkfield Microscope
• Phase-contrast Microscope
• Fluorescence Microscope
• Electron Microscope
– Transmission
– Scanning
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How do microscopes make things look
bigger?
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Simple Magnifier
• Object O is placed
close to the lens
– rays converge but do
not intersect
– real image not formed
• The observer’s eye
follows rays back to
the point of apparent
origin (I)
• (I) bigger than object
The Compound Microscope
• Rays pass first through the objective lens forming a
real, slightly enlarged, inverted image
• The second lens (eyepiece) acts as a simple magnifier
Remember Letter e lab?
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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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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Limitations of Light Microscope
• Radiation of a given wavelength can’t be used
to probe structural details much smaller than
its own wavelength
• Light Microscope
– limited to range of visible light
• 0.4 mm (violet) to 0.7 mm (deep red)
– bacteria & mitochondria (~0.5mm wide) smallest objects that
can be seen clearly
Tissue Prep Center
Microtome
Water bath with
sliced wax/tissue
Embedding Area
Tissue prep-Mount onto slides
Stains
• Help visualize details of specimen
– Vital Stains=Don’t harm living specimens (ex.
Methyl Blue)
– Lethal Stains=Kills Specimen (ex. Iodine)
Staining Samples
• “Fixing”- Chemically make tissue permanent
and attached to 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.
• Staining= color for emphasizing certain
structures or components.
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Dyes: Acids and Bases
• Basic dyes (cation):
– Ex. crystal violet, methylene blue and safranin
– 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.
– Great for cell shapes, sizes & capsules.
– Ex. eosin, nigrosin & India ink.
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Stains (Microscopy)
• Types
– Simple
– Differential
– Special
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Simple Stains
• Aqueous or alcohol solution of a single basic
dye.
– highlight microorganisms to determine cellular
shapes and arrangements.
– Ex. 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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Darkfield Microscopy
• 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.
• Most useful for extremely small living organisms that are
invisible in the light microscopes.
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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 documents
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Comparison Microscope
Split-image comparison of
firing pin imprints in coaxial
incident light
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Comparison Microscope
Real
Forgery
Split-image comparison of banknotes:
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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.
• It allows the detailed observation
of living organisms, especially
the internal structures.
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Bright field vs Phase Contrast
Polarizing Microscope
• Based upon the rotation of polarized light.
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Polarizing Potato Starch
Fluorescence Microscope
• 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.
• Used primarily in a diagnostic
procedure called fluorescentantibody (FA) technique, or
immunofluorescence.
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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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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.
• 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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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 Electron Microscope
• This scanning electron microscope has a magnification range
from 15x to 200,000x and a resolution of 5 nanometers
Range of Readily Resolvable Objects
How the SEM Works
Conventional light microscopes use a series of glass lenses
to bend light waves and create a magnified image.
• The Scanning Electron
Microscope creates the
magnified images by
using electrons instead
of light waves
The SEM shows very detailed 3-dimensional images
at much higher magnifications than is possible with a
light
microscope. The images created without light waves
are rendered black and white
Samples have to be prepared carefully to withstand
the vacuum inside the microscope
• Biological specimens
are dried in a special
manner that prevents
them from shriveling.
• Because the SEM
illuminates them with
electrons, they also
have to be made to
conduct electricity
• How do you make a
mosquito conductive?
• SEM samples are
coated with a very thin
layer of gold by a
machine called a
sputter coater
The specimen is now prepared
The sample is placed inside the microscope's
vacuum column through an air-tight door
• Air is pumped out of the
column
• An electron gun [at the top]
emits a beam of high
energy electrons.
– travels downward through a
series of magnetic lenses
designed to focus the
electrons to a very fine spot
• Near the bottom, a set
of scanning coils moves
the focused beam back
and forth across the
specimen, row by row
• As the electron beam
hits each spot on the
sample, secondary
electrons are knocked
loose from its surface.
• A detector counts these
electrons and sends the
signals to an amplifier
• The final image is built
up from the number of
electrons emitted from
each spot on the
sample
TEM Pictures
Silver Nanoprisms
Gold Nanoparticle
Fiber Analysis
Who am I?
Who am I?
I’m a louse fly of a wallglider (an alpine bird)
SEM Images
Human Hair (1100X)
Diatom
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FT-IR Microspectrophotometer
Photocopier Toner Analysis
• important for establishing corroborative evidence
linking documents to specific locations in forensic
investigations of corporate crime
• Must be performed non-destructively
– can’t remove toner from paper
– physical size of specimen is very small
• microscope to find sample
• FT-IR to analyze the sample