Microscope 1
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Transcript of Microscope 1
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Scale
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Discovery of
Microorganisms Antony van
Leeuwenhoek
(1632-1723) first person to
observe and
describe
micro-organisms
accuratelyFigure 1.1b
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Lenses and the Bending of
Light light is refracted (bent) when passing
from one medium to another
refractive index a measure of how greatly a substance slows
the velocity of light
direction and magnitude of bending isdetermined by the refractive indexes of
the two media forming the interface
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Lenses
focus light rays at a specific placecalled the focal point
distance between center of lens andfocal point is the focal length
strength of lens related to focal
length short focal length more
magnification
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Figure 2.2
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The Light Microscope
many types
bright-field microscope
dark-field microscope
phase-contrast microscope
fluorescence microscopes
are compound microscopes
image formed by action of 2 lenses
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The Bright-Field
Microscope produces a dark image against a
brighter background
has several objective lensesparfocalmicroscopes remain in focus
when objectives are changed
total magnification product of the magnifications of the
ocular lens and the objective lens
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Figure 2.3
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Microscope Resolution
ability of a lens to separate or
distinguish small objects that are
close together wavelength of light used is major
factor in resolution
shorter wavelength greater resolution
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working distancedistance between the front surface of lens and surface of
cover glass or specimen
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Figure 2.5
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Figure 2.6
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The Dark-Field Microscope
produces a bright image of the
object against a dark background
used to observe living, unstainedpreparations
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Figure 2.7b
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The Phase-Contrast
Microscope enhances the contrast between
intracellular structures having slight
differences in refractive index
excellent way to observe living cells
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Figure 2.9
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Figure 2.10
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The Differential Interference
Contrast Microscope creates image by detecting
differences in refractive indices andthickness of different parts of
specimen
excellent way to observe living cells
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The Fluorescence
Microscope exposes specimen to ultraviolet,
violet, or blue light
specimens usually stained withfluorochromes
shows a bright image of the object
resulting from the fluorescent light
emitted by the specimen
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Figure 2.12
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Figure 2.13c and d
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Fixation process by which internal and external
structures are preserved and fixed inposition
process by which organism is killed andfirmly attached to microscope slide
heat fixing
preserves overall morphology but not internal
structures chemical fixing
protects fine cellular substructure andmorphology of larger, more delicate organisms
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Dyes and Simple Staining
dyes
make internal and external structuresof cell more visible by increasing
contrast with backgroundhave two common features
chromophore groups
chemical groups with conjugated doublebonds
give dye its color
ability to bind cells
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Dyes and Simple Staining
simple staining
a single staining agent is used
basic dyesare frequently used dyes with positive charges
e.g., crystal violet
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Differential Staining
divides microorganisms into groups
based on their staining properties
e.g., Gram stain e.g., acid-fast stain
C G C f
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Gram staining
most widely used differential
staining procedure
divides Bacteria into two groupsbased on differences in cell wall
structure
C i ht Th M G Hill C i I P i i i d f d ti di l
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Figure 2.14
primarystain
mordant
counterstain
decolorization
positive
negative
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Acid-fast staining
particularly useful for stainingmembers of the genusMycobacter ium
e.g., Mycobacter ium tuberculosiscausestuberculosis
e.g., Mycobacter ium lepraecauses leprosy
high lipid content in cell walls isresponsible for their stainingcharacteristics
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Staining Specific
Structures Negative staining
often used to visualize capsules
surrounding bacteria
capsules are colorless against a stained
background
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Electron Microscopy
beams of electronsare used toproduce images
wavelength ofelectron beam ismuch shorter than
light, resulting inmuch higherresolution
Figure 2.20
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The Transmission Electron
Microscope electrons scatter when they pass
through thin sections of a specimen
transmitted electrons (those that do
not scatter) are used to produce
image
denser regions in specimen, scatter
more electrons and appear darker
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Figure 2.23
EM
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Specimen Preparation
analogous to procedures used for
light microscopy
for transmission electronmicroscopy, specimens must be cut
very thin
specimens are chemically fixed andstained with electron dense material
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Other preparation methods
shadowing
coating specimen with a thin film of a
heavy metal freeze-etching
freeze specimen then fracture along
lines of greatest weakness (e.g.,membranes)
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Figure 2.25
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Ebola
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Fly head
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The Scanning Electron
Microscope uses electrons reflected from the
surface of a specimen to create
image
produces a 3-dimensional image of
specimens surface features
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Figure 2.27
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Newer Techniques in
Microscopy confocalmicroscopyandscanning probe
microscopy have extremely
high resolution
can be used toobserve individualatoms
Figure 2.20
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Confocal Microscopy
confocal scanning laser microscope
laser beam used to illuminate spots
on specimen computer compiles images created
from each point to generate a 3-
dimensional image
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Figure 2.29
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Figure 2.30
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Scanning Probe
Microscopy scanning tunneling microscope
steady current (tunneling current)maintained between microscope probe
and specimen
up and down movement of probe as it
maintains current is detected and used
to create image of surface of specimen
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Scanning Probe
Microscopy atomic force microscope
sharp probe moves over surface ofspecimen at constant distance
up and down movement of probe as it
maintains constant distance is detectedand used to create image