3. 3
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
4. 4
The light microscope
has several objective lenses
parfocal microscopes remain in focus when
objectives are changed
total magnification
product of the magnifications of the ocular
lens and the objective lens
5. . 5
The Bright-Field Microscope
produces a dark image against a brighter
background
uses a prism to send the light to both eyes
light passing through specimen is diffracted
and absorbed to make image
Staining is often necessary because very low
contrast
6. . 6
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
10. 10
The Phase-Contrast Microscope
A phase ring in condenser allows a cylinder
of light through in phase.
Light that is unaltered hits the phase ring in
the lens and is excluded.
Light that is slightly altered by passing
through different refractive index is allowed
through.
11. 11
The Phase-Contrast
Microscope
Light passing through cellular structures
such as chromosomes or mitochondria is
retarded because they have a higher
refractive index than the surrounding
medium.
Elements of lower refractive index advance
the wave. Much of the background light is
removed and light that constructively or
destructively interfered is let through with
enhanced contrast
12. 12
The Phase-Contrast
Microscope
Visualizes differences in refractive index
of different parts of a specimen relative
to the unaltered light
enhances the contrast between
intracellular structures having slight
differences in refractive index
excellent way to observe living cells
13. 13
The Differential Interference
Contrast Microscope or Nomarski
creates image by detecting differences in
refractive indices and thickness of
different parts of specimen
excellent way to observe living cells
14. . 14
The Differential Interference
Contrast Microscope or Nomarski
creates image by detecting differences in
refractive indices and thickness of
different parts of specimen.
excellent way to observe living cells.
a prism is used to split light into two
slightly diverging beams that then pass
through the specimen.
15. 15
The Differential Interference
Contrast Microscope or Nomarski
On recombining the two beams, if they
pass through difference in refractive
index then one retarded or advanced
relative to the other and so they can
interfere.
By changing the prism you can change
the beam separation which can alter the
contrast.
16. 16
The Differential Interference
Contrast Microscope or Nomarski
Also measures refractive index changes,
but for narrowly separated regions of
light paths-ie it measures the gradient
across the specimen
Gives a shadowed 3D effect
Optically sections through a specimen
18. 18
The Fluorescence Microscope
exposes specimen to ultraviolet, violet, or
blue light
specimens usually stained with
fluorochromes
shows a bright image of the object
resulting from the fluorescent light
emitted by the specimen
19. 19
The Fluorescence Microscope
Fluorescent dye- a molecule that absorbs
light of one wavelength and then re-emits it
at a longer wavelength
Can be used alone or in combination with
another molecule to gain specificity
(antibodies)
21. 21
Preparation and Staining of
Specimens
increases visibility of specimen
accentuates specific morphological
features
preserves specimens
22. 22
Fixation
process by which internal and external
structures are preserved and fixed in
position
process by which organism is killed and
firmly attached to microscope slide
heat fixing
preserves overall morphology but not internal
structures
chemical fixing
protects fine cellular substructure and
morphology of larger, more delicate organisms
23. 23
Dyes and Simple Staining
dyes
make internal and external structures of cell
more visible by increasing contrast with
background
have two common features
chromophore groups
chemical groups with conjugated double
bonds
give dye its color
ability to bind cells
24. . 24
Dyes and Simple Staining
simple staining
a single staining agent is used
basic dyes are frequently used
dyes with positive charges
e.g., crystal violet
25. . 25
Differential Staining
divides microorganisms into groups based on
their staining properties
e.g., Gram stain
e.g., acid-fast stain
26. 26
Gram staining
most widely used differential staining
procedure
divides Bacteria into two groups based on
differences in cell wall structure
28. 28
Acid-fast staining
particularly useful for staining members of the genus
Mycobacterium
e.g., Mycobacterium tuberculosis – causes
tuberculosis
e.g., Mycobacterium leprae – causes leprosy
high lipid content in cell walls is responsible for their
staining characteristics
30. 30
Staining Specific Structures
Spore staining
double staining technique
bacterial endospore is one color and
vegetative cell is a different color
Flagella staining
mordant applied to increase thickness of
flagella
31. 31
Electron Microscopy
beams of electrons
are used to
produce images
wavelength of
electron beam is
much shorter than
light, resulting in
much higher
resolution
Figure 2.20
32. 32
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
33. 33
The Scanning Electron Microscope
uses electrons reflected from the surface
of a specimen to create image
produces a 3-dimensional image of
specimen’s surface features
35. 35
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
36. 36
Confocal Microscopy
Fluorescence microscope
Uses “confocality” (a pinhole) to eliminate
fluorescence from out of focus planes
Minimum Z resolution=0.3µm
Because you can optically section through a
specimen, you can determine the localization of
probes in the Z dimension
You can also build 3D (4D) models of structures
and cells from the data
37. 37
Confocal Microscopy
Uses a laser to get a high energy point
source of light
The beam is scanned across the specimen
point by point and the fluorescence
measured at each point
The result is displayed on a computer
screen (quantitative data)
38. 38
Confocal Microscopy
Spinning disk confocal microscope
Illuminates the whole field “simultaneously
with a field of points
Captures images of the whole field at once
with a camera
Much faster than CSLM
Can be viewed through eyepieces
39. . 39
Confocal Microscopy
Two photon confocal microscope
A fluor like fluorescein normally absorbs a photon of about 480nm
and emits one at about 530nm
If fluorescein absorbs two photons of 960nm near enough to each
other in time so that the first does not decay before the second is
absorbed, it will fluoresce- 2 photon fluorescence
Confocal microscope with a laser that emits picosecond pulses of
light instead of a continuous beam is used
Advantage
960nm light penetrates farther into biological specimens
The density of light is very high at focal point, but low
elsewhere, so damage to cell is less
You don’t need a second pinhole because excitation only
happens at the focal point
40. 40
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
41. 41
Scanning Probe Microscopy
atomic force microscope
sharp probe moves over surface of
specimen at constant distance
up and down movement of probe as it
maintains constant distance is
detected and used to create image
