The document discusses various types of light microscopes and their uses. It describes brightfield, darkfield, phase contrast, and fluorescence microscopes. It also discusses specimen preparation techniques like fixation, staining, and the use of dyes. Newer high resolution techniques like confocal and scanning probe microscopy are able to visualize structures at the atomic level.

1.  History
 Parts
 Care
 Types
Lecture 2: Dr. Wonder

2. 2

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

7. 7
•working distance
— distance between the front surface of lens and surface of cover
glass or specimen

8. 8

9. 9
The Dark-Field Microscope
produces a bright image of the object
against a dark background
used to observe living, unstained
preparations

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

17. 17
Brightfield Phase Contrast
DIC Darkfield

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)

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required for reproduction or
display. 20
Endoplasmic Reticulum Stained with a synthetic dye that
dissolves in ER membranes

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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

27. 27
Escherichia coli – a gram-negative rod

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

29. 29
Staining Specific Structures
Negative staining
often used to visualize capsules
surrounding bacteria
capsules are colorless against a stained
background

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

34. 34
Newer Techniques in
Microscopy
confocal
microscopy and
scanning probe
microscopy
have extremely
high resolution
can be used to
observe individual
atoms

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

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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