The document discusses microscopy and different types of microscopes. It provides details on: - The basic principles of light microscopes, including how lenses produce magnified images. Compound microscopes use objective and eyepiece lenses to further magnify images. - Electron microscopes, which use electromagnetic lenses to focus electron beams for much higher magnifications than light microscopes. - Components of light microscopes like the condenser, objective, and eyepiece lenses. Additional details are given on magnification, resolution, and numerical aperture. - Different microscope techniques for enhancing image contrast or viewing living cells, including brightfield, darkfield, phase contrast, and fluorescence microscopes.

1. MICROSCOPY

2. • The technical field of using microscopes to
view objects that cannot be seen with the
naked eye is called microscopy.

7. © 2013 FEI
Historic Figures in Microscopy
Robert Hooke
(1635-1703)
Ernst Ruska
(1906-1988)
Ernst Abbe
(1840-1905)
Richard Feynman
(1918-1988)
Antony van Leeuwenhoek
(1632-1723)

9. When the object is at a distance greater
than twice the focal length from the lens, a
real, inverted, minified image occurs at a point
between
one and two times the focal length on the
other side of the lens
When the object is
at a point exactly twice the focal length, the
image is real, inverted and the same size
When the object is at a distance between one
and two times the focal length, the image is
real,
inverted and magnified, and occurs at a
distance greater than twice the focal length
When the object is at the focal point, the
image occurs at infinity

10. The basic compound microscope uses two
convex lenses in the arrangement shown in
Figure 5.4. The lens closest to the
object is called the ‘objective’. The lens
closest to the eye is called the ‘eyepiece’.
The microscope is adjusted so that the
object (a) is at a distance between one and
two times the focal length
The image (b) formed is therefore real,
magnified and inverted. This real image acts
as the object for the second lens, i.e. the
eyepiece. The eyepiece magnifies this
intermediate image, and produces a large
virtual image (c) which then becomes the
image for the eye or for the camera lens.
The final real image (d) is formed on the
retina of the eye or the photo film
The overall magnification of the instrument
is the product of the magnifications of the
objective and the eyepiece

11. • The objective is combination of convex and
concave lenses to produce the desired
magnification and eliminate distortions.
Similarly the eyepiece is built up of many
lenses.
• Nevertheless, the principles underlying the
magnifying systems remain as illustrated here

12. Types: two types
• Optical or light microscope: magnify approx.
1500 times
• Electron microscope: magnify approx.
200,000 times, only dead cells
• Light microscopes use a series of glass lenses to focus light in order to form an
image whereas electron microscopes use electromagnetic lenses to focus a
beam of electrons.
• Magnification is not the best measure of a microscope. It is the Resolution, the
ability to distinguish between two closely spaced points in a specimen
• 0.5 micrometers (mm) for light microscope
• 1 nanometer (nm) for electron microscope

15. Optical/ light microscope
• Many of the interesting features of biological systems are
too small to be seen with the naked eye
• When molecular detail is not required, the light microscope
is an ideal, and hence essential, instrument for a biologist
• Light microscopes use glass lenses to bend and focus light
rays and produce enlarged images of small objects.
• Light microscopy is easy to use and inexpensive.

16. Lenses and the Bending of Light
• When a ray of light passes from one medium
to another, refraction occurs—that is, the ray
is bent at the interface.

18. Simple microscope
• Consists of a single glass lens mounted in a metal
frame – a magnifying glass.
• The specimen requires very little preparation, and
is usually held close to the eye in the hand.
• Focusing of the region of interest by moving the
lens and the specimen relative to one another.
• The source of light - the Sun or ambient indoor
light.
• The detector - the human eye.
• The recording device - a hand drawing or an
anecdote.

19. Compound microscopes
• more than one glass lens in combination
• major components are the condenser lens, the objective lens
and the eyepiece lens
• The main components of the compound light microscope
include a light source that is focused at the specimen by a
condenser lens. Light that either passes through the specimen
(transmitted light) or is reflected back from the specimen
(reflected light) is focused by the objective lens into the
eyepiece lens
• image is either viewed directly by eye in the eyepiece or it is
most often projected onto a detector, for example
photographic film or, more likely, a digital camera

20. • A correctly positioned condenser lens
produces illumination
that is uniformly bright and free from glare
across the viewing area of the specimen
(Koehler illumination). Condenser
misalignment and an improperly adjusted
condenser aperture diaphragm are major
sources of poor images in the light microscope

22. Basic Types :
• Upright microscope: light source is below the
condenser lens and the objectives are above the specimen
stage
• Inverted microscope: light source and the condenser
lens are above the specimen stage, and the objective lenses
are beneath it
• Typically light sources are mercury lamps, xenon lamps, lasers
or light-emitting diodes (LEDs)

24. Objective lens
• For producing magnified
images
• Can be most expensive
component
• Available in different
varieties like
magnification (4 ,10 ,20
,40 ,60 , 100 ),immersion
requirements (air, oil or
water), coverslip
thickness (usually
0.17mm) and often
more-specialized optical
properties of the lens
(corrected from
chromatic aberration
and flatness of field)

25. Eye piece
• Eye piece or sometime
called ocular works in
combination with
objective lens to further
magnify the image
• Eyepieces usually
magnify by 10 X

27. Chromatic Aberration
• occurs when different wavelengths of light are separated and
pass through a lens at different angles
• This results in rainbow colors around the edges of objects in
the image
• This problem was encountered in the early
microscopes of van Leeuwenhoek and Hooke
• All modern lenses are now corrected to some degree in order
to avoid this problem.

28. Bright-Field Microscope
• It forms a dark image against a brighter background
• The light is transmitted through the sample and the image is
formed by the absorption of this light. Thus the image will appear
darker than the background which is the bright field.
• Contrast in brightfield images is usually produced by the color of
the specimen itself
• Light source- either a mirror or an electric illuminator
• has several objective lenses
– microscopes remain in focus when objectives are changed
• total magnification
– product of the magnifications of the ocular lens and the
objective lens

31. Properties
• Magnification
• Resolution
• Numerical aperture

32. Magnification
• The objective lens forms an enlarged real image
within the microscope, and the eyepiece lens further
magnifies this primary image.
• When one looks into a microscope, the enlarged
specimen image, called the virtual image, appears to
lie just beyond the stage about 25 cm away.
• The total magnification is calculated by multiplying
the objective and eyepiece magnifications together.
For example, if a 45 objective is used with a 10
eyepiece, the overall magnification of the specimen
will be 450.

33. Microscope Resolution
• Resolution is the ability of a lens to separate
or distinguish between small objects that are
close together.
• the ability to distinguish between two closely
spaced points in a specimen

34. Resolution
is defined as the act, process, or capability of distinguishing
between two separate, but adjacent objects or sources of light,
or between two nearly
equal wavelengths.
Resolving Power
is the ability to make points or lines which are
closely adjacent in an object distinguishable in
an image.

35. Contrast: Dependent on the amount of light
scattered by the object and the amount of
light scattered by the objects around it.
A white fox in snow is not visible because both fox
and the snow scatter light to the same degree.
Camouflage in war fare and Chameleon on the
trees.
Sensitivity: Depends on the absolute amount
of light scattered by an object.
A dark adopted eye can detect a pulse of visible
light containing ~50 photons (other devises can
detect smaller amount of light-higher sensitivity).

36. Numerical aperture
• is a measure of the ability of a lens to collect
light from the specimen
• is a number usually between 0.04 and 1.4
• is always marked on the lens
• Lenses with a low NA collect less light than
those with a high NA
• higher NA objectives yield the best resolution

40. • The maximum
theoretical resolving
power of a microscope
with an oil immersion
objective (numerical
aperture of 1.25) and
blue-green light is
approximately 0.2 um.
• d is resolving limit

41. • The working distance of an objective is the
distance between the front surface of the lens
and the surface of the cover glass (if one is
used) or the specimen when it is in sharp
focus.
• Objectives with large numerical apertures and
great resolving power have short working
distances.

43. The Dark-Field Microscope
• produces a bright image of the object against a dark
background
• Generally, An extra condenser lens is added so that the
rays of light fall obliquely on the object.
• used to observe living, unstained preparations like
viewing the outlines of objects in liquid media such as
living spermatozoa, microorganisms or cells growing in
tissue culture,
• A hollow cone of light is focused on the specimen in
such a way that unreflected and unrefracted rays do
not enter the objective

47. The Phase-Contrast Microscope
• enhances the contrast between
intracellular structures having slight
differences in refractive index
• excellent way to observe living cells

48. • In a microscope if the phase of the rays scattered
from the sample can be made to have a different phase from
the unscattered rays, then the image of the specimen will be
different in light intensity as compared to the background and
will be more clearly visible.
• This is because when two waves (e.g. the scattered rays and
unscattered rays) are in phase, they reinforce one another
and the total amplitude is larger
• This effect is used in a phase contrast microscope to image
thin transparent specimens.

53. The Differential Interference
Contrast Microscope
• creates image by detecting
differences in refractive indices and
thickness of different parts of
specimen
• excellent way to observe living cells

54. • The object beam passes through the
specimen, while the reference beam passes
through a clear area of the slide.
• After passing through the specimen, the two
beams are combined and interfere with each
other to form an image.

56. Fluorescence Microscope
• exposes a specimen to ultraviolet, violet, or
blue light and forms an image of the object
with the resulting fluorescent light.
• is currently the most widely used contrast
technique since it gives superior signal-to-
noise ratios
• most commonly used fluorescence technique
is called epifluorescence light microscopy,
where ‘epi’ simply means ‘from above’

57. • Flourochromes are small fluorescent molecules, which may be
linked chemically to materials of interest
• Fluorescein isothiocyanate (FITC) absorbs blue light and emits
green light. Rhodamine compounds absorb green light and
emit red light
• In clinical medicine, fluorescence microscopy is a powerful
technique to identify the presence of particular viruses in the
sample.
• In biology, it enables the mapping of the distribution of even
fairly small cell components

62. Preparation and Staining of
Specimens
• Living microorganisms can be directly
examined with the light microscope.
• But dead cells are to be fixed and stained.

63. Fixation
• Fixation is the process by which the internal and
external structures of cells and microorganisms
are preserved and fixed in position.
• It inactivates enzymes that might disrupt cell
morphology and toughens cell structures so that
they do not change during staining and
observation.
• A microorganism usually is killed and attached
firmly to the microscope slide during fixation.
• Heat-fixed or chemical fixation

64. Staining
Basic dyes
• methylene blue, basic fuchsin, crystal violet,
safranin, malachite green
• have positively charged groups (usually some
form of pentavalent nitrogen) and are generally
sold as chloride salts.
• Basic dyes bind to negatively charged molecules
like nucleic acids and many proteins.
• Because the surfaces of bacterial cells also are
negatively charged, basic dyes are most often
used in bacteriology.

65. Acid dyes
• eosin, rose bengal, and acid fuchsin
• Possess negatively charged groups such as
carboxyls (—COOH) and phenolic hydroxyls
(—OH).
• Acid dyes, because of their negative charge,
bind to positively charged cell structures.

66. Differential Staining
• Gram’s staining
• Acid-fast staining

68. Staining Specific Structures
• Negative staining-India ink or Nigrosin dye
• Spore staining-Schaeffer-Fulton procedure

69. Electron Microscopy

70. • Used when greatest resolution is required and
when living state can be ignored
• Revealed the ultra structure of the cells
• Two different types
• Transmission electron microscope (TEM):
electrons that pass through the specimen are imaged
• Scanning electron microscope (SEM): electrons
that are reflected back from the specimen
(secondary electrons) are collected, and the surfaces
of specimens are imaged
• Light source: electron gun

71. • Tungsten filaments emit electrons when high
voltage of 40000 to 100000V
• These electrons pass through hole in anode
forming electron beam
• Electron beam passes through stack of
electromagnetic lenses

72. Scanning probe microscope Charged particle microscope
Optical (light) microscope
Optical (light)
microscope
objective lens
light beam
specimen
light source
Scanning
probe
microscope
laser diode
X, Y piezoelectric
scanner
Z piezoelectric
scanner
cantilever
sample
(stationary)
mirror
multiple segmentphotodiode
(position sensitive detector)
Charged
particle
microscope
objective aperture
selected area aperture
condenser aperture
electron source
first condenser lens
second condenser lens
fluorescent screen
microcondenser lens
specimen (thin)
objective imaging lens
objective condenser lens
diffraction lens
intermediate lens
first projector lens
second projector lens
projector chamber
(Illustration of a TEM shown)

73. Comparing Microscopes
LIGHT MICROSCOPE ELECTRON MICROSCOPE
Use of vacuum No vacuum
Entire electron path from
gun to camera must be
under vacuum
The source of
illumination
The ambient light source is
light for the microscope
Electrons are used to “see” –
light is replaced by an electron
gun built into the column
The lens type Glass lenses Electromagnetic lenses
Magnification
method
Magnification is changed by
moving the lens
Focal length is charged by
changing the current through
the lens coil
Viewing the
sample
Eyepiece (ocular)
Fluorescent screen or
digital camera

74. KEY CONCEPT: Resolution
Resolution
is defined as the act, process, or capability of distinguishing
between two separate, but adjacent objects or sources of light,
or between two nearly
equal wavelengths.
Resolving Power
is the ability to make points or lines which are
closely adjacent in an object distinguishable in
an image.

75. Resolving Power of the Human Eye
What can we see?

76. Resolution & Magnification
scale

77. How is Resolution Affected by Wavelength?

78. KEY CONCEPT: The Electron
An atom is made up of:
Protons
Neutrons
Electron

79. CORE TECHNOLOGY: The Electron Gun
• Three main sources
of electrons:
– Tungsten
– LaB6 (lanthanum hexaboride)
– Field Emission Gun (FEG)
• Different costs and
benefits of each
• Each selected primarily
for their brightness

80. CORE TECHNOLOGY: Electromagnetic Lenses
electron beam
soft iron pole piece
electrical coil

81. CORE TECHNOLOGY: The Vacuum
• A vacuum is a region
of reduced gas pressure.
• Electron microscopes
use a vacuum to make
electrons behave
like light.

82. What is a Transmission Electron Microscope?
projector lens
electron source
condenser system
specimen (thin)
objective lens

83. Transmission Electron Microscope
• Electron beams behave like radiation and can be
focused much as light is in a light microscope.
• If electrons illuminate the specimen, the microscope’s
resolution is enormously increased because the
wavelength of the radiation is around 0.005 nm,
approximately 100,000 times shorter than that of
visible light.
• The transmission electron microscope has a practical
resolution roughly 1,000 times better than the light
microscope; with many electron microscopes, points
closer than 5 A or 0.5 nm can be distinguished, and the
useful magnification is well over 100,000X

84. TEM Aberration Correction
• Chromatic aberration is distortion that occurs when there is a failure of a lens to
focus all colors (wavelengths) to the same convergence point.
• Correcting the aberration is necessary, otherwise the resulting image would
be blurry and delocalized, a form of aberration where periodic structures
appear to extend beyond their physical boundaries.
• Recent improvements in aberration correction have resulted in significantly-
improved image quality and sample information.
• Spherical aberration occurs when parallel light rays that pass through the central
region of the lens focus farther away than the light rays that pass through the
edges of the lens.
• Result is multiple focal points and a blurred image.
Chromatic Aberration
Spherical Aberration

89. TEM Enables 3D Imaging
3D Imaging

90. Environmental Microscopy with TEM
FEI Titan ETEM
Nickel catalyst film on silica membrane
Nickel oxide particles in nitrogen gas

92. Sample preparation
• Fixation -chemicals like glutaraldehyde or osmium
tetroxide to stabilize cell structure
• Dehydration - organic solvents (e.g., acetone or
ethanol).
• soaked in unpolymerized, liquid epoxy plastic until it is
completely permeated, and then the plastic is
hardened to form a solid block
• higher the atomic number the greater the scattering and the
contrast. Thus heavy metals are used to add contrast in the EM, for
example uranium, lead and osmium
• the electron beam can only be produced and focused in a vacuum
therefore all water must be removed

93. • Thin sections are cut from this block with a
glass or diamond knife using a special
instrument called an ultramicrotome
• The specimen must be around 20 to 100 nm
thick
• Section should be thin because electron has low penetration
power

94. • Soaking thin sections with solutions of heavy
metal salts like lead citrate and uranyl acetate.
• Heavy osmium atoms from the osmium
tetroxide fixative also “stain” cells and
increase their contrast.
• The stained thin sections are then mounted
on tiny copper grids and viewed.

96. Scanning Electron microscope
• used to examine the surfaces of microorganisms
in great detail
• The SEM differs from other electron microscopes
in producing an image from electrons emitted by
an object’s surface rather than from transmitted
electrons.

98. What is Scanning Transmission Electron Microscopy?
STEM image of a 32nm semiconductor device
Elemental map of a 45 nm PMOS
transistor structure
EDX map of semiconductor device

99. What is a Scanning Electron Microscope?
vacuum
electron beam
impact area
electron source

101. Specimen preparation
• easy, and in some cases air-dried material can be
examined directly.
• Microorganisms must first be fixed, dehydrated,
and dried to preserve surface structure and
prevent collapse of the cells when they are
exposed to the SEM’s high vacuum.
• Before viewing, dried samples are mounted and
coated with a thin layer of metal to prevent the
buildup of an electrical charge on the surface and
to give a better image

104. Comparing SEM and TEM
TEM SEM
Imaging
Electrons must pass through and
be transmitted by the specimen
Information needed is
collected near the surface
of the specimen
Electron Beam Broad, static beams
Beam focused to fine point;
sample is scanned line by line
Voltages Needed
TEM voltage ranges from
60-300,000 volts
Accelerating voltage much lower; not
necessary to penetrate the specimen
Image Rendering
Transmitted electrons are
collectively focused by the
objective lens and magnified to
create a real image
Beam is scanned along the surface of
the sample to
build up the image
Interaction of the
beam electrons
Specimen must be very thin
Wide range of specimens allowed;
simplifies
sample preparation

106. FEI V400ACE Focused Ion Beam
What is a Focused Ion Beam? (FIB)
Cross-section of a semiconductor wafer imaged with a plasma FIB
Physical Failure Analysis
Platinum Nano-Wire
FIB-cut in steel v2a EE by 1nA to
1B milling-002 steel

107. What is a DualBeam™ System?
Bidens Ferulifolia Pollen on plant structures

108. Scanning tunneling microscope
• invented in 1980
• Magnifications of 100 million and allow
scientists to view atoms on the surface of a
solid.

110. • It has a needlelike probe with a point so sharp
that often there is only one atom at its tip.
• The probe is lowered toward the specimen
surface until its electron cloud just touches
that of the surface atoms.
• If a small voltage is applied between the tip
and specimen, electrons flow through a
narrow channel in the electron clouds
(tunneling current).

112. © 2013 FEI
Applications of Microscopy

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Industrial Applications

114. © 2013 FEI
Life Science Applications
Breast cancer cells
Sperm tails tangled up
in a seminiferous tubule

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Natural Resources & Energy

116. © 2013 FEI
Scientific Research