Microscopy techniques with principles and its applications

1. By
Dr. Baljinder S Gill
Department of Biotechnology
Panjab University
Chandigarh

2. An easy way to understand the microscope is by means of a comparison with a
slide projector.
magnification

3. A microscope is an optical instrument that uses a lens or a combination
of lens to produce a magnified image of an object; too small to be seen

6. Terms related to microscopy

7.  The media through which the light passes will
be able to slow down or retard the speed of the
light in proportion to the density of the
medium.
 Higher the density, greater the retardation.
 Light rays entering a sheet of glass at right
angle are retarded but their direction is
unchanged.

8.  If the light enters the glass at any angle other
than right angle, a deviation in the direction
will occur in addition to retardation, known as
Refraction.
 A curved lens will exhibit both refraction and
retardation.
 The extent of which is determined by angle of
incidence, refractive index and curvature of the
lens.

10. diffraction is the change in the direction and the intensities of group of waves after
passing by an obstacle or through an aperture, whose size is approximately the same
as the wavelength of the wave.
Dispersion is a phenomenon, in which separation of light into its constituent
wavelength occurs from entering a transparent medium. For example, white light
consist consists of more than one wavelength. They would be separated or they would
distribute when they pass through prism or certain other medium.
magnification is the process of enlarging an object only in appearance and not in
physical size
interference is the variation of wave amplitude that occurs when waves of the same or
different frequency come together. There could be constructive interference or there
could be destructive interference.
resolving power is defined as the distance separating 2 point objects within the
specimen that can just be distinguished from one another in the image

11. Focal point, focal plane, object distance, object plane, image distance,
image plane

12. Concave lens
Form reduced, erect and virtual, image
Converging Lenses - Object-Image Relations

13. Case 1: The object is located beyond 2F
inverted image
reduced in size
real image
Case 2: The object is located at 2F
larger than the object dimensions
image dimensions are equal to
the object dimensions
Case 3: The object is located between 2F
and F
Case 4: The object is located at F
no image is formed.
the refracted rays neither converge nor
diverge. After refracting, the light rays
are traveling parallel to each other and
cannot produce an image

14. Case 5: The object is located in front of F

15. Aberrations are when lenses fail to bring rays or all rays on the
given point on the object to a unique focus. That is called
aberrations. Now, when there is an aberration, then blurred or
not so clear images are formed. This is because all rays are not
focused at the same point and you do not get sharp image.
There are 2 main types of aberrations here. 1 is called the
chromatic aberration and another is called spherical
aberration.
Aberrations

16.  Light is composed of spectrum of colors, each
having a different wavelength, will be refracted
to a different extent, with blue being brought to
a shorter focus than red.
 This lens defect is known as chromatic
aberration and results in an unsharp image and
distorted edges.
 This is known as chromatic aberration and it’s
correction is known as achromatism.

18.  It is possible to construct compound lenses of
different glass elements to correct this.
 An achromat is corrected for two colors, red
and blue, producing a secondary spectrum of
yellow/green, which is in turn corrected by
adding more lens components like fluorospar,
three colors can be brought into focus – the
more expensive – Apochromat.

19.  Spherical aberration is caused by the virtue of
its curvature, where the light rays entering the
lens at the periphery are refracted more than
the light rays entering at the center of the lens,
and thus not brought to a common focus.
 These defects are also corrected by using a
combination of lens elements of different glass
and of different shape.

21. A convex lens is the simplest microscope.
A light ray parallel to the optical axis of the lens passes
through the focus of the lens while a ray passing through the
centre of the lens does not bend.

22. The objective has several major functions:
1. The objective must gather the light coming from each of the
various parts or points of the specimen.
2. The objective must have the capacity to reconstitute the light
coming from the various points of the specimen into the various
corresponding points in the image. (Sometimes called anti-points.)
3. The objective must be constructed so that it will be focused close
enough to the specimen so that it will project a magnified, real
image up into the body tube.
KEY PARTS OF MICROSCOPE

23. Classification According to Microscopy Method
"reflected darkfield objective (a circular-zone light path is applied to the periphery of an
inner lens)", "Differential Interference Contrast (DIC) objective (the combination of
optical properties with a DIC( Nomarski ） prism is optimized by reducing lens
distortions)", "fluorescence objective (the transmittance in the near-ultraviolet region is
improved)", "polarization objective (lens distortions are drastically reduced)", and
"phase difference objective (a phase plate is built in) are available
Classification According to Magnification
Classification According to Chromatic Aberration Correction
chromatic aberration correction is divided into three levels of achromat,
semiapochromat (fluorite), and apochromat according to the degree of correction. The
objective lineup is divided into the popular class to high class with a gradual difference
in price. An objective lens for which axial chromatic aberration correction for two colors
of C ray (red: 656,3nm) and F ray (blue: 486.1nm) has been made is known as Achromat
or achromatic objective..

24. In the case of Achromat, a ray except for the above two colors (generally violet g-ray:
435.8nm) comes into focus on a plane away from the focal plane. This g ray is called a
secondary spectrum. An objective lens for which chromatic aberration up to this
secondary spectrum has satisfactorily been corrected is known as Apochromat or
apochromatic objective. In other words, Apochromat is an objective for which the axial
chromatic aberration of three colors (C, F, and g rays) has been corrected.

26. These objectives are corrected chromatically for three colors: red, blue, and
green; they are corrected spherically for two colors. Apochromatic objectives
are the best for color photomicrography in white light. Because of their high
level of correction, such objectives usually have, for a given magnification,
higher numerical apertures

27. All three types of objectives project images that are
curved rather than flat. To overcome this inherent
condition, lens designers have produced flat-field
objectives which yield flat images. Such lenses are called
plan-achromats, or plan-fluorites, or plan-apochromats.

28. There are several main kinds of eyepieces: negative and positive. In
negative eyepieces, there are two lenses: the upper or eye-lens and the lower
or field lens. In their simplest form, both lenses are Plano-convex, with
convex sides “facing” the specimen. The simplest kind of negative eyepiece,
or Huygenian eyepiece, is found on most routine microscopes fitted with
achromatic objectives.
The other main kind of eyepiece is the positive eyepiece or Ramsden
eyepiece. Its eye lens and field lens may be cemented together and better
corrected than the simple Huygenian type. In the positive eyepiece, the
circular, fixed diaphragm is below the field lens.

29. Huygens eyepieces consist of two
plano-convex lenses with the plane
sides towards the eye separated by an
air gap. The lenses are called the eye
lens and the field lens. The focal plane
is located between the two lenses
The Ramsden eyepiece comprises two
plano-convex lenses of the same glass
and similar focal lengths, placed less
than one eye-lens focal length apar

32. CONDENSERS
A condenser is an optical lens which renders a divergent beam from a point
source into a parallel or converging beam to illuminate an object. In the context
of microscopy, the parallel illumination scheme is known as Köhler
illumination whereas the converging illumination scheme is known as critical
illumination.
Abbe condenser
This condenser is composed of two lenses a plano-convex lens somewhat larger
than a hemisphere and a large bi-convex lens serving as a collecting lens to the
first. The focus of the first lens is traditionally about 2mm away from the plane
face coinciding with the sample plane. A pinhole cap can be used to align the
optical axis of the condenser with that of the microscope

33. Aplanatic and achromatic condensers
An aplanatic condenser corrects for spherical aberration in the
concentrated light path, while an achromatic compound
condenser corrects for both spherical and chromatic aberration

34. • Light microscopes
–Bright field microscope
–Dark field microscope
–Phase contrast microscope
–Fluorescent microscope
DIFFERNNT TYPES OF MICROSCOPY

35. Bright-field microscopy
diffracted (diffracted by the specimen) and undiffracted
(light that transmits through the sample undeviated)
lights are collected by the objective lens.
"brightfield" is derived from the fact that the specimen is
dark and contrasted by the surrounding bright viewing
field.

37. Dark Field
illumination is a
technique used to
observe unstained
samples causing
them to appear
brightly lit against a
dark, almost purely
black, background.

38. Phase contrast microscopy
Little difference in contrast between cells and water. Provides
very high contrast as compared to the bright-field and dark-field
microscopic methods
Image is generated from both diffracted and undiffracted lights
Application
Useful for microbial mobility,
shape of living cell, detection
of endospores and inclusion
bodies.

39. Invented by the Dutch physicist Frederick Zernike in 1930
It converts the different degrees of retardation of light rays passing through a
translucent material into amplitude differences to create an image with increased
contrast.

40.  Objects with poor intensity can be resolved well.
 Image of the unstained object can be obtained
 It is widely used in microbiology and tissue culture
research to detect bacteria, cellular organelles and other
small entities in living specimens
 It allows the visualization of living unstained cells and
their organelles.
 For the detection of bacterial components such as
endospores and inclusion bodies containing poly β
hydroxy butyrate, polymetaphosphate, sulpher and other
substances

41. Fluorescence microscopy
Uses extrinsic fluorescent probes that can go inside the cell
and bind to the intracellular molecules with high specificity
4',6-diamidino-2-
phenylindole

42. xenon arc lamp or
mercury-vapor lamp
Epifluorescence microscopy

43. Single Cell Organism ( Tetrahymena) observed with:

44. Nomarski microscopy or Differential interference contrast
(DIC) microscopy or a polarization interference contrast
microscope.
improvement over phase contrast microscopy
light refraction by different parts of living cells

46. Polarising filter- plane-polarized light necessary for the
interference detection, (polarised at 45°)
Nomarski Prism or Wollaston prism or condenser prism,
this beam-splitting prism – split into two beams (two rays
polarised at 90° to each other, the sampling and
reference rays)
Objective Prism- recombines the separated beams into
elliptically polarized light
second polarizing filter- where the interference occurs that
generates the differential interference contrast
If there has been a phase shift between the two rays as they
pass through areas of different refractive index then
elliptically polarised light is the result.

47.  Invented by William Hyde Wollaston.
 Wollaston prisms - made of two layers of a crystalline
substance, such as quartz - due to the variation of
refractive index depending on the polarisation of the
light, splits the light according to its polarisation.
 It seperates randomly polaraised or unpolarised light
into two orthogonal linearly polarised outgoing beams.

48.  One of the wedges is identical to a conventional Wollaston wedge
and has the optical axis oriented parallel to the surface of the
prism.
 The second wedge of the prism is modified by cutting the crystal
in such a manner that the optical axis is oriented obliquely with
respect to the flat surface of the prism.
 The Nomarski modification causes the light rays to come to a focal
point outside the body of the prism, and allows greater flexibility so
that when setting up the microscope the prism can be actively
focused.

50. Differential interference contrast (DIC)
Specimen
(inhomogen phase object)
Phase
difference
Polarisator
linear
polarized
light
Prism
(Nomarski)
two vertical
polarized
waves
Analysator
DIC prism
(Nomarski)
linear polarized
light
(analysator vertical
vs. polarisator)
unpolarized
light

51.  DIC works by separating
a polarised light source into two
orthogonally polarized mutually
coherent parts which are spatially
displaced (sheared) at the sample
plane, and recombined before
observation.
 The interference of the two parts at
recombination is sensitive to their
optical path difference (i.e. the product
of refractive index and geometric path
length).
 Adding an adjustable offset phase
determining the interference at zero
optical path difference in the sample,
the contrast is proportional to the path
length gradient along the shear
direction.

52.  Light passes through a polariser and is reflected
downward toward birefringent crystals Wollaston prism .
Light is split into two mutually perpendicular polarsied
components that move at different velocities with an
angular divergence(d).
 After emerging from the prism and reflecting off the sample
-two beams recombine by passing once again through the
wollaston prism in the opposite direction.
 The reconstituted beams –passes through an analyser –
intensity changes observed .
.

53. Interestingly, if no specimen is in place and both light beams enter
the upper prism without any refractory differences, the effect of
the lower prism is exactly reversed by the upper prism and the
image field appears black, an effect known as extinction.
Disadvantages
DIC is unsuitable (in biology) for thick samples, such as tissue slices,
and highly pigmented cells. DIC is also unsuitable for most non
biological uses because of its dependence on polarisation, which many
physical samples would affect

54. Hoffman modulation contrast
Hoffman modulation contrast is similar to
DIC except that it uses plates with small
slits in both the axis and the off-axis of the
light path to produce two sets of light waves
passing through the specimen.

55. Confocal laser scanning microscope
Confocal" is defined as "having the same focus.“
The object and its image are "confocal."
focus light from the focal point of one lens to another point. The red
rays of light represent light from another point in the sample, which is
not at the focal point of the lens, but which nonetheless get imaged by
the lenses of the microscope

56. The microscope is able to filter out
the out-of-focus light from above and
below the point of focus in the object.
The confocal microscope eliminates
this out-of-focus information by
means of a confocal "pinhole"
situated in front of the image plane
which acts as a spatial filter and
allows only the in-focus portion of the
light to be imaged. Light from above
and below the plane of focus of the
object is eliminated from the final
image.

57.  The optical details of the confocal microscope are
complex, but the basic idea is simple.
 CLSM is classified under Single beam scanning
microscopy, as the specimen s illuminated and
scanned using only 1 light source
 MARVIN MINSKY awarded confocal patent focal
scanning microscope in 1957, US PATENT 301467

63.  PHOTOBLEACHING: Reaction Involve the
interaction of Fluorophore + Light & Oxygen,
destroys Fluorescence and yield a free
radicals, that cause death of live cells and
tissues.
 Monochromatic LASER beam is harmful.
 It is cost effective.

74. Transmission electron microscopy and scanning electron
microscopy. TEM ,the images are formed by the transmitted
electrons. It provides you the internal details of the
specimen in contrast SEM. Here, rather than transmitted
electrons, back scattered electrons are used for image
formation. SEM provides you the surface details.

76. •A high-voltage electricity supply
powers the cathode.
•The cathode is a heated filament, a
bit like the electron gun in an old-
fashioned cathode-ray tube t generates
a beam of electrons that works in an
analogous way to the beam of light in
an optical microscope.
•electromagnetic coil (the first lens)
concentrates the electrons into a more
powerful beam.
•Another electromagnetic coil (the
second lens) focuses the beam onto a
certain part of the specimen.
•The specimen sits on a copper grid in
the middle of the main microscope
tube. The beam passes through the
specimen and "picks up" an image of
it.
•The projector lens (the third lens) magnifies the image.
•The image becomes visible when the electron beam hits a fluorescent screen at the
base of the machine. This is analogous to the phosphor screen at the front of an old-
fashioned TV .
•The image can be viewed directly (through a viewing portal), through binoculars at
the side, or on a TV monitor attached to an image intensifier (which makes weak

78. Different components of electron gun include cathode. A cathode is also called
emission source or filament. This is maintained at negative potential difference to
anode almost like 50 to 100 kilovolt negative potential difference. The most
common filaments used are thin tungsten wire. This is bent into a v shape, that is
the most commonly used and cheap one as well. But, there are other types which
can give you better illumination or a fine focus. These are lanthanum hexaboride L
a B 6. The field emission source, now lanthanum hexaboride filament is a pointed
rod that provides a stable current from a small area than a simple tungsten
filament. It is almost ten times brighter than the tungsten filament. It has a lifetime
of 500 hours or longer. But, it requires a vacuum in order of 10 to the power of
minus 6 torr.

79. Another better source than your lanthanum hexaboride and simple tungsten filament
that is called field emission source. You must have heard about field emission electron
microscopes. This requires very high vacuum conditions like 10 to the power minus 9.
It operates at room temperature. It requires a single crystal of tungsten within
emitting region of approximately 10 nano metre brightness.
It is almost 1000 times that of standard tungsten filament. It employs double anode
first anode around 2000 volts. It extracts electrons from tip and the second anode
accelerates the electron down the column. It produces a very small probe size with
negligible energy spreads. It is very effective and very good source of electrons You
can say this is the best, one of best field you can say emitter or filament. Here,
emission source it is certainly more expensive as compared to a simple tungsten
filament.

80. Conventional triode e-gun has 3 components
1. The filament or cathode which is held at negative potential.
2. The Wehnelt or grid which is held at a few hundred volt.
3. The Anode which is held at earth potential.

81. There is another component which needs a mention here. That is called the Wehnelt
shield. The Wehnelt shield is positioned between cathode and anode. It is held at
potential difference of 100 to 200 volt negative to cathode. Remember it is negative to
cathode. What it does, is the function to create intense cloud of electrons in a very small
aperture. You can say it serves as an electron source. So electrons from cathode like kind
collects in an intense cloud of electrons at this particular aperture.
Wehnelt shield is connected to high voltage and to filament heating voltage by resistors.
Then there is anode which directs the beam of electrons. Now, transmission electron
microscope will employ two condenser lenses with an aperture. There will be an
aperture of around 100 to 200 hundred micrometer. This is placed between the second
lens and the specimen to reduce the spherical aberration. There will be deflection coils
which are used to shift and tilt beam and these are part of the illumination system.

82. The deflection experienced by a charged particle in a magnetic field is
given by the Lorentz force law F = q (v × B).
he effect of adding an electric field E is:
F = e[E + (v x B)]
This means that an electron moving almost parallel to the field in a
magnetic lens is forced to take a helical path.

83. The magnetic field is largely, but not completely, parallel to the direction of
the electron motion. The magnetic field in an electromagnetic lens can be
resolved into radial and axial components as shown in figure B. An
electron entering the lens does not experience the axial component but
gets deflected by the radial component of the magnetic field. This
deflection imparts a radial velocity component to the electron that takes a
spiral path while going down the lens. The radial component of the
electron causes the electron to respond to the axial component of the
magnetic field; the force thus experienced decreases the radius of the
spiral as shown in figure C and thereby resulting in a focused electron
beam.

84. THE SPECIMEN PREPARATION
The scattering of electrons by specimen component of specimen is proportional
to the matter present. ALSO call it mass thickness or atomic density (the
measure of number of atoms per unit area and their atomic density). Biological
materials which are mainly made up of atoms of relatively low atomic number
like carbon oxygen, nitrogen, hydrogen etc. These are therefore, not effective
scatterers of electrons. This is because they have relatively less atomic density.
Mass thickness or atomic density of a specimen has needs to be enhanced. To
enhance them we need to fix and stain the samples with solutions of heavy
metals

85. Much heavier metals is required to diffract or scatter the electrons. These
heavy metals bind differentially to different components of the specimen.
To obtain the contrast The parts which complex with the metal atoms will
not be allow the passage of electrons, The other parts where the electrons or
heavy metals are not there they will allow the passage of the electrons. The
screen is hit by the electrons due to the passage through the specimen. The
areas will look bright, but wherever the electrons have not been allowed to
pass through. The areas on the fluorescent screen will be dark. That is how
the contrast will be created and the image formation in the electron
microscope will take place.

86. Ultra high vacuum system is present in the electron microscope. Only non living
specimens can be observed here. Except for certain environmental microscopes where
they allow partially hydrated samples where the vacuum conditions are around 10 to
the power minus 2 torr. In high vacuum conditions, the hydrated samples, the living
samples are not allowed. That thickness of the specimen for transmission electron
microscopy must be less than 100 or 200 nanometre. This is because of poor
penetrating power of electrons. This is much thinner than light microscopy sections

97. Fixation
This is done to preserve the sample and to prevent further deterioration so that it
appears as close as possible to the living state, although it is dead now. It stabilizes
the cell structure. There is minimum alteration to cell morphology and volume.
Glutaraldehyde is often used as the fixative in TEM. As a result of glutaraldehyde
fixation the protein molecules are covalently cross linked to their neighbors.
Rinsing
The samples should be washed with a buffer to maintain the pH. For this purpose,
sodium cacodylate buffer is often used which has an effective buffering range of
5.1-7.4. The sodium cacodylate buffer thus prevents excess acidity which may
result from tissue fixation during microscopy.
Post fixation
A secondary fixation with osmium tetroxide (OsO4), which is to increase the
stability and contrast of fine structure. OsO4 binds phospholipid head regions,
which creating contrast with the neighbouring protoplasm (cytoplasm). OsO4 helps
in the stabilization of many proteins by transforming them into gels without
destroying the structural features. Tissue proteins, which are stabilized by OsO4
and does not coagulated by alcohols during dehydration.

98. Dehydration
The water content in the tissue sample should be replaced with an organic
solvent since the epoxy resin used in infiltration and embedding step are not
miscible with water.
Infiltration
Epoxy resin is used to infiltrate the cells. It penetrates the cells and fills the
space to give hard plastic material which will tolerate the pressure of cutting.
Embedding:
After processing the next step is embedding. This is done using flat molds.
7) Polymerization
Next is polymerization step in which the resin is allowed to set overnight at a
temperature of 60 degree in an oven.

99. 8) Sectioning
The specimen must be cut into very thin sections for electron microscopy so
that the electrons are semitransparent to electrons. These sections are cut on
an ultramicrotome which is a device with a glass or diamond knife. For best
resolution the sections must be 30 to 60 nm.
OTHER METHODS FOR contrast creation for electron
microscopy.
In negative stain microscopy, the electron beam primarily interacts with the
stain. The stains used most frequently are 1 to 2 percent solutions of uranyl
acetate or phosphor tungstic acid ammonium molybdate methyl amene
tungstate and uranyl formate. When stain is added to a sample, the stain
surrounds the sample but is excluded from the volume occupied by the sample;
hence the use of the term 'negative stain‘.
The background is stained, leaving the actual specimen untouched, and thus
visible. This contrasts with 'positive staining', in which the actual specimen is
stained.

100. a well-stained sample is uniformly covered by the stain. When the electron beam
(arrows) passes through the sample, it will be deflected by its interactions with the
sample and stain. Since the protein sample excludes stain, the deflection of the electron
beam through protein (center arrow) is less than that through stain rich regions (outer
arrows). Electrons that are highly deflected by the stain are then filtered out by the
objective aperture located below the sample. Depending on the size of the aperture, the
quantity of electrons that are culled out will vary and determine the contrast and
resolution of the image.

101. Metal shadowing is another widely used technique to increase the contrast of a certain
specimen by depositing thin coat of metal onto the surface . MS spraying on a coat of a
heavy metal (such as platinum) at an oblique angle, it is possible to examine the surface
of a specimen. This techniques creates a three dimensional view of the specimen with
the specmen itself appearing darker. It cast shadows . This whole thing is performed in
a vacuum evaporator containing one or more pairs of electrodes. The metal to be
evaporated is held between a pair of electrodes and heated by an electric current. This
whole thing is done at an angle of 45 degrees or less. Your metal is placed around 45
degree or less to the sample section. The metal which could be platinum or sometimes
tungsten together with carbon is deposited on surfaces. These are facing the filament
that is which are towards the filament side. While the opposite surfaces in specimen and
grid will remain unquoted and incapable of scattering electrons. What you have is the
sides which are quoted will scatter electrons and will not be seen in the florescent
screen. The sides which are not quoted that are shadows will be passed electrons, will
be passed from there and the florescent screen will be lighted up there

102. Now, the areas in shadow appear bright on viewing screen. While, the metal coated
areas will appear dark. This technique provides an excellent contrast for isolated
materials and produces a 3 dimensional effect. In many metal shadowing technique
there is another technique which could be used which is rotary metal shadowing. In this
rotary metal shadowing a thin film of metal is deposited on a specimen at very low
angles. If you could see in metal shadowing it was 45 degree or less.
Here, 8 to 10 degree, at 8 or 10 degree. The metal is deposited while the sample is being
rotated. The metal atoms deposit on structures projecting above the specimen surface.
They will be quoted so molecular size shapes and interactions can be determined by use
of this technique. Purified samples of nucleic acids proteins, protein interacting with
nucleic acids etcetera can be seen in here on appropriate substrate

105. Scanning electron microscopy is a type of technique that produces the image of a
specimen by scanning it. It is done with a focussed beam of electrons across a
rectangular area of the specimen or in a rester scan pattern. The electron beam interacts
with a specimen either emission of low energy or secondary electrons or back scattered
high energy electrons. It will provide signals carrying information about the specimen
surface topography and composition. One is that the cathode is maintained at a negative
potential difference to anode around 2 to 30 kilovolt rather than 10 to 100. That is one
difference. Then there are 3 condenser lenses are used in SEM and the specimen is not
placed. Rather a specimen is preplaced at the bottom rather than at in the middle of the
column. The third condenser lens is used to reduce further the size of the beam or probe
which is around 5 nanometre in diameter. As this lens is used to focus beam on
specimen and thus focus image it is often called as the objective lens. There is nothing

106. TEM has much higher resolution than SEM.
• SEM allows for large amount of sample to be analysed at a time whereas with
TEM only small amount of sample can be analysed at a time.
In TEM, pictures are shown on fluorescent screens whereas in SEM, picture is
shown on monitor.
• SEM also provides a 3-dimensional image while TEM provides a 2-dimensional
picture
The method used in SEM is based on scattered electrons while TEM is based on
transmitted electrons. The scattered electrons in SEM are classified as backscattered
or secondary electrons. However, there is no other classification of electrons in
TEM.
The scattered electrons in SEM produced the image of the sample after the
microscope collects and counts the scattered electrons. In TEM, electrons are
directly pointed toward the sample. The electrons that pass through the sample are
the parts that are illuminated in the image.

110. Sample preparation for SEM: A specimen to be analyzed by electron microscopy
has to be dry which most biological samples are not. As dehydration might
lead to structural changes, the specimens are first fixed to preserve their
structural features. Fixation is the first step and can be achieved using
chemical methods such as fixation with glutaraldehyde or physical methods
such as cryofixation in liquid nitrogen. The fixed specimens are then
dehydrated usually by exposing them to an increasing gradient of ethanol (up
to 100%). The specimens are then dried using critical point method. The dried
specimens are then coated with a conducting material usually gold to make
the surface conducting and cause it emit more secondary electrons. A SEM
image of human erythrocytes coated with gold is shown in figure 18.4

111. sample. Normally, the probe is a sharp tip, which is a 3-6 um tall
pyramid with 15-40nm end radius (Figure 1). Though the lateral
resolution of AFM is low (~30nm) due to the convolution, the
vertical resolution can be up to 0.1nm.

113. silicon or silicon nitride
optical interferometry, capacitive
sensing or piezoresistive AFM
cantilevers. These cantilevers are
fabricated with piezoresistive
elements that act as a strain gauge.
Using a Wheatstone bridge, strain
in the AFM cantilever due to
deflection can be measured, but
this method is not as sensitive as
laser deflection or interferometry.
CONTACT TIP CAUSE
DAMAGE, tip or sample is
mounted on a 'tripod' of three
piezo crystals, with each
responsible for scanning in the x,y
and z directions.

114. tube scanner can move the sample in the x, y, and z directions using a single
tube piezo. An advantage of the tube scanner is better vibrational isolation,
resulting from the higher resonant frequency of the single-crystal
construction in combination with a low resonant frequency isolation stage.
Depending on the interaction under investigation, the surface of the tip of the
AFM probe needs to be modified with a coating. Among the coatings used
are gold - for covalent bonding of biological molecules and the detection of
their interaction with a surface,diamond for increased wear resistance and
magnetic coatings for detecting the magnetic properties of the investigated
surface.
The surface of the cantilevers can also be modified. These coatings are mostly
applied in order to increase the reflectance of the cantilever and to improve
the deflection signal

115. AFM cantilever deflection measurement
1. Beam deflection measurement
In this method, laser light from a solid-state diode
is reflected off the back of the cantilever and
collected by a position-sensitive detector
(PSD) consisting of two closely spaced
photodiodes whose output signal is collected
by a differential amplifier. Angular
displacement of the cantilever results in one
photodiode collecting more light than the
other photodiode, producing an output signal
(the difference between the photodiode
signals normalized by their sum), which is
proportional to the deflection of the cantilever.

116. 2. Capacitive detection — Metal coated cantilevers can form a capacitor with another
contact located behind the cantilever. Deflection changes the distance between the
contacts and can be measured as a change in capacitance.
3. Piezoelectric detection — Cantilevers made from quartz(such as the qPlus
configuration), or other piezoelectric materials can directly detect deflection as an
electrical signal. Cantilever oscillations down to 10pm have been detected with this
method.
4. Laser Doppler vibrometry — A laser Doppler vibrometer can be used to produce
very accurate deflection measurements for an oscillating cantilever (thus is only
used in non-contact mode). This method is expensive and is only used by relatively
few groups.

117. In contact mode, the tip is "dragged" across the surface of the sample and the
contours of the surface are measured either using the deflection of the
cantilever directly or, more commonly, using the feedback signal required to
keep the cantilever at a constant position
In tapping mode, the cantilever is driven to oscillate up and down at near its
resonance frequency by a small piezoelectric element mounted in the AFM tip
holder similar to non-contact mode. However, the amplitude of this oscillation
is greater than 10 nm, typically 100 to 200 nm. The interaction of forces acting
on the cantilever when the tip comes close to the surface, Van der Waals forces,
dipole-dipole interactions, electrostatic forces, etc. cause the amplitude of this
oscillation to decrease as the tip gets closer to the sample.
NON CONTACT MODE, the tip of the cantilever does not contact the sample
surface. The cantilever is instead oscillated at either its resonant frequency
(frequency modulation) or just above (amplitude modulation) where the
amplitude of oscillation is typically a few nanometers (<10 nm) down to a few
picometers.The van der Waals forces, which are strongest from 1 nm to 10 nm
above the surface, or any other long-range force that extends above the surface
acts to decrease the resonance frequency of the cantilever

118. Scanning Tunneling Microscopy
the voltage bias will cause electrons to tunnel between the tip and sample,
creating a current that can be measured. Once tunneling is established, the tip's
bias and position with respect to the sample can be varied.
If the tip is moved across the sample in the x-y plane, the changes in surface
height and density of states cause changes in current. These changes are mapped
in images.
STM tips are usually made from tungsten metal or a platinum-iridium alloy
where at the very end of the tip (called apex) there is one atom of the material.
photon scanning microscopy (PSTM), which uses an optical tip to tunnel
photons;scanning tunneling potentiometry (STP), which measures electric
potential across a surface; spin polarized scanning tunneling microscopy (SPSTM),
which uses a ferromagnetic tip to tunnel spin-polarized electrons into a magnetic
sample,

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