Microscopy

Important
Resources
• Lectures were prepared from
1. [Link]
2. [Link]
Contents
• Microscopy
• Optical microscopy
• Fluorescence and Confocal Microscopy
• Scanning electron microscopy
• Atomic force microscopy
Evolving equipment

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cts/tem/titan/

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Typical images from
microscopy
https://
[Link]/photos/
zeissmicro/8695004301
[Link]
Scanning
_electron_microscope
Sun et al., Phys. Rev. Lett. 101,
156101 (2008)

Confocal microscopy: Scanning electron microscopy: TEM: Extrusion of a gold crystal

Neuronal cultures stained Pollen grains from a punctured graphitic shell
for showing axonal under electron irradiation.
transport pathway used
by Bornavirus for its
efficient spread in
neurons.

[Link]
ence/
Bacteria/[Link]

Atomic force microscopy: E. coli bacteria grown

on a glass coverslip, imaged in air. 5µm scan.
Optical microscopy

[Link]
ml

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Microstructural
characterisation
• Can establish a direct causal relationship
• Structure-property relationship
• Structure-sensitive and structure insensitive
properties
o Structure insensitive: elastic constants (E, G, K,υ), thermal
constants, specific gravity
o Structure sensitive: Yield strength
Characteristic Information
• Morphology, size
• Transparency/opacity
• Color (Reflected and transmitted)
• Dispersion of Refractive Indices
• Refractive Indices
• Pleochroism
• Crystal system
• Birefringence
• Extinction Angle
• Fluorescence
• Melting Point, polymorphism, eutectics
Microstructural
characterisation
• Qualitative analysis
o Phase identification
o Microstructurl morphology
o Microchemical identification

• Quantitative
analysis
o Applied crystallography
o Stereology
o Microanalysis
Background
• Optics
• Lenses and image
formation
• Numerical aperture
• Resolution
• Types of optical
microscopy
o Bright field Vs. Dark field
o Polarised
o Interferometry
o Phase contrast
Background: Lenses and image
formation

• Remember: Using a magnifying glass as a burning glass

• The sun rays are concentrated at one point (focal point) and
the distance between the center of the lens to the focal point is
“focal length”.
Background: Lenses and image
formation

• Focal length varies on the curvature of

the lens. (smaller radius of curvature =
shorter focal length.
• Lens diameter is generally expressed in
relation to the focal length. In the field of
microscopy this parameter is called
aperture (also: numerical aperture NA).
• NA is the measure of its ability to
gather light and to resolve while
working at a fixed object/specimen
distance.
Background: Image
formation
The object has an infinite
distance to the lens:
Parallel rays from the
object to the lens are
assumed and redirected in
the lens to meet in the
focal point.

Object is situated at a
relatively large distance
(e.g. 100 x FL): An
image: Is produced that is
smaller than the object
(approx. one 100th of the
size of the original object).

Object is located at a 2FL in

front of the lens:
Image is created of the object
which is the same size as the
Background: Image
formation

Object is situated in
front of the focal point
but within the range of
twice the focal length:
An image is generated
which is larger than the
object.

Object is located at the

focal point of the lens:
A virtual image, is
generated. The rays will
leave the lens in a parallel
manner. No image can be
found unless we use
another optical system
Background: Image
formation

1)Place the object between F and 2F, which results in a magnified

image real image. This microscope lens is called the objective.
2)A second lens picks up this image exactly in its front focal point and
generates a beam of parallel rays, (not a real image). This optical
element is called eyepiece. The human eye is able to handle this parallel
beam and generates an image onto its retina.
Refractive index
• Calculated from the ratio of the
speed of light in a vacuum to that in
a second medium of greater density.
• Wavefront is refracted upon entering
the second medium if the incident
wave is oblique to the surface.
• The incident angle is related to
the refraction angle by Snell's law

• n1 > n2 the angle of refraction is always larger than the

angle of incidence.
• n2 > n1 the angle of refraction is always smaller than the
angle of incidence.
Refractive index
• Refractive index is an important variable
in calculating NA
NA = n Sinθ

• Increasing the refractive index by replacing the

imaging medium from air (refractive index =
1.000) with a low-dispersion oil (refractive index =
1.515)
Spherical Aberrations
• Causes the specimen
image to appear hazy or
blurred and slightly out of
focus.
• Light waves passing through
the periphery of an
uncorrected convex lens
are not brought into focus
with those passing through
the center.
• This produces a variety of
different focal points along
the optical axis.
 Spherical Aberrations

• Waves passing near the center: slight refraction: waves passing

near the periphery: greater refraction.
• Peripheral waves come to a shorter focus than do rays traveling
through the central or axial area.
• The best focus will be somewhere between the focal planes of the
peripheral and axial rays, an area known as the disc of least
confusion
Chromatic Aberrations
• Refractive index of the glass varies the
wavelength, hence the focal length is
only constant for a monochromatic
light .
• Visible light is not brought to a
single focus, blue light (short
wavelength) focussed at a greater
distance than red light.
• Inability of a lens to not bring a beam
of white light into sharp focus, but
rather to a region of finite zone is
referred as Chromatic aberration.

• Achromats: fully corrected for chromatic aberration at two wavelength

(blue and red) and spherical aberration for green.
• Plan achromats: not only central but periphery remains focussed
(image recording). Can be fully corrected for all wavelength
 Numerical
Aperture
• A measure of lenses ability to gather light and
resolve fine specimen detail at a fixed object
distance.
• A longitudinal slice of this cone of light shows the
angular aperture, a value that is determined by
the focal length of the objective
• The angle µ is one-half the angular aperture(A) :
NA = n Sin μ
• n is the refractive index of the imaging
medium between the front lens of the
objective and the specimen cover glass (1.00
to 1.51).

[Link]
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 Resolving power
• First conventional microscope developed by Antoni
van Leeuwenhoek (x200)
• Focus on improving the magnification and
quality of images
• Ernst Abbe (1873): relationship between wavelength
of light and resolving power of microscope

0.612
𝑑 𝜆
𝑛𝑠𝑖𝑛
=
𝛼
d = resolving power of a microscope (resolution)
λ = wavelength of the illuminant
n = index of refraction of the medium between the specimen and the objective lens
α = half angle in radian of the cone of light from the specimen plane accepted by the objective
Optical microscope: resolution

• Light from the specimen  objective reconstituted as an image 

points appear as small Airy patterns.
• Caused by diffraction or scattering  light passes through the
minute parts and spaces in the specimen.
• The limit up to which two small objects are still seen as separate entities
is used as a measure of the resolving power of a microscope.

dxy  
2(nSin)
2
d z (nSin)2
Resolutio
n
• Shortest distance
between two points
on a specimen that
can still be
distinguished
• The limit of
resolution of a
microscope
objective refers to its
ability to distinguish
between two closely
spaced Airy disks in
the diffraction
pattern

[Link]
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formulas/[Link]
Resolution

• Note: at high magnification, an image may appear un sharp

but still be resolved to the maximum ability of the objective.
• NA influences the resolving power of an objective, but the
total resolution of the entire microscope optical train is
also dependent upon the numerical aperture of the
substage condenser.
• The higher the numerical aperture of the total system,
the better the resolution.

• Resolution = λ / 2NA
• Resolution = 0.61 λ / NA
• Resolution = 1.22 λ / (NAobj + NAcond)
Optical microscope: resolution

• When two image points are far away  easy to recognize

• Distance is reduced  a limit point is reached when the principal
maximum of the second Airy disk coincides with the first
minimum of the first Airy disk.
• 2 brightness maxima that are separated by a valley. The
intensity in the valley is reduced by approximately 20 percent
compared with the two maxima.
• This is just sufficient for the human eye to see two separate
points, a limit that is referred to as the Rayleigh criterion.
Resolution
• These equations are based upon a number of factors to
account for the behaviour of objectives and condensers, and
should not be considered an absolute value of any one general
physical law.
• Low specimen contrast and improper illumination may serve to
lower resolution
• The real-world maximum value of r (about 0.25 µm @
wavelength of 550 nm) and a numerical aperture of 1.35 to
1.40 are not realized in practice.
Resolution

@ 550
nm
Optical limitation
• Abbe’s formula highlights the
restriction of using light
microscope for higher
magnification
• Optical limitation:
o Best possible NA could not objects
smaller than 0.5λ.
o Advanced lens manufacturing techniques
led to resolving power of ~0.250
μm
o New light source was required to see
smaller objects.
 Emergence of electron
microscopy
• End of 19 century JJ Thompson demonstrated
th

o Origin of electron sources

o Possibility of forming electron beam using electrical and magnetic fields

• De Broglie (1924): relationship between wavelength

and moving electron

ℎ
𝜆
𝑚
=
𝑣
λ = wavelength of the moving particle
h = Planck’s constant
m = mass of electron
v = velocity of the electron
Resolution

• The wavelength of light is

an important factor in the
resolution of a microscope.
• Shorter wavelengths yield
higher resolution (lower values
for r) and visa versa.
• Under most circumstances,
microscopists use white light
generated by a tungsten-
halogen bulb to illuminate the
specimen.

@ NA =
0.95
Depth of Field
• Emphasis is placed on point-
to- point lateral resolution in
the plane perpendicular to
the optical axis.
• Axial (or longitudinal) resolving
power of an objective, which is
measured parallel to the
optical axis.
• Referred as depth of field.
Depth of Field

!!!
!
dtot = Depth of field,
λ = wavelength of illuminating
light, n = refractive index of
the medium NA = Objective
numerical aperture.
e = smallest distance that can be resolved by a detector that is placed in the
image plane of the microscope objective, whose lateral magnification is M.
Depth of Field

• Notice that the diffraction-limited depth of field (the first

term in the equation) shrinks inversely with the square of the
numerical aperture, while the lateral limit of resolution is
reduced in a manner that is inversely proportional to the first
power of the numerical aperture.
• Thus, the axial resolution and thickness of optical sections that
can be attained are affected by the system numerical
aperture much more so than is the lateral resolution of the
microscope.
Components of a microscope
• Eyepiece: brings the image into
focus for the eye (2x-10x)
• Turret:
• Objective lens: collects light from
the sample, characterised by
magnification (5x-100x) and NA (0.14-
0.7)
• Focus knob:
• Stage:
• Light source:
• Condenser: focuses light from the
illumination onto the sample
• Mechanical stage
 Bright field Vs. Dark field
• Brightfield microscopy: Uses light to
illuminate the specimen  light is
gathered in the condenser  shaped
into a cone and is focused on the plane
of the specimen.
• In order to view a specimen under a
brightfield microscope, the light rays that
pass through it must be changed enough
in order to interfere with each other (or
contrast) and therefore, build an image.

• Darkfield microscopy: Condenser forms

a hollow cone of light.
• Objective lens sits in the dark hollow of this
cone and light travels around the objective
lens, but does not enter the cone shaped
area.
• The entire field of view appears dark when
there is no sample on the microscope
stage. Presence of a sample appears bright
against a dark background.
Dark field
microsco
py
The light at the apex
of the cone is focused
at the plane of the
specimen; as this light
moves past the
specimen plane it
spreads again into a
hollow cone.

The objective lens sits

in the dark hollow of
this cone; although
the light travels
around and past the
Dark field
microscopy
• Entire field appears dark when there is no sample on the microscope stage
• The oblique rays cross the specimen (unstained, non-light absorbing) and
are diffracted, reflected, and/or refracted by optical discontinuities (such as
the cell membrane, nucleus, and internal organelles) allowing these faint
rays to enter the objective. Specimen appears bright on an otherwise black
background.
• In terms of Fourier optics, darkfield illumination removes the 0th order
(unscattered light) from the diffraction pattern formed at the rear focal plane
of the objective.
• This results in an image formed exclusively from higher order
diffraction intensities scattered by the specimen.
• Care should be taken in preparing specimens as features above and below
the plane of focus can also scatter light and compromise image quality (for
example, dust, fingerprints). In general, thin specimens are better because
the possibility of diffraction artifacts is reduced.
Phase contrast
microscopy
• Need for Phase Contrast Microscopy
o Biological samples
o Live imaging
o Avoid staining of samples
o Dynamics of biological processes can be observed and recorded.

• PCM translates minute variations in phase into corresponding

changes in amplitude, which can be visualized as differences
in image contrast.

• Diffraction from the specimen must occur for the phase

contrast microscope to work.
 Phase contrast microscopy
• Phase Objects:
Unstained
specimens that do
not absorb light
because they
slightly alter the
phase of the light
diffracted by the
specimen.

Typical Values:
• Air: RI ≈ 1.00
• Water: RI ≈ 1.33
• Living cells: RI ≈ 1.36-1.38
• Glass: RI ≈ 1.52
• Immersion oil: RI ≈ 1.52
Case (a): Undisturbed Light Wave
Refractive Index: RI = n (same as the surrounding medium)
Effect on Light: No change in amplitude or phase
Wave Behavior: Light passes through completely unaffected
Visibility: Nothing to see - wave continues unchanged
Real Example: Perfect vacuum or perfectly matched optical
medium

Case (b): Amplitude Specimen

Refractive Index: RI = n (same as medium)
Key Characteristic: Absorbs or scatters light
Effect: Reduces wave amplitude while maintaining phase
Visibility: Easily visible - appears darker than background
Real Examples: Stained cells, pigmented materials, metals
Why It's Visible: Creates contrast through amplitude differences
that our eyes can detect
Case (c): Phase Specimen (90° shift)

Refractive Index: RI > n (higher than medium)

Phase Shift: φ = 90° = λ/4 (quarter wavelength)
Amplitude: Unchanged - same brightness
Visibility: Completely invisible in normal microscopy
Real Examples: Living cells, glass, transparent polymers, water droplets
The Problem: Pure phase objects are invisible because human vision
cannot detect phase differences

Case (d): Combined Amplitude and Phase Specimen (180° shift)

Refractive Index: RI > n (significantly higher than medium)
Phase Shift: φ = 180° = λ/2 (half wavelength)
Effect: Major phase reversal, may also affect amplitude
Visibility: Visible due to extreme phase difference
Real Examples: Thick glass objects, dense biological structures
Interaction of Light Waves with
Phase Specimens
• An incident wavefront divides into two components upon
passing through a phase specimen.
o Un-diffracted or an un-deviated (zeroth-order) planar wavefront (S-wave)
o Deviated or diffracted spherical wavefront (D-wave) is also produced

• After leaving the specimen plane, D and S-waves enter the

objective front lens element and are subsequently focused to
combine through interference and produce a resultant
particle wave (P-wave).
P=D+S
OPD is the product of two terms: the thickness (t) and the difference
in refractive index (n).
- OPD can often be quite large even though the thickness of the
object is quite thin.
- When the refractive indices of the specimen and the surrounding
medium are equal, OPD is zero even for thick specimens.
Phase contrast microscopy
• We can only detect intensity and color differences, the
phase changes due to objects must be converted to
intensity differences.
• Works on the phase differences between objects in a
specimen and in the surrounding medium.
• This method utilizes the phase difference introduced
between
scattered and unscattered rays
Polarised light microscopy

• Contrast-enhancing technique that improves the quality of the

image obtained with bi refringent materials
• High degree of sensitivity and can be used for both
quantitative and qualitative studies (for anisotropic specimens)

Glass ceramic Microscope image made Microscope image made

transmission microscope with polarised light with polarised light and
image made with full wave plate
unpolarised light

[Link]
[Link]
Polarised light
• Illumination produces light waves whose electric field vectors vibrate in
all planes that are perpendicular with respect to the direction of
propagation.

• Light passing through a polariser is modified so that the light

transmitted vibrates in only one direction - this is polarised light. This
direction is parallel to the planes of the polarising material.
• Our eyes lacks the ability to distinguish between randomly oriented
and polarized light, and plane-polarized light can only be detected
through an intensity or color effect.
• Vertical polarizing direction of the first polarizer will pass only
the waves having vertical electric field vectors.
• This wave is subsequently blocked by the second
polarizer, (Horizontal polarizing direction).
• Crossed polarization: Using two polarizers oriented at right angles with
respect to each (fundamental to the concept of polarized light
microscopy).
Polarised light: isotropic samples

• For an isotropic material, for example a cubic crystal, or an

amorphous material, light vibrates equally easily in all directions.
These materials do not affect polarised light. If an isotropic material is
examined between crossed polars, extinction occurs, and the image
appears dark.
Polarised light: Anisotropic samples
• Anisotropic crystals refract
light by splitting the
incoming rays into two
components that take
different paths and emerge
as separate light rays.
• Occurs because of the
arrangement of atoms in the
crystalline lattice. Because
the precise geometrical
ordering of the atoms is not
symmetrical with respect to
the crystalline axes,
• Light rays passing through the
crystal can experience
different refractive indices,
depending upon the
direction of propagation.
Polarised light: Anisotropic samples

• Ordinary ray (O-ray)and an extraordinary ray

(E-ray). These rays vibrate in fixed planes at
right angles to each other.
• O-rays travel with the same velocity in
all directions and the E-rays travels with
a direction-dependent velocity.
• When the O-rays and E-rays emerge
from the crystal the phase of one set of
rays is retarded with respect to the
other.
• This retardation depends on the difference
in velocities of the two rays and the
thickness of the specimen.
• Such a crystal is said to exhibit birefringence
.
Polarised light: Anisotropic samples
Polarised light: Anisotropic samples
• The two transmitted rays interfere, and the effect produced depends
on the phase difference between the O-rays and E-rays and their
amplitudes at the analyser.
• Extinction occurs when the optical path difference between the O-
ray and the E-ray is a whole wavelength.
Polarised light: Anisotropic samples

• When white light is used, anisotropic crystals may appear

coloured when viewed between crossed polars, due to
interference effects between rays emerging from the analyser.
• Certain wavelengths, and therefore certain colours, will be
extinguished due to destructive interference.
• Colours seen depend on the birefringence of the crystal, its
thickness, and the orientation of the section relative to the
optic axis.
• The relation between retardation, birefringence and thickness
can be seen on a Michel-Levy chart.
Polarised light microscopy:
Applications

• Monosodium urate (Gout) crystals grow in elongated prisms that have a negative optical
sign of birefringence, which generates a yellow interference color when the long axis of
the crystal is oriented parallel to the slow axis of the first order retardation plate.
• Rotating the crystals through 90 degrees changes the interference color to blue.
• In contrast, pseudo-gout pyrophosphate crystals, have similar elongated growth,
exhibit a blue interference color when oriented parallel to the slow axis of the
retardation plate and a yellow color when perpendicular.
Polarised light microscopy:
Applications

• Nylon Fibers - Observations under plane-polarized light reveal

refractive index differences between a nylon fiber and the mounting
medium, and the presence of opacifying titanium dioxide particles.
• The image under crossed polarizers reveals second and third order
polarization colors and their distribution across the fibers indicate that
this is a cylindrical fiber.
• The use of the quartz wedge enables the determination of optical
path differences for birefringence measurements.
Polarised light microscopy:
Applications

• Upon nucleation, synthetic polymer chains often arrange

themselves tangentially and the solidified regions grow radially.
• These can be seen in crossed polarized illumination as white
regions, termed spherulites, with the distinct black extinction
crosses.
• When these spherulites impinge, their boundaries become polygonal.
This can be clearly seen in crossed polarizers but not under plane-
polarized light.
• The banding occurring in these spherulites indicates slow cooling of
the melt allowing the polymer chains to grow out in spirals.
Polarised light microscopy:
Applications

• Examination of geological thin sections using polarizing microscopy can reveal about how
the rock was formed.
• Phyllite, a metamorphic rock, clearly shows the alignment of crystals under the effects of
heat and stress.
• The crossed polarizers image reveals that there are several minerals present, including
quartz in gray and whites and micas in higher order colors.