The Principles of Microscopy

1. The Light Microscope

2. Microscope
 "Microscope" was first coined by members of the first
"Academia dei Lincei" a scientific society which
included Galileo
 “An instrument that produces an enlarged image of
an object”
Light microscope: uses a “light source” to
illuminate the specimen
 Simple or Compound
 Upright or Inverted
 Phase contrast or Dark field or Interferance
 Köhler Illumination
 Fluorescence Illumination

3. Earliest Microscopes
 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscopes
 1660 - Marcello Malpighi, considered the father
embryology and early histology, was one of the first great
microscopists - observed capillaries in 1660
 1665 - Robert Hooke (1635-1703)- book Micrographia,
published in 1665, devised the compound microscope most
famous microscopical observation was his study of thin slices
of cork. He wrote:
“. . . I could exceedingly plainly perceive it to be all perforated
and porous. . . these pores, or cells, . . . were indeed the first
microscopical pores I ever saw, and perhaps, that were ever
seen, for I had not met with any Writer or Person, that had
made any mention of them before this.”

4.  1673 - Antioni van Leeuwenhoek (1632-1723) Delft,
Holland, worked as a draper (a fabric merchant); he
is also known to have worked as a surveyor, a wine
assayer, and as a minor city official
 Created a “simple” microscope that could magnify to about 275x,
and published drawings of microorganisms in 1683
 His simple microscope could reach magnifications of over 200x
with simple ground lenses
 However compound microscopes were mostly of poor quality and
could only magnify up to 20-30 times
 Discovered bacteria, free-living and parasitic microscopic
protists, sperm cells, blood cells, microscopic nematodes
 In 1673, Leeuwenhoek began writing letters to the Royal Society
of London - published in Philosophical Transactions of the
Royal Society
 In 1680 he was elected a full member of the Royal Society, joining
Robert Hooke, Henry Oldenburg, Robert Boyle, Christopher
Wren

5. Secondary Microscope
 1740-1772: George Adams Sr. made many
microscopes but he was predominantly just a good
manufacturer not inventor
 ~1730: a barrister names Chester More Hall
observed that flint glass (newly made glass)
dispersed colors much more than “crown glass”
(older glass)
 He designed a system that used a concave lens next to
a convex lens which could realign all the colors
 This was the first achromatic lens.
 George Bass was the lens-maker who actually made
the lenses, but he did not divulge the secret until
over 20 years later to John Dolland who copied the
idea in 1759 and patented the achromatic lens.

6.  1827: Giovanni Battista Amici,
built high quality microscopes
and introduced the first
matched achromatic microscope
in 1827
 He had previously (1813)
designed “reflecting
microscopes” using curved
mirrors rather than lenses
 He recognized the importance
of cover-slip thickness and
developed the concept of “water
immersion”
© J.Paul Robinson

7. Limitations faced by cell biologists
 Exceedingly small dimensions of the cells and cell
organelles
 Transparent nature of cell and its components
 Can be overcome by
 Increasing the resolving power of microscope
 Increasing the contrast of cellular structures to
counteract the transparency of the cell

8. Abbe’s Law
 Ernst Abbe together with Carl Zeiss published a paper in 1877
defining the physical laws that determined resolving distance
of an objective which is now known as Abbe’s Law
“minimum resolving distance (d) is related to the
wavelength of light (λ) divided by the Numeric Aperture,
which is proportional to the angle of the light cone (θ)
formed by a point on the object, to the objective”
 Abbe and Zeiss developed oil immersion systems by making
oils that matched the refractive index of glass
 They were able to make the a Numeric Aperture (N.A.) to the
maximum of 1.4 allowing light microscopes to resolve two points
distanced only 0.2 microns apart (the theoretical maximum
resolution of visible light microscopes).

9. Resolution
 Limit of resolution: The minimum distance between
two points which allows their discrimination as two
separate points:
where:
 0.61 is a geometrical term, based on the average 20-20
eye,
 λ = wavelength of illumination,
 N.A. = Numerical Aperture: a measure of the light
gathering capabilities of an objective lens.
N.A. = n sin α
Where:
 n = index of refraction of medium,
 α = semi-angle of aperture subtended by the lens
 Sin α cannot exceed 1
 RI for most optical material does not exceed 1.6
Maximal NA of lenses (using oil immersion) is about 1.4

10. Factors Affecting Resolution
 Resolution improves if the
 wavelength (λ) of illumination is shorter
 R.I (n) is larger
 α is larger
 The eye is more sensitive to blue than violet

11. Magnification
 Ability of a microscope to produce an enlarged image
 An object can be focussed generally no closer than 250
mm from the eye (depending upon how old you are!)
 This is considered to be the normal viewing
distance for 1x magnification
 Young people may be able to focus as close as 125 mm
so they can magnify as much as 2x because the image
covers a larger part of the retina - that is it is
“magnified” at the place where the image is formed

12. Magnification
 The overall magnification is given as the product
of the magnification of lenses and the distance
over which the image is projected:
where:
 D = projection (tube) length (usually = 250 mm);
 M1, M2 = magnification of objective and ocular.
 250 mm = minimum distance of distinct vision for
20/20 eyes

13. Depth of Focus
 DOF or the vertical resolution: This is the ability
to produce a sharp image from a non-flat surface
 Depth of Focus is increased by inserting the objective
aperture (an iris that cuts down on light entering the
objective lens)
 However, this decreases resolution

14. Noteworthy…
1. All microscopes are similar in the way lenses work and
they all suffer from the same limitations and problems.
2. Magnification is a function of the number of lenses and
their individual magnifications.
3. Resolution is a function of the ability of a lens to gather
light.
4. Apertures can be used to affect resolution and depth of
field if you know how they affect the light that enters
the lens.
5. Simple microscopes could attain around 2 micron
resolution, while the best compound microscopes were
limited to around 5 microns because of chromatic
aberration.

15. Inverted Microscope

16. Specialized LM Techniques
 Bright & Dark field Microscopy
 Phase Contrast Microscopy & Differential
Interference Contrast Microscopy: Convert phase
differences to amplitude differences; Enhancement of
Contrast
 Fluorescence Microscopy: mainly organic materials
 Confocal Scanning Optical Microscopy (new)
 Three-Dimensional Optical Microscopy: Inspects and
measures sub-micrometer features in semiconductors and
other materials
 Hot- and Cold-stage Microscopy: Examines melting,
freezing points and eutectics, polymorphs, twin and
domain dynamics, phase diagram
 In situ microscopy E-field, stress, etc.
 Special environmental stages-vacuum or gases

17. Bright Field Microscopy
 The condenser is used to focus light on the specimen through an
opening in the stage
 After passing through the specimen, the light is displayed to the
eye with an apparent field that is much larger than the area
illuminated
 Typically used on thinly sectioned materials
 Drawback: Bright field illumination does not reveal differences in
brightness between structural details - i.e. no contrast
 Structural details emerge via phase differences and by
staining of components
 The edge effects (diffraction, refraction, reflection) produce
contrast and detail
Incandescent
Source
Condenser Specimen
Objective
Lens
Occular or
Eye piece
Eye

18. Contrast
 Contrast is defined as the difference in light
intensity between the specimen and the adjacent
background relative to the overall background
intensity
Image contrast, C is defined by:

19. Dark Field Microscopy or
Ultramicroscopy
 Light is scattered at the boundaries between
regions of different refractive indices
 An opaque disc is placed underneath the condenser
lens, so that only light that is scattered by objects on
the slide can reach the eye
 Ordinary condenser is replaced by one that
illuminates object obliquely (Dark-field condenser)
 No direct light enters the objective
 Instead of coming up through the specimen, the light is
reflected by particles on the slide
 Object appears bright because of the scattered light
 Everything is visible regardless of color, usually bright
white against a dark background
 Smaller objects can be detected but resolution is poor

20. OM images of the green alga Micrasterias

21. Phase Contrast Microscopy
 Although biological structures are highly
transparent to visible light, they cause phase
changes or retardations in the transmitted
radiations
 As the light wave impinges on a NON-ABSORBENT,
TRANSPARENT, REFRACTIVE material that has RI
different from that of the medium, amplitude is not
affected but velocity changes
 If the RI of the material is higher than the medium,
velocity is RETARDED
 As the wave emerges from the medium, velocity is
restored but retardation is maintained
 If material is ABSORBENT, amplitude is reduced

23. Principle
 In PC Microscope, small differences are intensified
 Lateral light passing through the objective is advanced or
retarded by 1/4th wavelength w.r.t. the central light
 Annular phase plate introduces a variation of ¼
wavelength in the back focal plane of the objective
 The phase effect results from the interference
between the direct geometric image produced by the
central part of the objective and the lateral image
that has been advanced or retarded by ½ wavelength
 Bright image: Negative contrast: the 2 sets of rays are
added; object appears brighter than the surrounding
 Dark image: Positive contrast: the 2 sets of rays are
subtracted making the image darker than the
surrounding

24. Uses…
 Minute phase changes in the object are amplified and
translated into amplitude changes
 But reveals only sharp differences
 Transparent object appears in various shades of grey
 PCM is used routinely to observe living cells and
tissues
 Particularly useful for in vitro studies of cell cycle
progression, etc.

25. Interference Microscopy (DIC)
 Similar principles as the PCM but gives quantitative data
 Used to detect small continuous changes in RI
 The variations of phase can be can be transformed into
vivid color changes
 Nomarski Interference Microscope: a special variation in
which the image obtained gives a “relief effect”
 Particularly useful for study of mitosis in live cells in vitro

26. Contrast and Illumination
 Brightness-contrast arises from different degrees of
absorption at different points in the specimen
 Color-contrast can also arise from absorption when the
degree of absorption depends on the wavelength and varies
from point to point in the specimen
 Phase contrast arises from a shift in the phase of the light as
a result of interaction with the specimen
 Polarization-dependent phase contrast arises when the
phase shift depends on the plane of polarization of the
incident light.
 Fluorescence contrast arises when the incident light is
absorbed and partially reemitted at a different wavelength.

28. Modern Microscopes
 Early 20th Century Professor Köhler developed the
method of illumination still called “Köhler
Illumination”
 Köhler recognized that using shorter wavelength light
(UV) could improve resolution
 Köhler illumination creates an evenly illuminated field
of view while illuminating the specimen with a very
wide cone of light
 Two conjugate image planes are formed – one contains
an image of the specimen and the other the filament
from the light

29. Electron microscope
 An electron microscope is a type of microscope that
uses a particle beam of electrons to illuminate the
specimen and produce a magnified image.
 Electron microscopes (EM) have a greater resolving power
than a light-powered optical microscope
 Like a beam of light, a stream of electrons also have
corpuscular and vibratory character
 Wavelength of electrons is about 100,000 times shorter than
visible light (photons), λe = .005 nm; λl = 550 nm
 EM can achieve better than 50 pm resolution and
magnifications of up to about 10,000,000x
 Ordinary non-confocal light microscopes are limited by
diffraction to about 200 nm resolution and useful
magnifications below 2000 x

30.  The electron microscope
uses electrostatic and
electromagnetic "lenses"
to control/deflect the
electron beam and focus it
to form an image
 These lenses are analogous
to, but different from the
glass lenses of an optical
microscope that form a
magnified image by
focusing light on or
through the specimen

31.  Cathode or Thermionic Gun:
Electron source – A metal
filament placed in vacuum and
heated emits electrons
 Condenser: a magnetic coil that
focuses the electrons on the
plane of the object
 Objective: 2nd magnetic coil that
deflects electron beam from the
object giving a magnified image
 Projection lens or Occular: 3rd
magnetic lens that receives and
magnifies the image from the
objective
 Final magnified image formed
on a fluorescent screen

32.  Image in EM is principally formed due to electron
scattering
 Electrons colliding with the atomic nuclei in the
object are dispersed and may fall outside the aperture
 Elastic dispersion: image on the fluorescent screen
results from absence of electrons blocked by the
aperture
 Inelastic dispersion: results from multiple collision
 Electron dispersion is a function of
 Thickness of the objective
 Molecular packing of the objective
 Depends on the atomic number of the atoms in the
object

33.  Electron microscopes are used to observe a wide range
of biological and inorganic specimens including
microorganisms, cells, large molecules, biopsy
samples, metals and crystals
 Industrially, the electron microscope is primarily used
for quality control and failure analysis in
semiconductor device fabrication

35. Transmission electron
microscope (TEM)
 The original form of electron microscope,
the Transmission electron microscope (TEM) uses
a high voltage electron beam to create an image
 The electrons are emitted by an electron gun commonly
fitted with a Tungsten filament cathode as the electron
source
 Electron beam is accelerated by an anode typically at +100
keV (40 to 400 keV) with respect to the cathode
 Focused by electrostatic and electro-magnetic lenses
 Transmitted partially through the specimen (that is in part
transparent to electrons) and partially scattered out of the
beam by the opaque part of the specimen

36.  Emerging electron beam carries information about the
structure of the specimen that is magnified by
the objective lens system of the microscope
 Magnified “electron image” (caused by spatial variation)
is viewed by projection onto a fluorescent viewing
screen coated with a phosphor or scintillator material
such as zinc sulfide
 The image can be photographically recorded by exposing
a photographic film or plate directly to the electron beam
 High-resolution phosphor may be coupled by means of a
lens optical system or a fibre optic light-guide to the
sensor of a CCD (charge-coupled device) camera
 The image detected by the CCD may be displayed on a
monitor or computer.

37.  Resolution of the TEM is limited primarily by spherical
aberration
 Advanced aberration correctors have been able to
partially overcome spherical aberration to increase
resolution
 Hardware correction of spherical aberration for
the high-resolution transmission electron
microscopy (HRTEM) has allowed the production of
images with resolution below 0.5 Angstrom (50 pm) at
magnifications above 50 million times
 The ability to determine the positions of atoms within
materials has made the HRTEM an important tool for
nano-technologies research and development

38. Scanning electron microscope
 Electron beam of the SEM does not at any time carry
a complete image of the specimen
 The SEM produces images by probing the specimen with a
focused electron beam that is scanned across a
rectangular area of the specimen ( Raster Scanning)
 At each point on the specimen the incident electron beam
loses some energy, and that lost energy is converted into
other forms
 Heat
 Emission of low energy secondary electrons
 Light emission (cathodoluminescence) or
 X-ray emission.

39.  The display of the SEM maps the varying intensity of any of
these signals into the image in a position corresponding to
the position of the beam on the specimen when the signal
was generated
 Generally, the image resolution of an SEM is ~1 order of
magnitude poorer than that of a TEM
 SEM image relies on surface processes rather than
transmission
 It is able to image bulk samples up to many cm in size
 Depending on instrument design and settings has a great
depth of field
 Can produce images that are good representations of the
three-dimensional shape of the sample.
 Environmental scanning electron microscope (ESEM)
can produce images of sufficient quality and resolution with
the samples being wet or contained in low vacuum or gas
 This greatly facilitates imaging biological samples which are
unstable in the high vacuum of conventional electron
microscopes.