Physics of light and Microscopy

N.Sangeetha
IInd M.D.S
 There are more animals
living in the scum on the
teeth in a man’s mouth than
are men in a whole
kingdom.
- Antony van Leeuwenhoek
 LIGHT

WHAT IS LIGHT?
 It's a kind of energy called "electromagnetic (EM)
radiation“.
 Light travels very fast, travelling at about 300,000
km ( 186,000 miles) per second, nothing can travel
faster.
 light radiates in all directions from its source.

 Each radiating ray, unless something interferes, travel in

straight line to infinity.
At this speed it can
go around the world 8
times in one second.
PROPERTIES OF LIGHT
 WHITE LIGHT

What is white light.

 Regular light from the sun or from a light bulb
that really contains all the colours of the rainbow.

 But you have to split

the light to see this
with a prism.
 what's a laser?
› A laser is a special source of light of only one pure color
(or WAVELENGTH). You can't break up laser light into
other colors.
 Absorption

› When light passes through an object the intensity is

reduced depending upon the color absorbed.
Control

Absorption

B & G absorbed

No blue/green light
 Diffraction

› Light rays bend around edges - new wavefronts

are generated at sharp edges - the smaller the
aperture the lower the definition
 Diffraction
 The property of light waves to bend around the
corners.
 Example -Dust seen in a beam of sunlight.
 Dispersion

› Separation of light into its constituent

wavelengths when entering a transparent
medium .
 AMPLITUDE:

 Refers to brightness of the light.

 Diminishes with increasing distance from the light
source.
 WAVELENGTH:
 The distance between the apex of one wave and the next
one.
 Wavelength determines the colour.
 Most light sources emit energy in a wide range of
wavelength.
 FREQUENCY:

 Number of waves per second.

 Frequency of light wave remain constant.
COHERENT LIGHT
 Two rays of light from the same source,
having the same frequency are said to be
coherent, and when recombined, will double
in amplitude or brightness if they are in phase
with each other ( CONSTRUCTIVE
INTERFERENCE).
 If however they are out of phase with each other,
destructive interference will occur.
a.This figure represents the
wave form of a light
ray.
b.The rays are identical but
one is ¼^ out of phase
with the other and they
interfere but with no
increase in amplitude.
 This shows one ray
now ½^ out of phase
with the other, and
they cancel each
other out.
 This is maximum
destructive
interference and no
light is seen, resulting
in maximum contrast.
 If one ray is brighter than the
other ( increased amplitude)
and is still ½^ out of phase ,
then the difference in
amplitude can be seen, while
maintaining maximum
interference. This position is
that which occurs in the phase
contrast microscope.
 REFLECTION

 Reflection of a light ray occurs when the incident

ray rebounds from the surface.
  The Law of Reflection

Angle of incidence = Angle of reflection

In other words, light gets reflected from a surface at

____ _____ angle it hits it.

The
same !!!
 RETARDATION

 Media through which light is able to pass will slow

down or retard the speed of the light in proportion to
the density of the medium.

 The higher the density, the greater the degree of

retardation.
 Rays of light entering a sheet of glass at right angles
are retarded in speed but their direction is
unchanged.
 REFRACTION

 If the light enters the glass at any other angle, a

deviation of direction will occur in addition to the
retardation and this is called refraction.
 Refraction

He sees the
fish here….

But it is really here!!

 A curved lens will exhibit both
retardation and refraction the
extent of which is governed by:
a) The angle at which the light
strikes the lens-the angle of
incidence
b) The density of the glass-its
refractive index and
c) The curvature of the lens.
 REFRACTIVE INDEX (RI).

When light passes from one medium into a denser it is

refracted towards the normal, which is calculated as
the ratio of the angles of incidence and refraction,
and is used in
the design and
constructions of
lenses.
 The higher the density of the medium, the higher
the RI.
 RI = speed of light in vacuum
speed of light in the medium

 RI of Air 1.00
water 1.30
glass 1.5
 Boundary behavior of light
 Reflection & refraction occurs.
 Both involve change in direction of the wave but refraction
involves a change in medium.
 Point of incidence – point where the incident ray strikes.
 Normal line – line drawn perpendicular to the surface
 Angle of incidence – between incident ray and normal.
 Angle of reflection – between reflected ray and normal.
 Angle of refraction between refracted ray and normal
 Boundary behavior of light
 Refraction of light – when light passes from less
dense to more dense medium – light ray bends
towards the normal.
 When light passes from more dense to less dense
medium – light ray bends away from the normal.
 Amount of bending depends on indices of refraction
of the two medium – described by Snell’s law.
 LENSES

 A lens is an object, usually glass, which is bounded by

one or two spherical surfaces.

 Lenses bend light in useful ways.

 Most devices that control light have one or more lenses

in them
There are TWO basic simple lens types:

1) CONVEX or POSITIVE lens

2) CONCAVE or NEGATIVE lens
 CONVEX

• CONVEX or POSITIVE lenses will

CONVERGE or FOCUS light and can
form an IMAGE

 CONCAVE
 CONCAVE or NEGATIVE lenses will
DIVERGE (spread out) light rays
 Image formation

 Parallel rays of light entering a

simple lens are brought together
by refraction to a single point, the
PRINCIPAL FOCUS or
FOCAL POINT, where a clear
image will be formed of an
object.
 The distance between the optical
center of the lens and the
principal focus is the FOCAL
LENGTH.
 Images produced by convex
lens

 1. an object placed at
twice the focal length
from a convex lens
produces real,
inverted image the
same size as the
object.
 when the object is
placed nearer to the
focal point it
produces a magnified
, real, inverted image.
 An object placed
inside the focal point
of a convex lens
produces a
magnified, virtual,
upright image.
 An object placed at
the focal point of a
convex lens produces
an image which
appears to be at
infinity.
 Images produced by concave lens:

 The images produced by a concave lens are

always virtual, upright and reduced in size
regardless of the location of the object along the
principal axis.
 IMAGE QUALITY

 When light is composed of all the

spectral colors and on passing
through a simple lens, each
wavelength is refracted to a
different extent, with blue being
brought to a shorter focus than
red. This lens defect is called
CHROMATIC ABERRATION and
results in an unsharp image with
colored fringes.
 It is possible to construct
compound lenses of different glass
elements to correct this fault.
 ACHROMATISM:

 Correction of chromatic abberation .

 This method will correct a thin positive lens for any

two colours, leaving a small error in the intermediate
colours( secondary spectrum).
 ACHROMAT&APOCHROMAT
 An ACHROMAT is
corrected for two colors,
blue and red, producing a
secondary spectrum of
yellow/green, which in
turn can be corrected by
adding more lens
components- the more
expensive
APOCHROMAT.
 Magnification,
 Microscopes are used to magnify objects.

 Through magnification, an image is made to appear

larger than the original object.

 Objects are magnified to be able to see small details.

 Resolving power

 Despite the importance of magnification in visualizing

tiny objects or cells, an additional optical property
essential for seeing clearly is Resolution RESOLVING
POWER.

 Resolution is the capacity of an optical system to

distinguish or separate two adjacent objects or points
from one another.
 For example, at a certain
fixed distance (about 10
inches), the lens in the
human eye can resolve two
small objects as separate
points just as long as the
two objects are no closer
than 0.2mm apart.
 It is important to note that the resolving power
and magnification are two different things.
N.A. / NUMERICAL APERTURE –

 In theory, microscope magnification could be

increased to almost infinite levels, but magnification
in itself is meaningless unless the image is sharp.

 The decisive factor in determining the distinctness

of the image is the resolving power of the objective.
 N.A

 The resolving power, in turn, is determined by the

numerical aperture of the objective.

 The N.A. value actually represents the brightness and the

resolving power of an objective.

 The higher the N.A., the more is the resolving power of

the lens.
 NA = n x sin u

 Where ‘n’ is the refractive index of the

medium between the coverglass over the
object and the front lens of the
objective, for example air, water or
immersion oil.
 ‘u’ is the angle included between the
optical axis of the lens and the
outermost ray which can enter the front
lens.
 The most important thing to remember is that a higher
numerical aperture number will provide better resolution.

 In order for the oil immersion lens to arrive at its

maximum resolving capacity, a drop of oil must be
inserted between the tip of the lens and the specimen on
the glass slide.
 Since oil has the same optical
qualities as glass, it prevents
refractive loss that normally
occurs as peripheral light
passes from the slide into the
air; this property effectively
increases the numerical
aperture.
 DEFECTS OF LENS

 For a microscope to be efficient, it must not only

produce magnified image, but one which will be
clear and well defined.
 The images formed by a single lens may be
distored by several causes.
 DIFFERENT TYPES OF LENS
DEFECTS:

 Coma
 Chromatic abberation
 Spherical abberation
 Field curvature
 Distortion
COMA:
 Results from different
magnifications
occurring in the various
lens zones leading to
object points away from
the principal axis
forming short comet
like images.
 CHROMATIC ABBERATION:
When white light is split into component parts,
each part vibrates to a different degree, producing to the eye
a different colour. These colours (red, orange, yellow, green,
blue, indigo and violet) are known as the primary spectrum,
and are seen in the rainbow, or through a spectroscope.
 It will be seen that the
vibrations of the red light
are twice the length of those
of violet light.
 The colours with shorter
wavelength, such as blue
and violet, being affected to
a greater degree than those
having a longer wavelength,
such as red and orange.
SPHERICAL ABBERATION:

 Occurs when a wide beam of light enters a lens.

 Parallel rays entering the lens at its periphery have a
larger angle of incidence and are deviated to a greater
degree than those rays close to the principal axis so that
the peripheral rays are focused at points slightly closer to
the lens than the focal points, resulting in a distortion of
the image.
 Spherical aberration
is circular and
concentrated at the
center:
FIELD CURVATURE:

 Makes a flat object appear curved.

 As a lens is naturally curved, the image field also
becomes curved with peripheral objects being focused
at points slightly closer to the lens than those close to
the principal axis so that when the center of the field is
in focus , the periphery appears blurred and viceversa.
 DISTORTION:

 Results from different magnification occurring

within the different parts of the lens.
 If the magnification of a square object increases
with distance from the centre, it will appear to
have concave sides.
 If it decreases with distance from the centre of
the object , then the image will have bulging
sides.
 COMPOUND
MICROSCOPE
 Today’s compound
microscope were the
addition of a second
magnifying lens system ,
a LAMP in the base to
give off the visible light
and illuminate the
specimen, and a special
lens called the
CONDENSER that
converges or focuses the
rays of light to a single
point on the object.
COMPOUND MICROSCOPE
LIGHT:
 Light from the lamp
is directed into the
first major optical
component, the
substage condenser,
either directly or by a
mirror or prism.
 The light source should be
 Uniformly intense
 Completely flood the back lens of the condenser
when the diaphragm is open.
 Should make the object appear as though it is self
illuminous.
 2 methods of illumination

 Nelson method: A homogenous light source is

used. No lamp condenses are used. The light
source is focused by moving sub stage condenser
up and down.
 Kohler’s method: light source need not be
homogenous, but lamp condensers are essential.
This method is commonly used with compound
microscope.
CONDENSER:
 The main purpose of
the condenser is to
focus and concentrate
the available light
into the plane of the
object.
Diaphragm:
 All condensers have an
aperture diaphragm with
which the diameter of
the light beam can be
controlled.
 Adjustment of the iris
diaphragm will alter the
size and volume of the
cone of light focused on
the object.
 The correct setting for the diaphragm is
when the numerical aperture of the
condenser is matched to the numerical
aperture of the objective in use
 If the diaphragm is closed too much, the image
becomes too contrast and refractile, whereas if
the diaphragm is left wide open, the image will
suffer from glare due to extraneous light
interference.
 In both cases the resolution of the image is poor.
 OBJECTIVES
 The type and quality of the
objective having the greatest
influence on the performance of
the microscope as a whole.

 Within the objectives there may

be lenses and elements from five
to 15 in number, depending on
image ratio, type and quality.
Objectives
 The main task of the objective is to collect the
maximum amount of light possible from the
object, unite it and form a high quality magnified
real image, some distance above.
EYEPIECE
 These are the final stage in the
optical path of the microscope
their function is to magnify the
image formed by the objective
within the body tube, and
present the eye with a virtual
image.
Negative Vs Positive
eyepiece
 Negative eyepiece - focus is within or between the
lenses of the eyepiece.
› lower or field lens collects the image – converts to slightly
smaller image at field stop or field diaphragm - upper lens
- enlarged virtual image.

 Positive eyepiece - focus is outside the eyepiece lens

system.
› field stop or field diaphragm is outside the eyepiece -
virtual image from the objective is focused and magnified
by the entire eyepiece.
 In order to be most effective, a microscope
should provide adequate
a. magnification,
{ the ability to enlarge objects}
b. resolution or Resolving power
{ the ability to show detail}
c. clarity of image.
 Magnification of the object
or specimen by a
compound microscope
occurs in two phases.

1.the objective lens

2.the ocular lens or eyepiece.
 tube length
 Magnification= focal length

 The optical tube length is standardized to

160mm.
 Thus focal length of a standard 10 x objective is
160/10= 16mm.
Total Magnification:
 To figure the total magnification of an image that
you are viewing through the microscope is really
quite simple. To get the total magnification take
the power of the objective (4X, 10X, 40x) and
multiply by the power of the occular lens.
 Care of the microscope

 Hold a microscope firmly by the stand only. Never grab it

by the eyepiece holder

 Hold the plug (not the cable) when unplugging the

illuminator.

 Since bulbs are expensive, and have a limited life, turn

the illuminator off when you are not working.

 Always make sure the stage and lenses are clean before
putting away the microscope.
 NEVER use a paper towel, a kimwipe, your shirt, or any material other than good
quality lens tissue or a cotton swab (must be 100% natural cotton) to clean an
optical surface.

 Be gentle! You may use an appropriate lens cleaner or distilled water to help
remove dried material. Organic solvents may separate or damage the lens
elements or coatings.

 Cover the instrument with a dust jacket when not in use.

 Focus smoothly; don't try to speed through the focusing process or force anything.
For example if you encounter increased resistance when focusing then you've
probably reached a limit and you are going in the wrong direction.
 The oil immersion objective should be wiped free of oil after use and care should
be taken to avoid contamination of dry objectives by oil.

 Persistent dust may become difficult to remove and , like fingerprints or grease
marks on eyepieces, requires proper cleaning.

 A useful cleaning mixture can be made up of seven parts ether and three parts
alcohol, or special commercial lens cleaning fluid may be employed.

 Xylene and other solvents should never be used as these can lead to partial
dissolution of the lens cement.
 Cleaning of mechanical components
 Cleaning of Mechanical Components: 10% alcohol or
commercial glass and surface cleaning product & piece of
terry cloth.
 Body tube, stage, fine and coarse adjustment & outer rims of
oculars – moist & dry cloth.
 The stand and table area - small vacuum cleaner with a
flexible hose and soft brush attachment.
 Cleaning of Optical Components

 Inspection of the lens surface – for abrasive particles, films,

smudges, or stains using magnifying glasses of 2x/3x.
 Removal of non-attached particles - by gently blowing air
across (not perpendicular to) the lens surface or rubber bulb
or balloon.
 Avoid any use of compressed air cans for lens cleaning.
 Removal of attached particles - thin bamboo skewer or
wooden toothpick. After breathing with mouth open use these
skewers with lateral force.
 Cleaning of Optical Components
 Removal of water-soluble films - lens tissue and moisture
produced by slowly breathing onto the lens. Frayed end of
tissue tube is used to clean the lens surface - circular motion
starting at the lens center and working outward.
 Removal of non-water soluble films - commercial lens
cleaning fluids.
References
Thank you