MICROSCOPY

DR PINKY PAVITHRAN
1ST YEAR PG
ORAL AND MAXILLOFACIAL
PATHOLOGY
CONTENTS
 Definition
 History of microscope
 Terminologies used in microscope
 Components of microscope
 Cleaning and maintenance
 Conclusion
 Reference
 DEFINITION
AN OPTICAL INSTRUMENT THAT USES A LENS OR
A COMBINATION OF LENSES TO PRODUCE
MAGNIFIED IMAGES OF SMALL OBJECTS ,
ESPECIALLY OF OBJECTS TOO SMALL TO BE SEEN
BY THE UNAIDED EYE
 HISTORY OF MICROSCOPE
 The early pioneers in the history of microscope are Digges of
England and Hans and Zacharias Janssen (1590) of Holland
,Robert Hooke (1665),John Marshaal(1700), Martin Forbenius
Leddermuller(1768)Louis Jablot(1755), Mayen(1747)

 Antony van Leeuwenhoek is known as the inventor of

microscope

 Early microscopes were simple microscopes but with advances

of science compound microscope were built
TERMINOLOGIES USED IN
MICROSCOPE
 LENS
1. A lens is the name given to a piece of glass or other transparent
material, usually circular having two surfaces ground and polished
in a specific form in order that rays of light passing through it shall
either converge (collect together) or diverge (separate)

2. Positive Lens: A lens is called positive when it causes light rays to

converge to form a real image

3. Negative Lens :A lens is called negative when it causes light rays to

diverge or scatter and positive or real image will not be seen
AMPLITUDE
 Amplitude refers to the strength of the energy or
brightness of the light

When light passes through any medium the

amplitude diminishes to a greater or lesser
degree depending upon the medium
WAVELENGTH
 The distance between the apex of one wave and the next is the wavelength
and is measured in nanometers
 Wavelength determines color
 Most light sources emit light in a wide range of wavelengths ,some parts at
differing amplitudes
FREQUENCY
 The number of waves per second is referred to
as frequency
 The frequency of a light wave remains constant

1. Coherent rays : Individual rays of identical

frequency from the same source are called
coherent , these rays may combine or
interfere with each other in an observable way

2. Non coherent rays : Rays from different

sources or of different frequencies are said to
be non coherent
RETARDATION AND REFRACTION

 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

 The effect of lens on a ray of light depends primarily on the density of glass (or
other material )which reduces the speed at which light travels through it .

 In a dense medium the light rays are retarded

 The change in direction of light passing obliquely from one medium to another of
different optical density, this is called refraction

 A curved lens exhibit both retardation and refraction , the extent of which is
governed by

1. The angle at which the light strikes the lens – the angle of incidence
2. The density of the glass – its refractive index and
3. The curvature of the lens
• The bending of the light rays (refraction) is due to the
fact that one part (B) strikes the surface of glass first (B1)
and is retarded while the other part (A) is still travelling
at normal speed thus causing the rays to bent and its
direction is altered
 The angle by which the rays are deviated within
the glass or other transparent medium is called
the angle of refraction and the ratio of the sine
values of the angle of incidence(i) and
refraction (r) gives a figure known as refractive
index (RI) of the medium
RI = SIN i/ SIN r
REFRACTIVE INDEX

 Ratio of speed of light in one medium (air) to that in another

medium (lens) is called refractive index

 So RI is defined as ratio of velocity of light in air to the velocity of

light in that substance ( velocity of light in one medium to velocity of
light in another medium )

 Greater RI Higher the density of the medium

 Refractive index of
1. AIR : 1.00
2. WATER :1.30
3. GLASS :1.50
 As a general rule light passing from one medium into a more dense
medium is refracted towards the normal and when passing into a
less dense medium is refracted away from normal

 The angle of incidence may increase to the point where the light
emerges parallel to the surface of the lens, beyond this total internal
reflection will occur and no light will pass through
PRINCIPAL FOCUS

 Parallel rays of light entering a simple lens are brought together by

refraction to a single point is called 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
CONJUGATE FOCI

 In addition to the principal focus ,a lens also has other pairs of points
on either side of the lens

 Conjugate foci are a pair of points on the principal axis of the lens
such that an object at one of the points will produce an image at
the other point
 The conjugate foci vary in position and as the object is moved nearer the lens
the image will be formed further away at a greater magnification and inverted
 This is the real image
 The objective lens of the microscope forms the real image (at intermediate
image plane)
 If the object is placed yet nearer the lens ,within the principal focus , the image is
formed on the same side as the object, but it will be a ghost image which can
be seen only by looking through the lens and cannot be focused on a screen
.The image formed is enlarged and right way up
 This is called virtual image
 This is formed by the eyepiece of the microscope of the real image projected
from the objective
 This appears to be at a distance of approximately 25cm from the eye around the
object stage level
 Figure illustrates the formation of both images(virtual and Real image) in the
upright compound microscope as is commonly used in histopathology
IMAGE QUALITY
Defects of a lens
Aberrations :
 Classification(1)
a. Chromatic aberrations
b. Monochromatic aberrations
 Spherical aberration
 Marginal astigmatism
 Coma
 Distortion
 Curvature of field
 Classification(2)
a. In focus and out of focus
b. Wide beam and narrow beam
c. On axis and off axis
d. Low degree and high degree
 Myopia,hypermetropia and
astigmatism
 Spherical,chromatic,coma,diffracti
on,distortion
Chromatic aberration
 When white light is passed through a prism it is split into a spectrum of color
ranging from red (wavelength 700nm)through orange yellow green blue indigo
and violet (wavelength 350nm)
 VIBGYOR
 A biconvex lens also splits white light into its component colors , the blue light
being refracted more than the red so that it comes to a focus nearer to the lens

 Those non convergence of the colored components of white light to a common

focus is termed as chromatic aberration
Correction

 By combination of lens element

 Since different types of glass have different optical properties , chromatic
aberration can be corrected to within useful limits by using a two component
lens
 A positive lens (of greater magnifying power than is finally required )is combined
with a negative lens made of glass producing a greater chromatic aberration
,but with same refractive index
 The negative lens corrects the chromatic aberration in the positive lens and only
partially neutralizes its magnifying power
 This method will correct a thin positive lens for any two colors leaving a small error
in the intermediate colors (secondary spectrum)
 This type of lens is known a AN ACHROMATIC LENS
 SO an achromatic lens or achromat is a lens that is designed to limit
the effects of chromatic and spherical aberration. Achromatic
lenses are corrected to bring two wavelengths into focus on the
same plane
 If fluorspar is incorporated in the glass of the achromatic lens, three
colors can be brought to one focal point
 Such lenses are known as APOCHROMATIC LENSES

Thus Apochromatic lens is

one in which chromatic aberration is corrected for
three colors and the spherical aberration for
two colors.
 Spherical aberration
 It is the defect of a single lens due to the fact that it has a curved surface
 Since the angle at which light rays enter and leave the surface of a lens varies
with each part of the lens ,those rays passing through the periphery (AA) will be
refracted to a greater degree than those travelling through the central area
 Spherical aberration is the foggy appearance of the outer part of the field of
view of a lens , and is caused by the non convergence of rays to a common
focus

 Correction:

 corrected by making combination of lens, elements of different glass using a

powerful positive lens and partially neutralizing its magnifying power with a
negative lens made of glass having a greater relative aberration
Astigmatism

 Rays that propagate in two perpendicular planes have different

focal points
 As a result the image appears stretched in one direction at one
plane of focus and stretched in another direction at another plane
of focus
Corrections

 Astigmatism may arise due to imperfect lens surfaces or

misalignments
 So using lens with perfect surface or proper alignment reduces
astigmatism
 Stigmators which apply correcting fields are used in electron
microscope for correcting astigmatism
COMPONENTS OF MICROSCOPE
 A simple microscope is composed of one or several lenses mounted close
together

 A compound microscope is composed of two widely separated lenses or set of

lenses , capable of producing greatly enlarged images
 The standard microscope is composed of two main parts

1. The microscope proper: incorporating the body tube with the

objective at one end and the eyepiece at the other

2. The stand: which includes the supporting adjusting and illuminating

apparatus
Optical components

 Eyepiece
1. Eyepieces are the final stage off the optical path of the
microscope

2. The function is to magnify the image formed by the objective

within the body tube and present the eye with the virtual image,
apparently in the plane of the object being observed ,usually this is
an optical distance of 250mm from the eye
• Early types of eyepiece ,were subject to aberrations, especially of
color. Compensating eyepieces were designed to overcome those
problems and can be used with all the modern objectives

1. Negative eyepiece : here the focus is within (between)the lenses of

the eyepiece

2. It is composed of two lenses, the lower or field lens collects the

image that would have been formed by the objective (virtual
image plane) and cones it down to a slightly smaller image at the
level of the field stop (or field diaphragm)within the eyepiece

3. The upper lens(Eye lens) then produces an enlarged virtual image

which is seen by the microscopist

4. An engraved scale placed in the field stop will be superimposed (in

focus)on the image
Positive eyepiece

 Here the focus is outside the eyepiece lens system, for this reason it
may be used in simple microscope
 The field stop diaphragm is outside the eyepiece from which the
virtual image (from the objective)is focused and magnified by the
entire eyepiece
 A scale placed on the field stop will be superimposed (in focus)on
the image formed by the objective
 Huygenian eyepieces
1. Designed by Huygens for telescope
2. Commonly used in microscopy
3. Negative ,undercorrected
4. Best suited for use with achromatic
objectives
 Ramsden eyepiece
1. Positive oculars
2. Lower lens has its plane side towards the object
3. Most of the compensated eyepieces are of this type
4. Usually have doublet or triplet lenses
 Wide field eyepieces
1. Gives large flat field of view
2. Used in biological laboratory

 High eye point oculars

1. For persons who were spectacles

 Compensating eyepiece
1. Primarily designed for use with apochromatic objectives
2. The apochromatic objectives are undercorrected for the magnification of light
of various colors and compensating(overcorrected)eyepiece are designed to
rectify this
3. They are recommended for all modern objectives
4. English speaking countries mark them “comp", while Germans as “K”
 Field of view
 Some eyepiece are marked with their field of view number from which can be
calculated the actual diameter of the specimen being viewed (field of view
number divided by the magnification of the objective , equals the field of view in
millimeter )
OBJECTIVES:

 These are the most important piece of the microscopes equipment

 The type and quality of the objective has the greatest influence on
the performance of objective

 The objective screws into the lower end of the body tube by means
of a standard thread ,thus all objectives are interchangeable

 The main task of the objective is to collect maximum amount of

light possible from the object ,unite it and form a high quality
magnified real image some distance above

 Objectives are computed for an optical tube length (length

between objective and eyepiece)of 160mm or 170mm by most of
the manufacturers
Numerical aperture

 The ability of an objective to resolve detail is

indicated by its numerical aperture and not by its
magnifying power

 The amount of detail that is revealed by a lens

depends not on its magnification but on the size of
cone of light that can be collected from object

 The numerical aperture (NA) is expressed as a

figure and will be found engraved on the body of
the objective
 Numerical aperture an optical constant is defined as the product of the
refractive index of the medium between the lens and object ,and sine of the
angle formed between the optical axis of the lens and the outermost ray which
can enter the front lens

NA=n sin u
 n= RI of the medium between lens and object
 U=angle between optical axis of the lens and the outermost ray which can enter
the lens
 Numerical aperture depends upon wave length i.e. resolution increases with
decreasing wavelength
 The wider the cone of light greater the NA
 In theory the greatest possible angle would be if the surface of the front lens
coincided with the specimen giving a value for U of 90degree
 In the above formula ,with air (RI=1.00)as the medium and a value for U of 90
degree
 NA= n sin90
=1 sin90
=1 1

Thus ; NA=1
 Theoretically with air as medium maximum NA is 1 but this is not possible since there
must always be some space between the surface and the value of 90 is not
obtainable

 In practice maximum NA attainable with a dry objective is0.95

 For water and oil immersion objectives theoretically values for NA are 1.30 and 1.50
respectively

 In practice values of 1.20 and 1.40 are highest obtainable

 Theoretically highest NA for* dry lens = 1.0

*oil = 1.50
*water =1.30
Resolving power

 The best image is not the largest but the clearest

 The power of lens to reveal detail is referred to as resolving
power or resolution of the lens
 Resolving power may be defined as the ability to reveal
closely adjacent structural details as being actually
separate and distinct
 The resolving power of a microscope is largely dependent
upon the angle of light entering the objective
 Oil conserves many light rays which would be otherwise lost
by refraction
 Resolution is restricted by two factors
1. The NA
2. The wavelength of light employed

R = 1.2λ/2NA
or
R = 0.61λ/NA
 Smaller this value ,higher the resolving power of
the microscope
 The resolution is higher for blue light than red light.
 Types of objective :
 Achromatic objectives : Most widely used for routine
purpose (red and blue
 Apochromatic objectives : Highly corrected for lens aberrations (spherical, coma
ad so on), chromatically corrected for deep blue red and green, spherically
corrected for blue and green
 Fluorite objectives( neofluor) or semi apochromatic: Fluorite or semi
apochromatic objectives have fluorite incorporated into the lens system
1. Gives better color correction
2. Corrected for three wavelengths of light (blue green and red)
3. Quality of image midway between the achromat and apochromat
 Spherical Chromatic Field
Objective Type
Aberration Aberration Curvature

Achromat 1 Color 2 Colors No

Plan Achromat 1 Color 2 Colors Yes

Fluorite 2-3 Colors 2-3 Colors No

Plan Fluorite 3-4 Colors 2-4 Colors Yes

Plan Apochromat 3-4 Colors 4-5 Colors Yes

 Plan achromat objectives:
1. Gives perfect flat field
2. Recommended for photomicrography

 Polarizing objectives:
1. stain free objectives
2. For polarizing microscope

 Phase objectives:
1. Contain a phase plate for use in phase contrast microscopy
2. They are designated Ph with a number which refers to the matching annulus
 Color codes
1. Microscope
manufacturers label
their objectives with
color codes to help in
rapid identification of
the magnification
2. In addition to color
coding other
information is also
embossed on the
objective
 Markings
1. All objectives are engraved with the information needed to obtain
maximum performance
2. Plan = planachromat
40 =40X magnification
160 = tube length 160mm
0.65 =NA
O.17 =Size of cover glass recommended
Cover glass thickness

 The cover glass thickness is only important if high power ‘dry ‘

objectives are being used

 When no 1 cover glasses should be used or an objective with a

correction collar may be employed which allows a range of
thickness of coverslip from 0.12 – 0.22mm to be used
 Nose piece
1. In most modern microscopes up to six
objectives are mounted on resolving
nosepiece
2. For rapid change of all objectives they
should be at focus and they should focus
the same central area of the section
when brought into the position
3. Such nosepieces are known as “par-
focal” and “par-central”
4. A carrier or nosepiece for a number of objectives is
usually fitted at the lower end of the body tube

5. It rotates on a central pillar and is designated by a number

of objectives it carries for example double triple and quadruple
nosepiece
 Body tube

1. Its present above the nose piece

2. Three main forms are available

 Monocular
 Binocular
 Combined photo binocular

3. The last sometimes has a prism system allowing 100 percent of the light to go
either to the observation eyepieces , or to the camera located on the vertical part,
and sometimes has a beam splitting prism dividing the light,20% to the eyes and
80% to the camera .This facilitates continuous observation during photography
 Provision is made on binocular tubes for adjustment of the
interpupillary distance, enabling each observer to adjust for
the individual facial proportions

 Body tube rarely contains a drawtube ,by which the distance

between the eyepiece and the objective may be varied

 Objectives are made to be used with specific mechanical

tube length eg:160mm
 Stage :
1. At the lower end of the limb supporting the
body tube and adjustments is a platform or
stage on which the objects to be examined
are placed

2. The stage may be plain or mechanical

3. Mechanical stage will give even steady

movement of the object in two directions by
means of two micrometer threads

4. Stage is usually adjusted to take a 3*1 inch

slide and moves over an area approximately 3
½*4½ inches so that a whole slide may be
examined

5. Most mechanical stages are fitted with a

Vernier scale for recording the position of the
slide in each direction and they may be very
useful if a particular field is to be found quickly
at a later re examination
 Illuminating apparatus

1. The substage

 Below the stage and usually attached

to it , is an adjustable substage which
can be moved up and down by a
helical screw or rack and pinion

 This consists of
 Condenser
 Iris Diaphragm
 Filter carrier
 Mirror
 Flat on one side and concave on other
 Condensers and Iris diaphragm

 Light from the lamp is directed into the first major optical component the substage
condenser , either directly or by a mirror or prism

 The main purpose of the condenser is to focus or concentrate the available light
into the plane of object

 Within comfortable limits ,the more the light at the specimen ,the better is the
resolution of the image

 The two lens Abbe condenser is commonly used but is not very efficient

 Condensers can be adjusted vertically in order to allow for varying heights

 The condenser should have a same NA as the objective with which it is being used
 All condensers have an aperture diaphragm (Iris
diaphragm) with which the diameter of the light
beam can be controlled

 Adjustment of the iris diaphragm will alter the size and

the volume of the cone of light focused on the object

 If the diaphragm is closed too much, the image

becomes too contrasty and refractile , whereas if the
diaphragm is left wide open the image will suffer from
glare due to extraneous light interference

 In both cases resolution of the image is poor

 Under no circumstances the iris
diaphragm be closed or lower the
condenser to reduce the intensity of
the light .Light absorbing filters can be
used.

 Many condensers are fitted with a

swing out top lens. This is turned into
light path when higher power
objectives are in use .It focuses the
light into a field more suited to the
smaller diameter of the objective
front lens

 Swing out top lens is used with the

lower power objectives to illuminate
the whole field
 The filter carrier
 The filter carrier is usually a recessed metal ring pivoting on a screw to facilitate
the easy removal of filters

 The mirror
 The two sided mirror is plane on one side and concave on the other and is fitted
about 4 inches below the stage . Concave mirror causes the light rays to
converge together and form an image
 Light source

 Light is an essential part of the system

 Earlier sunlight was the usual source

 A progression of light sources has developed from oil lamps to the low voltage
electric lamps of today . These operate via a transformer and can be adjusted to
the intensity required

 Larger instruments have their light sources built into them

 Illumination
 The source of illumination should be
 uniformly intense
 Should completely flood the back lens of the condenser with light
when the lamp iris diaphragm is open and
 Make the object appear as though it were self luminous

1. Critical illumination 1. Kohler illumination

2. Simple equipment 2. Photography
3. Separate light source is 3. Used with eyepiece
used removed
There are two universally recognized methods to correct illumination
 Nelson method (critical illumination)
 For this method light source should be homogenous and no lamp
condenser used
 Normally employed with a bare light source
 Light source should be focused on the object plane by racking the
substage condenser up and down
 In this method nelson or critical illumination the light source and the
object are in the same plane and in focus
 Kohler method :
 For this method to be used light source does not
have to be homogenous , but a lamp condenser
is essential to project an image of the lamp
filament on to the substage iris diaphragm and in
turn condenser aperture diaphragm is focused at
the back focal plane of the objective and can be
examined with the eyepiece removed

 In this system the lamp condensing lens (which is

evenly illuminated)functions as the light source
.this method must be used with compound lamps
and should always be used for photomicrography

 In kohler illumination the light source is focused on

the aperture diaphragm and the field diaphragm
and object are in focus
 Magnification
 Total magnification is the product of the magnification values of the objective
and eyepiece providing the system is standardized to an optical tube length of
160mm

 For variation of the tube length magnification depends upon

 Focal length of the objective

 Distance between the focal plane of the objective and the image it produces
 The magnification of the eyepiece

 Magnification = Tubelength / Focal length of objective *eyepiece

magnification
 When the magnification is marked on an objective it is only corrected when used
at the standard tube length . Total magnification is then calculated by
multiplying the magnification of the objective by the magnification of the
eyepiece

 The magnifying power of the eyepiece is marked on it and magnification of the

image produced is as follows

Magnifying power Magnification

X4 4 TIMES
X6 6 TIMES
X10 10 TIMES
 The magnifying power of each objective is shown by a figure engraved on the
sleeve of the lens as follows
 Magnifying Power Magnification
x10 10 times
x40 40 times
x100 100 times
Notes :
 Distance between front lens of the objective and the object slide (when the
image is in focus))
 Objectives Working distance
X10 5 – 6 mm
X40 0.5 – 1.5 mm
X100 1.5- 0.20 mm

 Recommended thickness of coverslip: 0.17mm

 Red ring : objective used with immersion oil
 Use only fine adjustment knob when using high power objective
 Cedar wood oil is widely used when microscope examination is carried out
under oil immersion lens, since RI of cedarwood oil is same as that of glass i.e.
1.515
 Paraffin oil or mineral oil should not be preferred since RI is not correct and it
often penetrates inside the objective
Cleaning and maintenance

 Daily cleaning routine

 Weekly cleaning routine
The ideal posture for viewing a microscope . An upright comfortable
posture with operators spine and arms correctly aligned for long
observation periods (center)
 CONCLUSION
 The light microscope is a very powerful tool for understanding the
structure and function of tissues, and it is widely used in biomedical
science courses, as well as in research and diagnostic laboratories.
Understanding the capabilities and limitations of the light
microscope is important if one is to get the best results from
microscopy. The investigation of cell structure has also been
considerably extended by the developments in electron
microscopy and fluorescence microscopy. In a nutshell, microscope
is the best device used to view extremely tiny particles such as
bacteria.
 Reference
 CELLULAR PATHOLOGY TECHNIQUE, MICROSCOPY – CFA CULLINGS
 BANCROFTS THEORY AND PRACTICE OF HISTOLOGICAL
TECHNIQUE,LIGHT MICROSCOPY –JOHN D BANCROFT, ALTON D FLYOD
 TEXT BOOK OF ORAL PATHOLOGY ,MICROSCOPE – ANIL GOVINDRAO
GHOM
.
 Microscope basics.
Sluder G1, Nordberg JJ. 2013;114:1-10. doi: 10.1016/B978-0-12-407761-4.00001-4
Author information
1
Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.