1. -DR. PARIKSHYA SHRESTHA
-P.G. 1ST YEAR
-DEPT OF ORAL PATHOLOGY AND MICROBIOLOGY
-RAJARAJESWARI DENTAL COLLEGE AND HOSPITAL
MICROSCOPE - I
2. CONTENTS
INTRODUCTION
HISTORY
TYPES OF MICROSCOPE
COMPONENTS OF MICROSCOPE
LIGHT AND ITS PROPERTIES
LENS
COMPONENTS OF MICROSCOPE IN DETAIL
MAGNIFICATION AND ILLUMINATION
MICROMETRY
CLEANING AND MAINTAINANCE OF MICROSCOPE
DARK GROUND MICROSCOPY
POLARIZING MICROSCOPY
PHASE CONTRAST MICROSCOPY
INTERFERENCE MICROSCOPY
DIFFERENTIAL INTERFERENCE CONTRAST MICROSCOPY
REFERENCE
3. Introduction
The word microscope is derived from two Greek words ‘mikro’
meaning ‘small’ and ‘scopien’ meaning ‘to view’.
Thus, it is an instrument which enables us to view small objects
by magnifying it and making it possible to be seen by the viewer.
The science of investing small objects using such an instrument
is called Microscopy
4. History of microscope
1590 – Hans and Zachcharias Janssen of Holland claimed to
have invented microscope
1609 – Galileo Galileli developed a compound microscope with
convex, concave lens
1572-1633 – compound microscope with two convex lens, invented
in Rome by Carnelius Drebbel
1625 – Giovanni Faber coined the term microscope
5. History of microscope
1674 – Anton van Leeuwenhoek made and used a simple
microscope to view biological specimens.
6. Types of microscopes
Basically two types of microscope :
1. Simple Microscope : A simple microscope consists of a
single lens or a magnifying glass.
2. Compound microscope : A compound microscope consists
of two or more lenses.
Compound microscope can be further divided according to
different microscopic methods used .
10. LIGHT
Visible light is that portion of electromagnetic spectrum
that can be detected by human eyes, having wavelength
ranging from approx 400 nm to 800nm
Light travels in straight lines. Light travels at different
speeds in air and in glass. Its path can be deflected or
reflected by means of mirrors or right angle prisms.
11. LIGHT
RETARDATION AND REFRACTION
If light enters sheet of glass at right angle, light is retarded in
speed but direction is unchanged.
If light enters the glass at any other angle, a deviation of direction
will occur in addition to retardation and this is called refraction.
12. LIGHT
Light is slowed or retarded and “bent” or refracted
when it passes through air and enters a convex lens, gets
refracted when it leaves the convex lens and reenters air.
13. LIGHT
The curved lens will exhibit both retardation and refraction,
the extent of which is governed by :
a) Angle of incidence : angle at which light strikes the lens
b) Refractive index
c) Curvature of the lens
Angle of refraction : the angle to which the rays are deviated
within the glass or other transparent medium
14. LIGHT
Refraction depends on the optical density of medium from
which lens is made, which is indicated by refractive index
(RI)
RI = velocity of light in air/velocity in subatance
Refractive index is of great value in the computaion and
design of lenses, microscope slides and coverslip and
mounting media.
RI of air = 1.00, water = 1.30, glass = 1.50
15. LIGHT
Total internal reflection : light passing from glass into air
emerges parallel to the surface of lens if the angle of
incidence is increased. If the angle is too great, (critical
angle), the rays do not emerge but are totally internally
reflected.
16. LENS
LENS
Piece of glass or other transparent material, usually circular,
having the two surfaces ground and polished in a specific form
in order that rays of light passing through it shall either
converge or diverge.
Two types of lens:
Positive lens
Negative lens
17. LENS
Positive lens
Thicker at centre
Causes light rays to concentrate or converge to form a real
image
Negative lens
Thinner at centre
Light passing will diverge or scatter and real image are
not seen
18. LENS
Focus
When the lens concentrates the light rays to form a clear sharp
image of an object, the object is said to be in focus.
The term ‘focus’ or ‘principal focus’ are used to indicate the
position in which a lens will form a sharp, clear picture of a
distant object.
Conjugate foci : in addition to principal focus, a lens also has
conjugate foci; these are two points one on each side of the lens,
in one of which a clear image will be formed on a screen of an
object placed in the other.
20. LENS
Image formed
1. Real Image
Formed by Objective lens of microscope
Image is inverted, at greater magnification
Can be seen on a screen
21. LENS
2. Virtual image
Image formed within principal focus on same side of object
Image appears the right way up and enlarged
Cannot be focused on screen
Formed by eyepiece of the microscope of real image
projected from the objective
22.
23. Defects of Lens
1. Chromatic aberration
Colours of white light, each refracted to different degree
Shorter wavelength, greater degree of refraction
White light entering a lens, on emerging forms a different
point of focus for each component colours, blue being
focused at a point nearer the lens than red
Results in unsharp images with colored fringes
24. Defects of Lens
Correction : It is known as achromatism
Done by using two component lens
Positive lens is combined with a negative lens
construction of compound lens of different glass element
a) Achromatic lens : will correct a thin positive lens for any two
colour
b) Apochromatic lens : fluorspar is incorporated, three colors can
be brought to the one focal point and the amount of chromatic
aberration visible in image is negligible.
25. Defects of Lens
2. Spherical aberration
Due to entry of light rays into curved lens at its periphery
Refracted more that at a centre and thus not brought to a
common focus
Blurred image is formed
Correction : same pattern used for chromatic aberration ie
using a powerful positive lens and partially neutralizing its
magnifying power with a negative lens made of glass having a
greater relative aberration
27. Eyepiece
Function - to magnify the image formed by the objective
within the body tube, and present the eye with a virtual
image.
Limits the field of view as seen by the eye
Usually 5x, 7x, 10x, 20x, 40x type magnification are used.
Can be used to correct the residual errors in objective lens.
Undercorrected : blue ray refracted to greater degree than red;
blue fringe
Overcorrected : orange fringe seen at edge of field diaphragm
29. Eyepiece
Positive eyepiece :
With this, the focus is outside the
eyepiece lens system
Field stop is outsidethe eyepiece,
from which the virtual image is
focused and magnified by entire
eyepiece.
Ramsden eyepiece : lower lens has
its plane side towards object. These
are preferred for micrometer
eyepiece as they impart less
distortion to scales
30. Eyepiece
Negative eyepiece :
With this, the focus is within the
eyepiece lens system
Lower or field lens collects the image
that would have been formed by the
objective and cones it down to a
slightly smaller image at the level of
the field stop within eyepiece; upper
lens then produces an enlarged
virtual image that is seen by eye.
Huygenian eyepiece : these are
undercorrected and are best suited
for use with achromatic objectives.
31. Eyepiece
Other types :
Compensating eyepiece
Pointer eyepiece
Micrometer eyepiece
Double demonstration eyepiece
High focal point eyepiece
32. Objective
These are screwed into lower end of body tube by means of
standard thread, thus are interchangable.
Designated by their focal length, dependent on tube length
Consists of lenses and elements 5-15 in number, depending
on ratio, type and quality
33. Objective
The main task of objective is to collect maximum light
possible from the object, unite it and form a high quality
magnified image some distance above.
Every objective has a fixed working distance, focal length,
magnification and numerical aperture (NA) .
35. Objective
The working distance is the distance between an object in focus
and the front of the lens system of the objective.
The focal length in the compound lens it is the distance between
an object in focus and a point approximately halfway between the
component lenses of the objective.
Total Magnification is product of magnification values of
eyepiece and objective in a standard microscope.
36. Objective
Color Codes
Microscope manufacturers label their objectives with color codes
to help in rapid identification of the objective.
Table : Color codes used for objectives
Magnification Color Code
4× Red
10× Yellow
40× Light Blue
100× White
37. Objective
Coverglass thickness
is important if high-power 'dry’ objectives are being used, when
No. 1 coverglasses should be used, or an objective with a
correction collar may be employed which allows a range of
thickness or coverslip from 0.12 to 0.22 mm to be used (usually
0.17mm)
oil-immersion objectives do not have coverglass restrictions since
they will have the same refractive index as the immersion oil.
38. Objective
Resolution
The ability of the lens to distinguish fine structural details in a
specimen is known as the ‘resolving power’. It is the smallest
distance between two dots or lines that can be seen as separate
entities.
It depends on the wavelength of light (λ) and the Numerical
Aperture of the lens.
It is calculated as:
Resolution = 1.2 λ/2NA
39. Objective
Numerical Aperture
This ability is of an object to resolve detail is expressed in terms
of numerical aperture(NA)
Numerical aperture depends primarily on the extreme range of
the divergent rays that can be made to enter the lens (angular
aperture) and secondarily on the refractive index of the medium
between the object and the objective.
The relation between numerical aperture, angular aperture and
refractive index is:
NA = n× Sin u
n=refractive index of medium between lens and object
sin u = the sine half the angle of aperture
40. Objective
Effect of high NA
whilst a high numerical aperture increases the resolution
of an objective, it has the following disadvantages:
a) it reduces the depth of focus, i.e. the ability to focus on more
than one layer of an object at the same time, and
b) it reduces the flatness or the field, so that the edges are out of
focus.
41. Objective
Types of Objectives
In most modern microscopes objectives are usually made up
more than one lens.
This series of lenses is used to overcome certain limitations in
the lenses.
42. Types of Objective
Achromatic: Corrected for two colors red and blue. It is the most
widely used for routine purposes.
Fluorite: Corrected for yellow green color. Green light is
brought to a shorter focus and violet light to a longer focus.
Apochromat: All colors are brought to same focus. It is fully
corrected for three colors. More highly corrected, often
incorporating fluorite glass. These lenses are used especially for
photomicrography and for screening cytological smears.
Plan-achromat: Although histological sections are flat the
image produced by the microscope is not flat. It is saucer
shaped; it is not possible to focus the whole of the field sharply
at any one time. This aberration is corrected using flat-field
objective, also called as plan-achromat lenses.
44. Body tube
Attached to limb, standard 160mm length
Nosepiece/carrier for objectives fitted at lower end,
designated by number of objectives (2/3/4 based on
magnification needed.
Three main forms : monocular, binocular and combined
photo-binocular
45. Body tube
Binocular
Microscope
Binocular tubes have provision made for
the adjustment of the interpupillary
distance, enabling each observer to adjust
for the individual facial proportion
46. Adjustments
a) Coarse adjustment knob
enables the stage and substage to be moved rapidly up and
down
b) Fine adjustment knob
enables the stage and substage to be moved slowly and
accurately
Works by micrometer screws, levers and cams
47. Object Stage
A rigid platform above the condenser which supports the
glass slide is object stage.
48. Object Stage
It has an aperture in the
center through which the light
can pass to illuminate the
specimen on the glass slide.
The stage holds the slide
firmly and allows the slide
movements with a mechanical
vertical and horizontal
adjustment screws.
49. Object stage
The mechanical stage is graduated with Vernier scales and the x
and y movements assist the operator to return to an exact
desired location in the specimen.
Traveling range in most of the microscopes is 76 mm(X) 30 mm
(Y).
50. Illuminating apparatus
Substage :
below the stage, attached to it and adjustable
consists of:
1. Condenser
2. Iris-diaphragm
3. Filter carrier
4. Mirror
51. Condenser
Light from the lamp is
directed into the sub stage
condenser either directly or
from a mirror or prism.
The main purpose of the
condenser is to focus or
concentrate the available
light into the plane of the
object
52. Condensers
Condensers should have the same numerical aperture as
objective.
The ideal condenser should form a perfect image of the light
source.
Three types of condensers are used :
Abbe Condenser
Aplanatic Condensers
Achromatic Condensers
53. Iris diaphragm
Also called aperture diaphragm
Used to control the cone of light entering the condenser
Intensity should always be reduced by using filters and not
by closing the diaphragm
Adjustment of this iris diaphragm will alter the size and
volume of the cone of light focused on the object.
54. Iris diaphragm
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.
55. Iris diaphragm
In both cases the resolution of the image is poor.
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.
56. Filter carrier
Usually a metal ring, pivoting on a screw to facilitate
the easy removal of filters
57. Mirror
Plano concave mirror, fitted about 4 inch below stage
Concave side have focus from object
Plane mirror must always be used with condenser
Built-in light source have mirrors fixed at the base.
59. Magnification
Magnification of lens will depend on its conjugate foci, i.e. the
distance from object to the lens and that from lens to image.
Magnification is the product of the magnification of the
objectives and eyepieces and is dependent on following factors:
1. Focal length of objective
2. Distance between focal plane of objective and image it produces
3. Magnification of eyepiece
Magnification =Tube length × Eyepiece magnification
Focal length of the objective
60. Illumination
Artificial illumination supplied by an electric filament lamp is
most commonly employed.
Source of illumination should be:
1. Uniformly intense
2. Should completely flood the back lens of the condenser with
light when the lamp iris diaphragm is open
3. Make the object appear as though it were self-luminous
In light microscope two different types of illuminations are used.
• Critical illumination
• Kohler illumination
61. Setting up microscope
Nelsonian method
Light source should be
homogeneous and no lamp
condenser
employed with bare light
source
Light source should be
focused on the object plane
by racking the substage
condenser up or down
62. Setting up microscope
Kohler illumination
Non homogeneous light
source
Lamp condenser is essential
to project an image of lamp
filament onto the substage
iris diaphragm
Lamp condensing lens
functions as light source
Used with compound
microscope
63. Micrometry
The standard unit of measurement in microscopy is a
micrometer(μm), which is 0.001mm
To measure microscopic objects an eyepiece micrometer scale is
used in conjugation with stage micrometer.
A. Eyepiece micrometer
Usually a disc on which arbitary scale is engraved
Placed inside Huygenuan eyepiece, resting on the field stop.
Gives sharp image of scale and have a greater eye clearance
B. Stage micrometer
Consists of a 3 x 1 inch slide on which a millimeter scale is
engraved in 1/10 and 1/100 graduations
64. Cleaning and Maintainance
A. Daily cleaning routine
Should be dusted daily and outer surface of lens of objective
polished with lens tissue or cotton wool
Top lens of eyepiece polished to remove dust or fingerprints
and microscope set up for correct illumination. If dust still
present, eyepiece may need to be dismantled and both lenses
cleared.
Substage condenser and mirror are cleaned in a similar
manner
Removal of chemically active and sharp pieces of grits and
foreign material if present
65. Cleaning and Maintainance
B. Weekly cleaning routine
Slides of adjustment, stage, substage wiped with cloth (in
xylene damp)
Lens system checked
Chip blower can be used for cleaning eyepiece and objective
66. Handling the Microscope
Carry it with 2 hands-one on the arm and the other under the
base.
Use lens paper (ONLY) to remove any oil from the 100X lens.
Once oil has been added to the slide, do not move back to the
40X lens to focus: oil should never get on this lens. If this
happens, it will be very difficult to get all of the oil off.
Turn the coarse adjustment knob so that the stage is far from the
lens.
Place the microscope back into the correct spot in cabinet, with
the arm toward us, making sure that the 10X low power lens is
in place, pointing towards the stage-not the 100X oil immersion
lens. The lens could hit against the stage and get scratched.
68. Dark Field
Fine structures can often not be seen in front of a bright
background as visibility is dependent on contrast between
object and the background.
69. Dark ground microscopy:
the oblique light is thrown upon the object which does not
enter through the objective, they appear as self illuminous
objects on dark background.
Only the reflected or scattered light forms the image of the
object.
70. Objectives and condensers:
Objectives must have a lower
numerical aperture than the
condenser
Low power- black
paper/glass inserted into filter
carrier.
central rays are cut off and
peripheral rays from the
condenser passes through the
object but do not enter the
objective; the only light
entering the objective will be
that scattered by the object
71. Objectives and condensers:
High power-special condenser
Oil immersion must be used between the objective and object to
ensure maximum reflected light from the object enters the
objective
Fixed-focus condenser: commonly used- thin glass slides and
coverslips( ideal no:1)
72. Setting up the microscope:
Thin preparation- thin slide- coverslip
Adjust light direct/through the condenser
Place a drop of oil immersion on lower side of slide and also on top
lens of the condenser
Move the rack up untill both surfaces meet without forming air
bubbles
If correctly focused a small point of light will illuminate the object
on a dark background(low power)
High power: place drop of oil on the coverslip and focused
73. Advantages and disadvantages
Advantages:
Finer structures can be seen clearly hence can be best used
for spirochetes.
Disadvantages:
Misleading impression of size
When stained , difficult to see
Need thinner sections without any refractory material like
oil, water droplets, air bubbles etc..
75. POLARIZING MICROSCOPY
PRINCIPLE:
Light rays when passed through a crystal are retarded in speed.
Being unevenly dense, the crystal will retard the rays to a different
degree hence the rays will be refracted or bent to differing degrees.
This is known as DOUBLE REFRACTION OR BIREFRINGENCE
The direction of vibration of the emergent rays will be at right
angles to each other.
76. Principle:
A ray of light entering such a crystal will be converted
into two rays which will emerge at two different points.
The emergent light rays will be polarized(one ray-on
single direction, second ray- single direction and right
angle to the first ray)
77. ISOTROPIC: substances through which light can pass in any
direction and at the same velocity , not able to produce polarized
light.
DICHROISM: A phenomenon given rise to by some substances and
crystals which can produce plane polarized light by differential
absorption.
PLEOCHORIC FILMS: Dichroic crystals are suspended in thin plastic
films and oriented in one direction .They can absorb all the colors
equally.
78. NICOL PRISM
It is composed of a crystal made of Icelandic spar slit in half
and the halves cemented together with Canada balsam.
Light rays having passed through it would emerge vibrating
in a single plane.
The single direction in which the light is vibrating when it
emerges is known as the optical path of the prism.
79. POLAROID DISCS
They are glass or celluloid covered discs with the ability to
polarize light
Act as a single crystal of herpathite embedded in nitro cellulose
and mounted between plastic sheets which is not only
birefringent but has the ability to absorb the ordinary ray which
would be refracted out of a Nicol prism
Only allows the extraordinary light to be transmitted.
80. MAIN COMPONENTS OF THE MICROSCOPE
The two polarizer's used are:
POLARIZER
ANALYSER
84. POLARIZING MICROSCOPY
In a polarizing microscope, a polarizing filter is placed
between light source and specimen.
The second polarizer called analyzer is placed above
specimen between objective and eyepiece.
85. POLARIZING MICROSCOPY
If the vibration directions
of the object correspond to
the vibration of the
polarizer when the
polarizer and analyzer are
at right angles there is
absence of light through
eyepiece.
86. Appearance of object depends on interference of the two
rays recombined in the analyzer which depends on phase of
difference between the two rays which in turn depends on the
difference in the two refractive indices of the crystal and on its
thickness
If the vibration direction do not correspond then the rays of
light transmitted by the object will be resolved in analyzer and
object appears bright on a dark background.
88. TYPES OF BIREFRINGENCE
INTRINSIC OR CRYSTALLINE
FORM
STRAIN
POSITIVE
NEGATIVE
QUARTZ AND COLLAGEN-POSITIVE BIREFRINGENCE
POLAROID-
DISCS,CALCITE,URATES,CHROMOSOMES-NEGATIVE
BIREFRINGENCE
89. SIGN OF BIREFRINGENCE
The ray passing through a medium of high RI is called slow and if
it passes through a medium of low RI it is called fast.
If the slow ray is parallel to the length of the crystal, or fiber
birefringence is positive.
If the slow ray is perpendicular to the long axis of the structure,
birefringence is negative.
Determined by the use of a compensator either above the
specimen or below the polarizer at 45° to the direction of
polarized light.
90. The compensator or specimen is rotated till the slow direction of
the compensator is parallel to the long axis of the crystal or fiber.
The field is now red and if the crystal is blue the birefringence is
positive
If the slow direction of compensator is parallel to the fast
direction of the crystal, it appears yellow and has negative
birefringence.
91. APPLICATIONS
It is used in the detection and observation of:
Artifacts like formalin pigment
Crystals of urate ,pyrophosphates etc
Lipids, myelin etc
Bone structure
Proteins like collagen,amyloid,keratin
Charcot-Leyden crystals, muscle striations etc.
93. PHASE CONTRAST MICROSCOPY
It is a technique which enables us to see very transparent
objects, which are almost invisible by ordinary transmitted light,
in clear detail and in good contrast to their surroundings, and to
see very small differences in thickness and density within the
objects.
This is accomplished by converting these slight differences in
refractive index and thickness into changes of amplitude.
94. PRINCIPLE
A ray of light is made of waves travelling together in a straight
line. When two such waves travel together,they are said to be in
phase. Such a ray will appear bright to the observer
If one of the waves is held up or made to change the path, they
will no longer travel together and are said to interfere with each
other, differing in their intensity
95. A special condenser and objective control the illumination in a
way that accentuates the differences in densities.
It causes light to travel different routes through the various
parts of the cell
The result is an image with differing degrees of darkness and
brightness collectively called contrast
96. Interference
CONSTRUCTIVE INTERFERENCE
Light rays are in phase
Amplitude or brightness is ‘doubled’ when recombined
DESTRUCTIVE INTERFERENCE
Light rays are incoherent
½λ out of phase
No light is seen
Maximum interference
97.
98. PARTS OF A PHASE CONTRAST
MICROSCOPE
ANNULUS:
Made of opaque glass
Has a hollow clear ring
Can be centered by means of centering screws
PHAZE PLATE OR Z PLATE
Clear glass disc with a circular trough etched in it to half the depth of disc
The light passing through the trough has a phase difference of 1/4ƛ
compared to the rest of the plate
Also contains a neutral density light absorbing material to reduce
brightness of direct rays.
99. PARTS OF A PHASE CONTRAST
MICROSCOPE
HIGH INTENSITY COMPOUND LAMP
Usually used with a mercury green filter
AUXILLARY TELESCOPE
Used in place of an eyepiece for examining the back focal
plane of the objective
101. WORKING OF THE MICROSCOPE
Annulus is placed in the condenser and the phase plate is placed
in the objective
It allows only a small ring of light to pass into the microscope
The phase plate has a circle engraved on it which should match,
with the ring of light coming in from the annulus through the
condenser.
102. Some rays of light will pass through unaltered while some
rays will be retarded or diffracted by approximately 1/4ƛ.
On passing through a phase plate the diffracted ray is
retarded further by 1/4ƛ and will now interfere with the
direct light ray.
The total retardation of diffracted rays is now 1/2ƛ and
interfere will produce image contrast thus revealing even
small details.
103. APPLICATIONS
For examining unstained bacteria
For examining wet preparations of specimens
For examining faecal preparations for trophozoites or amoebae
In searching for trypanosomes in blood and other body fluids
104.
105. INTERFERENCE MICROSCOPY
generates mutually interfering beams which produce the
contrast. It is this feature which enables very small phase
changes to be seen and measured.
The two rays which eventually combine to produce image are
formed by a plate of birefringent material placed immediately
above the condenser.
These two rays having passed through the object plane are
recombined by a similar plate of birefringent material below the
front lens of the objective.
106. One ray passes through a point
in the object and the other
through an area adjacent to it.
Each point in the final image is a
compound one made up of two
mutually interfering rays.
A special Wollaston prism is
added to the condenser to split
the beam of light and also to
recombine the two dissimilar
beams.
107. APPLICATIONS
To study individual parts of living cells with maximum
resolution of detail
To estimate dry mass when it is applied as a highly accurate
optical balance
To assess section thickness of specimen.
108. DIFFERENTIAL INTERFERENCE
CONTRAST MICROSCOPY
Designed by Nomarski hence also called as Nomarskis
microscope.
Relies on the interference of a pair of wave fronts to
generate contrast.
109. It comprises of:
-a polarizer
-a condenser with a modified Wollaston prism
-a beam splitting slide
This slide consists of a modified Wollaston prism oriented at
45° to anattached analyzer,mounted in an adjustable carriage
and accommodated in the analyzer slot between the
objective and the eyepiece.
110. Working of the microscope
Polarized light passes through the prism below the
condenser
The prism below the condenser acts as a compensator
Every interference fringe of the upper prism is correlated
with an interference fringe of the same order but opposite
sign in this compensator
111. The two rays pass in turn through the condenser the
object and the objective before passing through the
second prism and analyzer.
The upper prism can be moved laterally enabling the rays
to be displaced laterally or sheared before being
recombined in the analyzer when they undergo
interference.
This produces ‘interference contrast’ and together with
rotation of the polarizers enhances the three-dimensional
(3D) effect in the image.
112.
113. ADVANTAGES
Wide variety of interference colors can be used
Improved image contrast
No phase halo included
Lateral shearing of rays is reduced so that excellent three
dimensional images can be produced.
114. USES
As an infinitely variable phase contrast microscope,
individual parts of living cells can be studied.
As a highly accurate optical balance, used for estimating dry
mass down to 1x10 gm
Quantitative measurement of phase change or optical
path difference
Studying live and unstained biological samples such as a
smear from a tissue culture or individual water borne celled
organism
115.
116.
117. References
Textbook of oral pathology – Jaypee brothers, 1E; Anil Ghom,
Shubhangi Mhaske
Histology A Text and Atlas - With Correlated Cell and Molecular
Biology, 7E (2015) ; Wojciech Pawlina
Bancroft’s Theory and Practice of Histological Techniques ,7th edition
CFA Culling’s Histological techniques
Essentials of Microbiology; Surinder Kumar ,1st edition, 2016
MICROSCOPE Basics and Beyond, Revised edition 2003, Mortimer
Abramowitz
Hinweis der Redaktion
The object AB to be magnified is placed just outside the principal focus of the objective so that its real image is formed on the other side of objective. The image formed is real, inverted and magnified. The image due to the objective acts as the object for the eye piece. The position of the eyepiece is so adjusted that the image lies within the focus of eyepiece Fe. Eyepiece acts as magnifying glass and to forms the final image is virtual, erect and magnified.
Microscope manufacturers label their objectives with color codes to help in rapid identification of the magnification and any specialized immersion media requirements.
This is very helpful when you have a nosepiece turret containing 5 or 6 objectives and you must quickly select a specific magnification. Some specialized objectives have an additional color code that indicates the type of immersion medium necessary to achieve the optimum numerical aperture.
To check the setting for a particular specimen (where the coverslip thickness is unknown) first focus upon a high contrast area, then determine whether changing the collar setting increases or decreases the contrast.
The main purpose of the condenser is to focus or concentrate the available light into the plane of the object i.e. the condenser collects the maximum possible light reflected by the mirror or the inbuilt light source and condenses or converges it to a very small area at the position of the specimen (Fig. 1.2).
Rheinberg illumination, a form of optical staining, is a striking variation of low to medium power darkfield illumination using colored gelatin or glass filters to provide rich color to both the specimen and background.
Rheinberg illumination, a form of optical staining, is a striking variation of low to medium power darkfield illumination using colored gelatin or glass filters to provide rich color to both the specimen and background.