The document discusses the components and operation of optical microscopes. It describes the body tube, stages, illumination methods including Kohler and Nelson techniques, objectives, eyepieces, and other parts. It also covers magnification calculation, microscope setup, cleaning, measurement using micrometers, and different microscope types such as fluorescence, polarization, and electron microscopes. The document provides detailed information on the structure and use of optical microscopes.

2. MECHANICAL COMPONENTS
3.The body tube
Nose piece or carrier

3. 4. Supporting structure

4. 5. Adjustments
(a) Coarse adjustment
(b) Fine adjustment

5. 6. STAGE

6. The stage is of two types:
 Fixed stage
 Mechanical stage
A mechanical stage takes a 3X1 inch slide and moves over an
area approx. 31/2 X 11/4 inches for whole slide examination
Special stages are there to take very large slides and petri
dishes. Circular rotating stages (polarizing microscopes)
Mechanical stages are fitted with Vernier scale.

7. Illuminating apparatus
The Substage
 The condenser- To focus the light on the object when using
objectives with a focal length of 16mm or less.
 Two lens Abbe condenser(Earnest Abbe 1870s)
 Three lens aplanatic
 Highly corrected achromatic condenser
 The iris diaphragm(aperture diaphragm)- To control the cone of
light entering the condenser.

9.  The filter carrier- A recessed metal ring with filter Decreases
intensity of light
 The mirror- flat on one side and concave on the other, to allow
light directed into the condenser from any angle, a built in light
source with a fixed mirror.

10. Illumination
Source of illumination:-
 Uniformly intense( 60 Watt pearl bulb)
 Should completely flood the back lens of the condenser
uniformly with light when the lamp iris diaphragm is open
 Make the object appear as though it were self-luminous

11. SETTING UP THE MICROSCOPE
Illumination by Nelson or Kohler methods
Nelson method-
 Light source should be homogenous and no lamp condensers
use.
 Bare light source
 The light source should be focussed on the object plane by
moving substage up or down.

12. Kohler method
 Light source does not be homogenous.
 Lamp condenser is used to project an image of lamp filament
on to the substage iris diaphragm.
 It is used with compound lamps and should always be used for
photomicrography.
 Cardinal rule for microscopist- Always rack the objective
down near the object before looking through the eyepiece and
then to focus on the object or the front lens of objective.

13.  Particularly when using oil immersion objectives which have
short working distance.
 In Nelson the light source and the object are in focus.
 In Kohler illumination the light source is focussed on the
aperture diaphragm, field diaphragm and object are in focus.

15. Magnification
 It depends on its conjugate foci; that is the distance from
the object to the lens and that from the lens to the image.
 The magnification of the microscope is the product of the
magnifications of the objective and the eyepiece

16. Magnification depends on the three factors:-
 The focal length of the objective
 The distance between the focal plane of the objective and the
image it produces
 The magnification of the eyepiece
Magnification= Tube length X Eyepiece magnification
Focal length of
objective

18. CLEANING AND MAINTENANCE
 The microscope should be dusted daily.
 Outer surface of the lenses of objective polished with lens tissue
or cotton wool
 Top lens of the eyepiece should be polished to remove dust or
fingermarks.
 Rotation of the eyepiece will show if any dust present.

19. Weekly Cleaning Routine
 The slides of the coarse adjustment, the mechanical stage and
the substage condenser should be wiped with a cloth dampened
with xylene to remove dust.
 The lens system should be checked and cleaned.
 Dust is removed from the back lenses of objectives by use of
rubber bulb.
 Interocular adjustments slides cleaned once a month

20. MICROMETRY
 Standard unit of measurement in microscopy is a
micrometre(µm)= 0.001mm.
 To measure microscopic objects
 An eyepiece micrometer scale is used in conjunction with stage
micrometer.
 The eyepiece micrometer scale is a disc engraved with an
arbitrary scale.
 Placed inside the Huygenian eyepiece.
 The stage micrometer consists of a 3X1 inch slide on which a
millimetre scale is engraved in 1/10 and 1/100 graduations.

21.  Insert a micrometer eyepiece scale and place the stage
micrometer on the stage.
 Select the objective to be used when measuring the object,
and focus on stage micrometer scale.
 Determine the no. of divisions of eyepiece scale equal to
an exact no. of divisions of stage micrometer scale.
 Remove the stage micrometer , focus on object to be
measured and determine no. of eyepiece divisions.

22.  100 stage divisions= 1mm= 1000µm
 100 eyepiece divisions = 10 stage divisions
 Therefore, 100 eyepiece divisions= 100µm
 Therefore, 1 eyepiece division= 1µm
 Therefore, 12 eyepiece divisions= 12µm
 The diameter of object= 12µm

23. RACK AND PINION MECHANISM
Screw and Lever mechanism

24.  Improving the spatial resolution of optical microscopes is
important for a vast number of applications in the life sciences.
 Optical microscopy allows intact samples and living cells to be
studied in their natural environment, tasks that are not possible
with other microscopy methods (e.g. electron microscopy)
 By using interference and structured light methods microscope
resolution has been improved to 100 nm, and with non-linear
methods a ten times improvement has been demonstrated to a
current resolution limit of 30 nm.

25.  Brightfield Microscopy is the most elementary form of
microscope illumination techniques and is generally used with
compound microscopes.
 The name "brightfield" is derived from the fact that the
specimen is dark and contrasted by the surrounding bright
viewing field. Simple light microscopes are sometimes referred
to as brightfield microscopes.

26. Flea Glasses
The next step toward the microscope was taken in the 1500s by
the development of a small tube fitted with two pieces of glass
such that the part nearer the eye was convex and the other
terminated in a flat piece of glass. This device was commonly used
to closely examine small creatures such as the flea. Thus, the "flea
glass" came into common use.

27. OTHER MICROSCOPES USED
Darkground Microscopy
 Most objects examined microscopically are naturally transparent.
 In general they reflect or scatter light rays.
 In darkground illumination, oblique light is thrown upon them
which does not enter the objective.
 They appear as self luminous objects on a dark background.
 Gives misleading impression of size, fine particles appears much
larger.

28. Fluorescence Microscopy
 In 1852, Stokes first used the word ’fluorescence’- describe the
reaction of fluorspar to ultraviolet light.
 In 1903 R.W.Wood devised a filter which absorb visible light and
transmit only ultraviolet light.
 In 1911, Lehmann described first fluorescence microscope.
 In 1935, Max Haitinger pioneered and developed the technique
of staining histological preparations and smears with fluorescent
dyes.

29.  Fluorescence- When quantum of light is absorbed by an atom or
molecule, an electron is boosted to a higher energy level. When
the displaced electron returns to its original ground state it may
emit a quantum of light.
 If the light is emitted only during time of exposure it is
fluorescence.
 If emission persists after the exciting light is cut off it is called
phosphorescence

30. Polarizing microscopy
 (applications, Wolman,1975)
 Artefacts. Formalin pigment, sutures, starch
 Crystals. Talc, urate, pyrophosphate, silica,etc.
 Lipids. Myelin
 Bone structure
 Protein. Collagen, amyloid, keratin
 Muscle striations, charcot-Leyden crystals

32. Phase-contrast microscopy (Zernicke, 1935)
 It is a technique to see very transparent objects, which are
invisible by ordinary transmitted light, in clear detail and in good
contrast to their surroundings
 To see very small differences in thickness and density within
object.

34. Interference Microscopy(Hale,1958)
 Classifies these as:-
 (a) multiple beam systems
 (b) double beam systems
Uses:-
 As an infinitely variable phase-contrast microscope with which
individual parts of living cells may be studied with maximum
detail, in wide variety of interference colors.

36.  ULTRA VIOLET MICROSCOPE
 Ordinary optical lens nearly opaque to ultra violet
 Lens system of it  quartz lens
 Resolution improved twice ( 0.1 micrometer)
 UV light also used in fluorescence microscopy.
 Some substance have property of emitting visible light when UV
focussed specimen glows  observed by fluorescence

37. ELECTRON MICROSCOPY
 Transmission electron microscope( TEM )
 Utilizes system analogus to light microscope.
 Illuminating sources  beam of high velocity electrons
accelerated in vacuum.
 beam passed through specimen  focussed in fluorescent
screen by series of electromagnetic fields.
 wave length of electrons depend on acceleration voltage used.

39.  Routinely  wave length of electron of order of 0.05 A
 Electric or magnetic field used as lens are not perfect & do not
have numerical aperture of optical lens.
 practical limit of resolution  2A ( 0.2 nm )
 usual limit  3.5 A ( 0.35 nm).
 Structure smaller than macro molecule can be visualized.
 Scanning electron microscopy ( SEM )  recent development
 Do not depend on electrons passing through specimen

40.  Beam of electrons bombards surface of specimen.
 As beam strikes a point on specimen deflected primary &
emitted secondary electrons from surface is collected by
detector.
 Signals from many such points form image  displayed on
cathode ray tube.
 Thin section of thick organs ( liver , kidney ) viewed by
transillumination with quartz rods.
 Tissues can be preserved in harmless liquid (serum , 0.85 %
NaCl )

41. REFERENCES
 Gear Nomenclature, Definition of Terms with Symbols. American
Gear Manufacturer Association p. 72. ISBN 1-55589-846-
7.OCLC 6556273
 Websites:
 nobelprize.org/Microscopes
 history-of-the-microscope.org
 microscopyu.com/articles/optics

42.  Cellular pathology Techniques, Culling : 4th edition
 Theory and Practice of Histological Techniques, Bancroft: 6th
edition
 Modern Microscopy (Elementary theory and practice), C.F.A
Culling

43. THANK YOU