Light microscope is one the most used instrument by a pathologist and they need to know how it functions and make the most of it

1. LIGHT MICROSCOPE
Dr.Vaani Lalitya

2. Objectives
Main topics addressed:
 The basics properties of light.
 Image formation.
 The light microscope and it’s components.
 Advantages and limitations of light
microscope

3. Light
 Visible light occupies a very narrow portion of
400 – 700nm between UV and Infrared
radiation in the electromagnetic spectrum.
 Electromagnetic energy is complex, which is
both wave like and particle like.
 The natural light we see is a complex mixture
of lights with different wavelengths.
 Therefore almost all light sources provide a
mixture of wavelengths of light.

5. Electromagnetic spectrum.

6. Light
 Natural light which is a
mixture of lights of
different wavelengths.
 Monochromatic
 Laser

7. Properties of Light
 Shorter wavelengths have higher energy for a
given brightness of light.
 Energy content of light is expressed as energy
level or amplitude based electron volts per
photon.
 Visible light has an energy of one electron
volt per photon and the energy increases as
one moves towards the violet and ultraviolet
range of the spectrum.

8. Properties of Light
 Approaching the soft X- Ray spectrum, the
energy levels reach 50-100eV per photon.
 It is this higher energy in the shorter
wavelengths of that is exploited to elicit
fluorescence in some materials.
 Emission spectrum.
 Color temperature.

9. Properties of light
 Some of the light is absorbed by the medium
through which it passes.
 This is seen as reduction in amplitude and
energy level.
 The medium through which the light passes
can also have an effect in the speed of light
which is known as Retardation.

10. Retardation
 The media through which the light passes will
be able to slow down or retard the speed of
the light in proportion to the density of the
medium.
 Higher the density, greater the retardation.
 Light rays entering a sheet of glass at right
angle are retarded but their direction is
unchanged.

11. Refraction
 If the light enters the glass at any angle other
than right angle, a deviation in the direction
will occur in addition to retardation, known as
Refraction.
 A curved lens will exhibit both refraction and
retardation.
 The extent of which is determined by angle of
incidence, refractive index and curvature of
the lens.

12. Refraction

13. Refraction

14. Refractive index
 Refractive index = Sin I
Sin R
Where I is the angle of incidence and R is angle of
refraction.
Higher the density of the medium, greater the RI.
 The RI of most transparent substances is known and
is of great value in the computation and design of
lenses, microscope slides and coverslips, and
mounting media.
 Air has a refractive index of 1.00, water 1.30, and
glass a range of values depending on type but
averaging 1.50.

15. Image formation
 Focal point.
 Focal length.
 Conjugate foci.
 Real image.
 Virtual image.

16. Focal point and focal length

17. Conjugate foci
 There are conjugate foci on either side of the
lens such that the object placed at one focus will
produce a clear image on the screen placed at
the other focus.
 The conjugate foci vary in position. As the object
is moved closer to the lens, image will be formed
further away, at a greater magnification and
inverted.This is real image.
 This is formed by the objective lens of the
microscope.

18. Real image formation

19. Conjugate foci
 If the object is placed yet nearer the lens,
within the principle focus, the image is
formed on the same side as the object, is
enlarged, the right way up, cannot be
projected on to a screen and can be seen
through lens.This is called virtual image.
 This is formed by the eye piece of the
microscope and it appears to be at a distance
of approximately 25cm from the eye, around
the object stage level.

21. Virtual image

22. Ray path through microscope

23. Chromatic aberration
 Light is composed of spectrum of colors, each
having a different wavelength, will be
refracted to a different extent, with blue
being brought to a shorter focus than red.
 This lens defect is known as chromatic
aberration and results in an unsharp image
and distorted edges.
 This is known as chromatic aberration and it’s
correction is known as achromatism.

24. Chromatic aberration
 It is possible to construct compound lenses of
different glass elements to correct this.
 An achromat is corrected for two colors, red
and blue, producing a secondary spectrum of
yellow/green, which is in turn corrected by
adding more lens components like fluorospar,
three colors can be brought into focus – the
more expensive – Apochromat.

25. Chromatic aberration

26. Spherical aberration
 Spherical aberration is caused by the virtue of
its curvature, where the light rays entering
the lens at the periphery are refracted more
than the light rays entering at the center of
the lens, and thus not brought to a common
focus.
 These defects are also corrected by using a
combination of lens elements of different
glass and of different shape.

27. Spherical aberration

29. LIGHT MICROSCOPE

30. A brief history….
 1590- Hans Lippershey and Zacharias Janssen have
made the first compound microscope.
 1609- Galelio Galilie developed a microscope with a
convex and concave lens.
 1665- Robert Hooke’s book called micrographia
documented a wide range of observations through
microscope.
 1674-AntonVan Leeuwenhoek
 1826- Joseph Jackson Lister developed achromatic
lens
 1860- Ernst Abbe discovers the Abbe sine condition.
 1931- Ernst Ruska starts to build the first electron
microscope.

31. Zacharias Janssen

32. Anton van Leeuwenhoek’s
microscope

33. Light microscopes
 Two basic configurations of light microscope:
 1. simple microscope
 2. compound microscope

34. Simple microscope
 Simple microscope is where a single lens or a
closely placed set of lens is used to magnify
the object.
 The magnification is usually limited.
 Eg. Magnifying glass and eyepiece of a
compound microscope.

35. Compound microscope

36. Light microscope - Introduction
 Also known as Optical microscope.
 The light microscope, so called because it
employs visible light and a system of lenses
to visualize small objects.
 Optical microscopes are the oldest design of
microscope and were probably invented in
their present compound form in early 17th
century.

37. Light Microscope
 Probably the most well-used and well-known
research tool in biology.
 The biggest challenge lies in
 A. obtaining sufficient contrast.
 B. finding the focal plane.
 C. obtaining good resolution.
 D. recognizing the subject when one sees it.

38. Light microscope

39. Standard light microscope
 Principally has two components:
 The microscope proper and
 The stand/supporting system.

40. Components of Compound
microscope
 Light source
 Condenser
 Objective stage
 Objectives
 Body tube
 Eye piece
 Magnification
 Supporting stand

41. Light source- Illumination
 Initially it used to be sunlight.
 Later oil lamps were used.
 Simple pearl bulb. Or high intensity lamp.
 Built-in light source for the recent
microscopes.
 Halogen bulbs.
 Neutral density filters to reduce excess
brightness.

43. Condensers
 The first major optical component.
 The main purpose of the condenser is to
focus the available light into the plane of the
object.
 Within comfortable limits, the more the light
at the specimen, the better is the resolution
of the image.
 Capable of vertical adjustments
 Adjusting the Iris diaphragm/aperture
diaphragm.

44. Iris diaphragm
 The correct adjustment is when the numerical
aperture of the condenser is matched with
the numerical aperture of the objective in
use.
 This is achieved, approximately, by removing
the eye piece, viewing the sub-stage iris
diaphragm in the back focal plane of the
objective, and closing it down to two-thirds
of the field of view.

45. Objective stage
 Above the condenser is the rigid stage with an
aperture(1-1 ½ inches) through which light may
pass. It has metal spring clips.
 The stage supports glass slide bearing the
specimen and therefore should be sturdy and
perpendicular to the optical path.
 In order to hold the slide firmly and to move the
slide easily and smoothly, a mechanical stage is
either attached or built in.
 Vernier scales are incorporated in most cases.
 Spherical stages.

46. The objective
 The objective screws into the lower end of the
body tube by means of a standard thread, thus
all objectives are interchangeable.
 They are usually designated by their focal length
rather than their magnifying power because
their actual magnifying power depends on the
tube length at which they are used.
 Some manufacturers label objectives with the
magnifying power of the lens where there is no
draw tube.

47. Objectives
 The type and quality of the objective has the
greatest influence on the microscope as a
whole.
 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 somewhere above.
 Fixed tube length systems(160mm-DIN Std;
And 170mm- Leitz only) have now been
replaced by infinity corrected objectives.

48. Objectives
 The ability of an objective to resolve detail is
indicated by its numerical aperture and not by
its magnifying power.
 Resolution is restricted by two factors: the
numerical aperture and wavelength of the
light employed.
 NA = n Sin u

49. Numerical aperture
 N = 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, and u is the angle
included between the optical axis of the lens
and the outermost ray that can enter the front
lens

50. Body tube
 Above the nose piece is the body tube
 Three main forms are available : monocular,
binocular, and the combined photo-binocular.
 Provision is made for the binoculars to adjust the
interpupillary distance.
 Altering the interpupillary angle may alter the
mechanical tube length thus altering the optical
path.
 This can be corrected by adjusting the individual
eye piece tubes or by a compensating
mechanism built into the tube.

51. Eye piece
 Final stage in the path of the optical path.
 Two most commonly used ones are
huygenian (achromatic objective) and
compensating eyepiece (apochromatic
objective).
 It is basically a simple microscope to observe
image formed by the objectives.
 It has two lens. Field lens(lower) and upper
lens.

52. Eye piece
 Their function is to magnify the image formed
by the objective within the body tube, and
present the eye with a virtual image,
apparently in the plane of the object being
observed; usually this is an optical distance of
250 mm from the eye

53. Magnification
 Total magnification is the product of the
magnification values of the objective and
eyepiece, provided the system is standardized
to an optical tube length of 160nm.
 The formula for magnification is
(Optical tube length/Focal length of objective) ×
Magnification of eyepiece.

54. Micrometry
 The standard unit of measurement in
microscopy is a micron, which is 0.001mm.
 An eyepiece micrometer scale is used in
conjunction with a stage micrometer.
 Eyepiece micrometer scale is divided into
1/10 and 1/100 graduations, is placed inside
Huygenian eyepiece, resting on a field stop.
 Kellner eyepieces have scale permanently in
position.

55. Micrometry
 The stage micrometer has a 3 x 1 inch slide on
which a millimeter scale is engraved in 1/10 to
1/100 graduations.
 Insert eyepiece and stage micrometer scales.
 Select an objective to be used and focus on stage
micrometer scale.
 Determine the no. of divisions on the eyepiece
scale equal to the no. of divisions on the stage
micrometer scale. Remove the stage micrometer
and focus on the object to be measured.

56. Micrometry
 Determine the no. of eyepiece divisions
exactly covered by the object.
 Assuming 100 eyepiece divisions were equal
to 10 stage divisions, and say for eg. the
object has covered 15 eyepiece divisions,
 100 stage divisions = 1mm = 1000 microns
 10 stage divisions = 100microns
 1 eyepiece division is equal to 1 micron,
 Then 15 eyepiece divisions = 15 microns.

57. Advantages of light
microscope
 Most widely used tool to study organic and
inorganic research.
 Cost effective.
 Simple setup with very little preparation
required.

58. Disadvantages
 Biological samples are often low contrast
with little natural pigmentation, so samples
usually need to be stained.
 Staining may destroy or introduce artefacts
Resolution is restricted to ~0.2 μm.

59. TLC!! Proper care of
microscope!
 Cleaning. Daily cleaning routine.
 Microscope – dusted daily, outer surface of
the lens should be wiped with lens paper or
well washed silk.
 Top lens of the eyepiece polished to remove
dust or fingermarks.
 Clean the substage and mirror in a similar
way
 Cover it when not in use.

60. References
 Bankroft’s theory and practice of Histological
techniques, chapter 3.
 Susan C. Lester’s manual of Surgical Pathology, Part
I, Chapter 9.
 Handbook of Histopathological technique by C. F. A.
Culling.
 https://en.wikipedia.org/wiki/Optical_microscope
 http://www.nobelprize.org/educational/physics/micr
oscopes/timeline/
 http://www.nature.com/nprot/journal/v7/n9/fig_tab/
nprot.2012.096_T1.html

61. Thank you…