a short seminar on light microscope and its parts. includes history and origin.

2. MICROSCOPE- II
Presented by:
Dr. SONAL AGGARWAL
MDS II Year

3. CONTENTS
• The basics properties of light.
• Image formation.
• The light microscope and it’s
components.
• Advantages and limitations of
light microscope

4. DEFINITION
•New latin microscopium, from micr- small +
scopium –scope
•An optical instrument consisting of a lens or
combination of lenses for making enlarged
images of minute objects

5. 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.

6. •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.

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

8. Light microscopes
Two basic configurations of
light microscope:
1. Simple microscope
2. Compound microscope

9. PARTS OF A
MICROSCO
PE
1. Base or metal stand
2. Pillars
3. Inclination Joint
4. Curved arm
5. Body tube
6. Draw tube
7. Coarse adjustment
8. Fine adjustment
9. Stage
10. Mechanical stage
11. Revolving nosepiece
1. Light source
2. Diaphragm
3. Condensor or sub stage condenser
4. Objective
5. Eyepiece or ocular

11. 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.

12. 12
ILLUMINATION - Lamp, sunlight,
battery operated lamp, 60 W bulb,
Quartz halogen light.
FILTERS - Blue, Green, Heat
absorbing filters, Barrier filters.

13. KOHLER’S ILLUMINATION
• Many microscopes utilize backlight illumination
compared to traditional direct light illumination.
• In Koehler illumination, incident light from an
illumination source, such as a light bulb, floods the
object under inspection with light from behind.
• It employs two convex lenses: the collector lens and
the condenser lens.
• This is important because it ensures the user is not
imaging the filament of the light bulb.
• brightfield illumination.

15. 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.

16. •There are three types of condensers as follows:
(a) Abbe condenser (Numerical aperture=1.25): It is
extensively used.
(b) Variable focus condenser (Numerical aperture =1.25)
(c) Achromatic condenser (Numerical aperture =1.40): It
has been corrected for both spherical and chromatic
aberration and is used in research microscopes and
photomicrographs.

17. 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.

19. 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.
• Vernier scales are incorporated in most
cases.
• Spherical stages.

20. 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.

21. 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.

23. 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 ø

24. Numerical
aperture
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 ø is the angle
included between the optical axis of the lens and the outermost ray that can enter the front
lens
NA = n x Sin ø

25. 25
Magnification

26. 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.

27. 27
• Forms magnified virtual & erect
image
• TYPES
(a) Monocular
(b) Binocular
(c) Trinocular
or
(a) Huygenian (negative)
(b) Ramsden (positive)
(c) Compensating
EYE PIECE

28. 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.
• 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

29. 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 :
(Optical tube length/Focal length of objective) ×
Magnification of eyepiece.

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

31. 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.

32. References
• Handbook of Histopathological technique by C. F. A. Culling.
• Bankroft’s theory and practice of Histological techniques, chapter
3.
• Susan C. Lester’s manual of Surgical Pathology, Part I, Chapter 9.
• http://www.nobelprize.org/educational/physics/microscopes/timelin
e/
• http://www.nature.com/nprot/journal/v7/n9/fig_tab/nprot.2012.096_
T1.html