MICROSCOPE

THE MICROSCOPE
 Lesson Objectives

• Identify the different Parts of A Microscope

• State and differentiate the types of Microscopy
• Compare magnification and resolution.
• Define parfocal and describe how it is used in Microscopy.
• Define Alignment and describe the process of aligning a
microscope.
• Demonstrate a good technique in using the microscope.
• Discuss and demonstrate proper care and handling of the
microscope.
Microscope
- is an instrument that produces enlarged images of small objects, allowing
the observer an exceedingly close view of minute structures at a scale
convenient for examination and analysis.

Microscopy
- the science of the use of microscopes
PARTS OF THE MICROSCOPE

Structures Basic to All Types of Microscope:

1. The Framework
2. Illumination System
3. Magnification System
4. Focusing System
The Framework

a. Base – is the firm foot on which the microscope rests. It is

usually horseshoe-shaped on monocular microscope

b. Arm – is the structure that supports the magnifying and

adjusting systems. It is also the handle by which the
microscope can be carried without damaging the delicate
parts.
● In cases of older models of monocular microscope the
arm is attached to the base by the Inclination Joint. It can
also be used for tilting the microscope if required for
observation in sitting position.
The Framework

c. Stage – is the horizontal platform, or shelf, on

which the object being observed is placed.

- Mechanical stage which most microscopes have

provides easier manipulation of the object being
observed using the mechanical controls or
adjustment screws which move the slide up,
down, left or right. Microscopic slides are held on
the stage by either simple side clips or by a
mechanical stage clip.
 
The Framework

ARM

STAGE

Inclination
Joint
Mechanical
Slide
Controls

BASE
Illumination System

a. Light Source – built in bulb (Halogen Bulb) or a mirror. The

two sided mirror provides necessary illumination through
reflection of natural or artificial light.
● On top of a built-in light source is an in-built filter holder to fit
the filter of desired quality:
- Blue filters are used to change the light from ordinary
electric bulbs into a more natural white light.
- Neutral density filters are used to reduce brightness without
changing the colour of the background
● The light intensity is controlled by a rheostat, dimmer switch, or
slide ensuring both adequate illumination and comfort for the
microscopist.
Illumination System

b. Condenser
– directs and focuses the beam
of light from the bulb onto the
material under examination. The
condenser position is adjustable; it
can be raised and lowered beneath
the stage by means of an
adjustment knob. When it is
correctly positioned, the image
field is evenly lighted.

● Microscopes generally use a

substage Abbé type condenser.
Illumination System

c. Iris Diaphragm
– also controls the amount of light passing
through the material under observation.

● Aperture Iris Diaphragm

– It is located at the bottom of the
condenser body. This consists of series of
horizontally arranged interlocking plates with
central aperture. It can be opened or closed as
necessary to adjust the intensity of the light by
means of a lever or dial.

● Field Diaphragm – It is located in the light

port in the base of the microscope, through
which the light passes up to the condenser.
Illumination System

CONDENSER

Diaphragm
Lever

LIGHT
SOURCE
 Magnification System

a. Ocular (Eyepiece) – a lens that magnifies the

image formed by the objective. The usual
magnification of the ocular is 10 (10x); however, 5x
and 20x oculars are generally available.

● Microscopes with two eye-pieces are called

Binocular microscopes. Those with only one
eye-piece are called Monocular microscopes.

● The distance between the two oculars

(interpupillary distance) is adjustable, as is
the focus on one of the oculars (diopter
adjustment).
Magnification System

b. Objectives – responsible for primary image formation and play a central

role in determining the quality of images the microscope is capable of
producing.

● The objectives are mounted on a revolving nosepiece, which is a

pivot that enables a quick change of objectives.

● Objectives are described or rated according to focal length, which is

inscribed on the outside of the objective. The Focal Length is a
physical property of the objective lens and is slightly less than the
distance from the object being examined to the center of the
objective lens. Practically speaking, the focal length of a lens is very
close in value to the working distance, the distance from the
bottom of the objective to the material being studied.
Magnification System
PARFOCAL - means that if one objective is in focus and change is made to
another objective, the focus will not be lost and require only minimum adjustment.

Most microscopes have three objectives, sometimes four, objectives.

Oil Immersion High Power Low Power Scanning

Objective Objective Objective Objective
Magnification System

i. Low Power Objective (LPO)

- This objective is used for initial scanning and
observation in most microscope microscope work.
This is also the lens employed for the initial focusing
and light adjustment of the microscope. Usually a
10x lens with 16-mm working distance.

- Very Low-power objective with 4x magnification

that is usually the fourth objective lens (Scanning
lens) in some microscope is used in the initial
scanning in the morphological examination of
histologic sections
Magnification System

ii. High Power Objective (HPO)

- Is used to study histologic sections and wet

preparations (e.g., urine sediment) in more
detail. Usually a 40x lens with 4mm working
distance.

- When using this objective the condenser

should generally be all the way up (or very
slightly lowered) and the light field slightly
closed, with aperture iris diaphragm for
maximum focus.
Magnification System

iii. Oil Immersion Objective (OIO)

- Oil immersion lens is routinely used for

morphologic examination of blood films
and microbes. OIO lens requires a
special grade of oil called “Immersion
oil” be placed between the objective and
the slide or cover glass.
- Generally a 100x lens with a 1.8 mm
working distance.
Magnification System

EYEPIECE

Revolving
Nosepiece

Objectives
Focusing System

a. Oculars are a component of the focusing system.

b. Body tube is a part of the microscope through which the light passes
to the ocular. This is the tube that actually conducts image.
-Draw Tube is a cylindrical structure on top of the body tube that holds
the ocular lenses.
c. Adjustment system enables body tube to move up or down for focusing the
objectives.

This usually consists of two adjustments:

● Coarse Adjustment gives rapid movement over a wide range and is used to
obtain an approximate focus.
● Fine Adjustment gives a very slow movement over a limited range and is
used to obtain an exact focus after coarse adjustment.
 Magnification System
OCULARS

Draw Tube
Body Tube

Coarse
Adjustment

Fine
Adjustment
MICROSCOPIC TERMS

 Total Magnification – it is the product of the

magnifications between the two magnifying lenses (the
objectives and the eyepiece).

Example: The total magnification of an object seen with a

10x ocular and a10x objective is 100 times (100x).
Magnification units are in terms of diameters, so 10x means
the diameter of an object is magnified to 10 times its original
size.
MICROSCOPIC TERMS

 Resolution
– indicates how small and how close individual objects
can and still be recognized.
 Alignment
– Done to get the best view in a microscopic
observation.
- When properly aligned, the microscope is adjusted
in such a way that the light path through the
microscope, from the light source to the eye of the
observer, is correct.
MICROSCOPIC TERMS

 Numerical Aperture (NA)

– the light gathering ability of a microscope objective. NA is a measure of
the number of highly diffracted image-forming light rays captured by the
objective. Higher values of numerical aperture allow increasingly oblique
rays to enter the objective front lens, producing a more highly resolved
image.

TERM MAGNIFICATION NA VALUE

Scanning 4x 0.1
LPO 10x 0.25
HPO 40x 0.65
OIO 100x 1.25
FUN FACT!

e
TYPES OF MICROSCOPE

1. Brightfield Microscope
- This is the commonly used type of microscope. In
brightfield microscopy the field of view is brightly lit so
that organisms and other structures are visible against it.
- It is mainly used with stained preparations. Differential
staining may be used depending on the properties of
different structures and organisms.

Loose Areolar
connective
tissue.
2. Darkfield Microscope

- In darkfield microscopy the field of view is dark and the organisms are
illuminated. A special condenser is used which causes light to reflect from
the specimen at an angle.
- It is used for observing bacteria such as treponemes (which cause
syphilis) and leptospires (which cause leptospirosis).

Treponema
pallidum under
Darkfield
Microscope.
3. Phase-Contrast Microscope

- This type allows the examination of live unstained

organisms. It is basically a brightfield microscope with
changes in the objective and the condenser.

- An annular diaphragm, or ring, is put into (or below) the

condenser. This condenser annulus is designed to let a
hollow cone or “doughnut” of a light to pass through the
condenser to the specimen. A corresponding absorption
ring is fitted into the objective.
.
- The net effect of phase contrast is to slow down the speed of light by
one-fourth of a wavelength.
- Objects with differences in refractive index, shape, and absorption
characteristics show added differences in the intensity and shade of
light passing through them. The end result is that the viewer can
observe unstained wet preparations with good resolution and detail.
4. Interference-Contrast Microscope

- This technique gives the viewer a three-dimensional (3D) image of

the object under study.

- As with phase contrast, interference contrast is especially useful for

wet preparations such as urine sediment, showing finer details
without the need for special staining techniques.

- For the interference-contrast microscope, the bright field

microscope is modified by the addition of a special beam-splitting
(Wollaston) prism to the condenser.
5. Polarizing Microscope

- Another useful adaptation of the brightfield microscope that uses a

polarizing filter placed between the light source (bulb) and the
specimen and a second polarizing filter (called an analyzer) placed
above the specimen, between the objective and the eyepiece (either
at some point in the microscope tube or in the eyepiece).

- A polarizer (or polarizing filter) may be defined as a sieve that takes

ordinary light waves, which vibrate in all orientations (or directions),
and allows only light waves of one orientation or aligned waves
(north-south or east-west) to pass through the filter.
- Compensated Polarized Microscope is a further modification of the
polarizing microscope that involves the use of a compensator. It is specially
useful clinically for differentiating between monosodium urate (MSU) and
calcium pyrophosphate dihydrate (CPPD) crystals in synovial fluid.

On the left side, the polars are aligned (not crossed), and on the right side, the
polars are crossed.
 6. Flourescence Microscope

- It is a further refinement of the darkfield microscope. It is basically a

darkfield microscope with wavelength selection.

- In fluorescence microscopy specimens are stained with

fluorochromes/ fluorochrome complexes. Light of high energy or short
wavelengths (from halogen lamps or mercury vapour lamps) is then
used to excite molecules within the specimen or dye molecules
attached to it. These excited molecules emit light of different
wavelengths, often of brilliant colours.
A. Caenorhabditis elegans GFP
gut lumen (Thomas Hannich,
UNIGE). B. Hela cell
endosomes (Dimitri Moreau,
UNIGE). C. Live cells in
alginate capsule (Ilaria Di
Meglio, UNIGE). D. Stable Hela
GFP membrane receptor
(Dimitri Moreau UNIGE).
7. Electron Microscope

- Provides a good resolution of up to 50,000x magnification.

- The principle of the electron microscope is the same as for the light
microscope. Rather than a beam of light, however, the specimen is
illuminated with a beam of electrons produced by an electron “gun.”
TYPES OF MICROSCOPE

1. Bright Field Microscope

2. Darkfield Microscope
3. Phase Contrast Microscope
4. Interference-Contrast Microscope
5. Polarizing Microscope
6. Flourescence Microscope
7. Electron Microscope