Chapter 2: Microscopy

LABORATORY

THE COMPOUND MICROSCOPE Putting the microscope away

→ Rotate the lowest-power objective into position.
Microscope → Remove the slide from the stage.
→ produce magnified visual or photographic images of → Clean the microscope surfaces free of dust or debris.
small objects. If immersion oil has been used, wipe it off the lens and
→ Gk. mikrós = “small” stage with lens tissue.
→ Gk. skopeîn = “to look or see” → Coil the power cord around the base of the
→ Light compound microscope microscope.
 uses visible light to illuminate the specimen, and → Replace the dust cover and/or return the microscope
passes that light through two separate lens to to its correct place in the cabinet.
magnify the image.
Parts of the Microscope

Support System

A F 1 Base or foot the overall support

B G 2 Arm or Limb supports the observation tube
C H 3 Revolving objective changer
D I nosepiece
E J 4 Stage holds the slide specimen in place
Care of the Microscope Magnification System
→ consist of a system of lenses
Transport and setting-up a microscope
→ mounted in two groups
→ Hold both sides around the
hole of the arm when
carrying the microscope
 do not carry two
microscopes at
one time
 do not hold the microscope by the stage (1)
1 Objective
or observation tube (2)
first group of lenses at the bottom of the tube, just
→ Don’t let the electric cord of the microscope dangle in
above the object
such a way as to hazard foot entanglement
→ Place the microscope in a stable flat surface. Keep the
workstation uncluttered
→ Microscope w/ rotatable observation tube may be
used in 2 ways:
 w/ microscope arm near the observer or
 w/ microscope arm away from the observer.
To change from one position to another, hold the 4x Scanning magnifies 4 times
observation tube firmly with one hand while loosening 10x Low-power magnifies 10 times
the observation tube adjustment clamp and rotating objective
the observation tube with the other hand to prevent it 40x High-power magnifies 40
from accidentally falling off from the microscope and objective times
causing damage to the ocular lenses. Don't forget to 100x Oil-immersion magnifies 100
tighten the clamp times
→ Check the lenses to make sure they are clean

Burton’s Microbiology 1
 Chapter 2: Microscopy
LABORATORY

3 Iris -a series of thin plates found

4x diaphragm inside the condenser
air is the imaging medium -has a central aperture which
10x Dry objectives
between the cover glass control the amount of light that
40x
100x Oil-immersion the tip of the OIO passes into the condenser.
objective lens is immersed in oil 4 Filter -allows the passage of light of
desired wavelength only
-Daylight blue filter is used to
2 Eyepiece or Ocular balance the light created by
tungsten or halogen microscope
second group of lenses at the top of the tube
lights.

Adjustment System

10x magnifies the image produced by the

objective 10 times
20x magnifies the image 20 times

A microscope may be:

▪ monocular – has only 1 eyepiece
▪ binocular – has 2 eyepieces; used most clinical
laboratories because both eyes are used view an
object, thus eyestrain

Magnification
→ refers to the ability of an optical system to enlarge an
image of a specimen
→ expressed as magnifying power - the number of
times the image of an object is enlarged
1 Mechanical used to move the object slide
Illumination System
stage control on the stage
knobs
(1a) – x-axis knob
 horizontal
(1b) – y-axis knob
 vertical
2 Observation enables the observation tube
tube of the microscope to rotate
adjustment
clamp
3 Coarse -largest screw
1 Light source -an electric bulb built into the
adjustment -used first to achieve an
/Illuminator microscope beneath the stage
knob approximate focus
-Microscopes that do not have
-rotated to bring the
built-in light bulb have a mirror
specimen as close as possible
that reflects rays from the
to the objective
sunlight or external lamp onto the
4 Fine -moves the objective more
object.
adjustment slowly
2 Condenser brings the rays of light
knob used to bring the object into
(illumination) to a common focus
perfect/precise focus
on the object to be examined.

Burton’s Microbiology 2
 Chapter 2: Microscopy
LABORATORY

5 Condenser used to raise the condenser → In a similar manner, the distance from the rear
adjustment for greater illumination or to principal plane to the image is termed the image
screw lower it to reduce the distance.
illumination
6 Iris diaphragm can be moved to close or open
ring the diaphragm, thus reducing
or increasing both the angle
and the intensity of the light.
7 Light intensity -adjusts the light
adjustment
knob
8 Interpupillary regulates the eyepieces
distance according to the distance → When object is situated two focal lengths in front of the
adjustment between your eyes so that you lens, the image is also two focal lengths behind the
observe a single image lens.
through the eyepieces. → The lens is performing 1:1 magnification, i.e., the
9 Diopter -compensates for the image size is the same as the object size.
adjustment difference in the eyesight → For a magnified image to be observed, the object
ring between the 2 eyes distance must be shorter than the focal length of the
-focuses first with the lens
right eye
10 Pre-focusing controls the mechanism for
knob preventing collision between
the specimen and the
objective

Principles of Light Microscopy

→ The microscope must accomplish three tasks: produce
a magnified image of the specimen, separate the
details in the image, and render the details visible to
→ A real image is an image which is located in the plane
the human eye or camera.
of convergence for the light rays (back focal plane)
that originate from a given object. If a screen is placed
Magnification
in the plane of a real image, the image will generally
→ Magnification is a function of the interaction between
become visible on the screen and appears inverted.
the visible light rays and the curvature of bi-convex
→ A virtual image is produced on the same side of the
lens, one that is thicker at the center than at the
lens as the object, such as the image produced by a
periphery
simple magnifying lens. It is formed on the retina of
the eye, therefore cannot be projected on a screen.
These images always appear upright.
→ Parasitologic work uses brightfield microscope
which is best for observing stained specimens and
living organisms.

→ When parallel rays of light pass through a bi-convex

lens, light rays are refracted (bent) and converge at
intermediary
one point called the focal point. image
→ The vertical plane in which the focal point lies is the
focal plane.
→ The distance from the center of the bi-convex lens to
the focal plane is known as the focal distance (focal
length).
→ The distance between the front principal plane of the final image
lens and the object is known as the object distance.

Burton’s Microbiology 3
 Chapter 2: Microscopy
LABORATORY

→ Light from illuminator passes the substage condenser,

which forms a well-defined light cone that is
concentrated onto the object.
→ Light is transmitted through the specimen and into the
objective which then projects a primary enlarged
image, called intermediate image, to a fixed plane
within the body tube.
→ The intermediate image becomes the “object” for the
eyepiece to produce a secondarily enlarged image,
called the final image.
→ When the human eye is placed above the eyepiece, the
lens and cornea of the eye “look” at the final image as
if it were 10 inches from the eye, near the base of the
microscope
→ Total magnification of the microscope is derived by
multiplying the magnification values of the objective
and the eyepiece. For instance, using a 10X objective
with a 10X eyepiece yields a total magnification of 100X
and likewise.

Resolution
→ ability of the optical system to separate two closely
adjacent objects into 2 distinct entities.
→ determines microscopic image clarity and richness of
detail
→ also expressed as resolving power, i.e., the shortest
between two objects so that they can be and still
appear separate; the smaller the value, the greater
the resolution.
→ The limit of resolution of unaided human eye is 0.1 mm
→ The maximum resolving power of a light microscope
with the highest magnification possible is 0.2 µm.
(0.0002 mm). It, therefore, cannot resolve structures
smaller than 0.2 µm.
not resolvable

resolvable
→ RP is expressed by the following mathematical
equation:

where:
 λ (lambda) = wavelength of light
 NA = numerical aperture
→ Wavelength of light.
→ Numerical aperture (NA).

Burton’s Microbiology 4