MICROSCOPY

DEFINITION OF TERMS:
• Resolving power/Resolution

• Working Distance

• Magnification

• Numerical Aperture

• Refractive Index
 Resolving Power/Resolution

• Capacity of the microscope to

separate clearly 2 points

• Determined by the shortest

wavelength of visible light and
maximum numerical aperture
 Working Distance

• Distance between the surface of the lens

and the surface of the cover glass or the
specimen when it is in sharp focus

• Objectives with large NA and great

resolving power have short working
distanceworking distances
 Magnification
• Ratio of image size to the actual size

• Objective Magnification X Ocular

Magnification
 Numerical Aperture
• Measure of the size or angle of the cone of light
entering the aperture of the lens

• Light-gathering capacity of the microscope

• A number that indicates the resolving power of a lens

system

• Derived mathematically from the refractive index(n) of

the optical medium (for air, n=1) and the angle of light
(u) made by the lens
NA= n X sin u
• The larger the NA, the higher the magnification
 Refractive Index
• Measure of the light-bending ability of the medium such
as glass or air

• Light bends as it passes from the glass slide into the air;
bending is due to the fact that the glass slide and air
have different refractive indices

• Immersion oil has the same refractive index as the glass;

immersion oil placed between the glass slide and the
OIO lens prevents the refraction or scattering of light
rays that would otherwise be lost to the objective lens
 The Properties of the Microscope Objectives
property scanner LPO HPO OIO
magnification 4X 10X 40-45X 90-100X
NA 0.01 0.25 0.55-0.65 90-100
Approximate focal length 40 mm 16 mm 4 mm 1.8-2.0 mm
Working distance 17-20 mm 4-8 mm 0.5-0.7 mm 0.1 mm
Approximate resolving 2.3 um 0.9 um 0.35 um 0.18 um
power with light of 450
nm

Scanner-shortest objective; largest lens; red ring; lowest magnification;

locates various structure

LPO- shorter; larger lens; yellow ring; lower magnification; for initial
focusing; gives general outline

HPO- longer; smaller lens; blue ring; higher magnification; gives details
of specimen

OIO- longest; small lens; white ring; highest magnification; examines

microorganisms
 Units of Measure
• When measuring microorganisms, we use the metric
system; has the advantage of easy conversion by a single
factor of 10

• The standard unit of length is the meter (m)

• Microorganisms and their structural components are

measured in even smaller units, such as micrometers
and nanometers

• Micrometer (um)= 10-6 m

• Nanometer (nm)= 10-9 m
• Angstrom (A)= 10-10 m or 0.1 nm
 TYPES OF MICROSCOPE
• Light or Optical Microscope
a. polarizing microscope
b. differential interference
c. phase-contrast
d. fluorescence
e. dark-field
f. bright-field
• UV Microscope
• Electron Microscope
a. TEM
b. SEM
 The Light Microscopes

• Uses visible light as a source of illumination

• Specimen appears against a dark or bright background

• Magnification: 2,000X; Resolution: 0.2 um

• Brightfield illumination is used for stained smears;

most common is the binocular compound microscope

• Unstained cells are observed using modified compound

microscopes
 The Nature of Light Waves

How are waves quantified?

Crest and trough
Amplitude - maximum excursion from its undisturbed or
relaxed position.
Waves travel at a speed, v. The number of crests that pass at a
specific point in space is called a wave’s frequency or f, and is
recorded in units of Hertz.
Period - the time it takes for one complete cycle, measured in
seconds. This is known as P.
Wavelength - the distance a wave travels during one complete
oscillation.
f = 1/P Wavelength() = speed x P or speed/f
 MECHANISMS OF LIGHT MICROSCOPE

The principle is based on the wave nature of light rays, and

the fact that light rays can be in phase (their peaks and
valleys match) or out of phase.

If the wave peak of light rays from one source coincides with
the wave peak of light rays from another source, the rays
interact to produce reinforcement (relative brightness)

However, if the wave peak from one light source coincides

with the wave through from another light source, the rays
interact to produce interference (relative darkness)
 POLARIZING MICROSCOPE
•Birefringence :
Capacity to change the direction of the axis of light

•Modification : with two filters

POLAROID : between the light source and condenser

ANALYZER : at the draw tube

•Applications : mineral elements

ash residues
spindle fiber
A POLARIZING MICROSCOPE
 PHASE-CONTRAST
• Distinguishing features: uses a special condenser containing an annular (ring-
shaped) diaphragm; the diaphragm allows direct light to pass through the
condenser, focusing light on the specimen and a diffraction plate in the objective
lens; direct and reflected or diffracted light rays are brought together to produce
the image

• Principle: Combination of in-phase and out-of-phase; different protoplasmic

constituents produce phase variations

• One set of light rays comes directly from the light source; the other set comes
from light that is reflected or diffracted from a particular structure in the
specimen

• When the 2 sets of light rays- direct rays and reflected or diffracted rays- are
brought together, they form an image on the ocular lens, containing areas that
are relatively light (in phase), through shades of gray, to black (out of phase)

• Application : facilitates detailed examination of the internal structures of living

specimens
 DIFFERENTIAL INTERFERENCE
• Like phase contrast, uses differences in
refractive indexes to produce images

• However, uses 2 light beams separated by

beamplitting prisms, adding contrasting
colors to the specimen

• Provides a colored 3D image

Differential Interference Contrast Microscope
 FLUORESCENCE MICROSCOPE

• Takes advantage of fluorescence, the ability to absorb light

at a shorter wl (ultraviolet) and emit it at a longer wl (visible)

• Uses an UV or near-UV source of illumination that causes

specimen to emit light or fluoresce; The specimen appears
luminescent bright object against a dark background

• the specimen can either be fluorescing in its natural f orm

or stained with fluorochromes
e.g. fluorochrome auramine for M. tuberculosis (glows yellow)
fluorescein isothiocyanate (FITC) for B. anthracis
(appears apple green)
 FLUORESCENCE MICROSCOPE cont’d

Parts: LIGHT SOURCE : Hg vapor lamp

: Quartz iodine lamp
OPTICAL SYSTEM : 2 barrier filters
1 dichroic beamsplitting mirror

Application: for fluorescent-antibody

techniques (immunofluorescence) to rapidly
detect and identify microbes in tissues or
clinical specimens
 DARKFIELD MICROSCOPE
• Uses a special condenser with an OPAQUE DISC that
blocks light from entering the objective lens directly

• Light reflected by the specimen enters the objective

lens, and the specimen appears light against a bright
background

• APPLICATION : examines living microorganisms that

are invisible in brightfield microscopy, do not stain
easily, or are distorted by staining; frequently used to
detect T. pallidum in the diagnosis of syphilis
 objective lens

stage

condenser lens

DARKFIELD MICROSCOPE CONFIGURATION

 CONFOCAL MICROSCOPE
• A computerized microscope that uses laser
light to illuminate one plane of a specimen at a
time

• Images are taken point-by-point and

reconstructed with a computer

• Application: obtains 2- or 3-dimensional

images of cells for biomedical application
CONFOCAL MICROSCOPE CONFIGURATION
 SCANNED-PROBE MICROSCOPE

• Uses probes to examine surface of a specimen at a

very close range without causing modification or
damage

• Application: 1. maps atomic and molecular shapes

2. characterizes magnetic and chemical
properties
3. determines temperature
 SCANNED-PROBE MICROSCOPE cont’d
Types:
1. Scanning Tunneling Microscope (STM)
- uses a thin metal (tungsten) probe
- provides incredibly detailed views of molecules such as
DNA
- resolution is greater than that of an EM

2. Atomic Force Microscope (AFM)

- uses a metal-and-diamond probe
- no special preparation is required
- produces a 3D image
 Detector

SCANNED PROBE MICROSCOPE CONFIGURATION

 UV MICROSCOPE

utilizes quartz, fluorite, and other ultraviolet-

transmitting lenses

the image is made visible by photography,

fluorescence of special glasses, or
television; in a scanning instrument the
receptor is a multiplier phototube.
UV MICROSCOPE
 ELECTRON MICROSCOPE

• Ultrastructure and subcellular structures

• UNIQUE FEATURES : e- beams (W filaments)

fluorescent screen/TV
monitor
electromagnetic field
 ELECTRON MICROSCOPE

• ADVANTAGES: high resolution; high magnification

• DISADVANTAGES : Requires
vacuum enclosed system
high voltage
mechanical stability
different way of specimen preparation
well-trained staff
Two Types of ELECTRON MICROSCOPE