MICROSCOPY

DEFINITION OF TERMS:
 Resolving power/Resolution
 Magnification
 Numerical Aperture
 Refractive Index
 Refraction
 Working distance
 Resolving Power

  Capacity of the microscope to SEPARATE

CLEARLY TWO POINTS
  ability of a lens to separate or distinguish small
objects that are close together
  wavelength of light used is major factor in
resolution
shorter wavelength ⇒ greater resolution
  Depends on the objective (NOT EYEPIECE!)
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 delivered by the
illuminating condenser lens to the
object plane and of the cone light
emerging from the object
 Light-gathering capacity of the
microscope
Refractive Index

 Measure of the light-bending ability of

the medium
Refraction


  
bending
of light away from the specimen
when light passes thru the glass slide
Working Distance

•  distance between the front surface of lens and surface of cover
glass or specimen
 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
 MECHANISMS OF LIGHT
MICROSCOPE

PARTS OF A LIGHT WAVE

(REVIEW)
IN-PHASE
OUT-OF-PHASE
Crest
Trough
Wavelength – distance between crests and troughs
IN-PHASE

- crest or troughs are aligned

OUT-OF-PHASE
 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
 PHASE-CONTRAST
  Combination of in-phase and out-of-phase
  Contrast on the light penetration between
thicker and thinner area occurs
  Principle : different protoplasmic constituents
produce phase variations
  Application : unstained cells
  excellent way to observe living cells
  enhances the contrast
between intracellular
structures having slight
differences in refractive
index

  uses special condenser

containing annular (ring –
shaped diaphragm)
 DIFFERENTIAL INTERFERENCE
  2 LIGHT BEAMS

beam splitting : between the light source and condenser

: detail under observation

beam combining : at the draw tube

: neutral area, beside, above
or below

  3D image is perceived
  creates image by detecting differences in
refractive indices and thickness of different
parts of specimen
  excellent way to observe living cells
FLUORESCENCE MICROSCOPE
  Uses stained specimen

  Uses light of one wavelength to illuminate the specimen

  PARTS
  LIGHT SOURCE : Hg vapor lamp (with HIGH
PRESSURE)
: Quartz iodine lamp
  OPTICAL SYSTEM :
2 barrier filters
1 dichroic mirror (beam-splitting mirror)
FLUORESCENCE MICROSCOPE
  BARRIER FILTERS

-One is located between

the light source and
condenser

-One is found in between

the objective and ocular

  DICHROIC MIRROR : near the

objective (NOT INSIDE THE
DRAW TUBE)

  MECHANISMS OF ACTIONS
OF THE PARTS

  APPLICATION : malaria and

protein structures
  exposes specimen to ultraviolet, violet, or blue light

  specimens usually stained with fluorochromes

  shows a bright image of the object resulting from the

fluorescent light emitted by the specimen
DARK-FIELD MICROSCOPE
  With OPAQUE DISC :
eliminates scattered
light

-uses a special
condenser with an
opaque disc that blocks
light from entering the
objective lens directly

  Employs STRONG
OBLIQUE LIGHT
  OUT-OF-PHASE
Mechanism
  APPLICATION :
Diagnosis of syphilis
  specimen appears light against a
dark background
  used to observe living, unstained
preparations
BRIGHT-FIELD MICROSCOPE

  Employs
VERTICAL
LIGHT

  IN-PHASE
Mechanism
  produces a dark image against a brighter
background
  has several objective lenses
  parfocal microscopes remain in focus when
objectives are changed
  Total magnification
  product of the magnifications of the ocular
lens and the objective lens
UV MICROSCOPE
  has higher resolving power than LM

  ADVANTAGE : provides natural contrast

  UNIQUE FEATURES : UV light

Reflector lens : QUARTZ
 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