MICROSCOPY

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

Resolving Power
y Capacity of the microscope to SEPARATE CLEARLY TWO POINTS y ability of a lens to separate or distinguish small objects that are close together y wavelength of light used is major factor in resolution shorter wavelength greater resolution y Depends on the objective (NOT EYEPIECE!)

Magnification
y Ratio of image size to the actual size

y Objective Magnification X Ocular Magnification

Numerical Aperture
yMeasure 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 yLight-gathering capacity of the microscope

Refractive Index
y Measure of the light-bending ability of the medium

Refraction
y

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
y Light or Optical Microscope a. polarizing microscope b. differential interference c. phase-contrast d. fluorescence e. dark-field f. bright-field y UV Microscope y Electron Microscope a. TEM b. SEM

MECHANISMS OF LIGHT MICROSCOPE

PARTS OF A LIGHT WAVE (REVIEW) IN-PHASE - crest or troughs are aligned

OUT-OF-PHASE

Crest Trough Wavelength distance between crests and troughs

POLARIZING MICROSCOPE
y Birefringence : Capacity to change the direction of the axis of light y Modification : with two filters POLAROID : between the light source and condenser ANALYZER : at the draw tube y Applications : mineral elements ash residues spindle fiber

PHASE-CONTRAST
y Combination of in-phase and out-of-phase y Contrast on the light penetration between thicker and thinner area occurs y Principle : different protoplasmic constituents produce phase variations y Application : unstained cells y excellent way to observe living cells

y enhances the contrast between intracellular structures having slight differences in refractive index y uses special condenser containing annular (ring shaped diaphragm)

DIFFERENTIAL INTERFERENCE
y 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

y 3D image is perceived

y creates image by detecting differences in refractive indices and thickness of different parts of specimen y excellent way to observe living cells

FLUORESCENCE MICROSCOPE
y Uses stained specimen y Uses light of one wavelength to illuminate the specimen y PARTS

y LIGHT SOURCE : Hg vapor lamp (with HIGH PRESSURE) : Quartz iodine lamp y OPTICAL SYSTEM : 2 barrier filters 1 dichroic mirror (beam-splitting mirror)

FLUORESCENCE MICROSCOPE
y BARRIER FILTERS -One is located between the light source and condenser -One is found in between the objective and ocular y DICHROIC MIRROR : near the objective (NOT INSIDE THE DRAW TUBE) y MECHANISMS OF ACTIONS OF THE PARTS y APPLICATION : malaria and protein structures

y exposes specimen to ultraviolet, violet, or blue light y specimens usually stained with fluorochromes y shows a bright image of the object resulting from the fluorescent light emitted by the specimen

DARK-FIELD MICROSCOPE
y With OPAQUE DISC : eliminates scattered light -uses a special condenser with an opaque disc that blocks light from entering the objective lens directly y Employs STRONG OBLIQUE LIGHT y OUT-OF-PHASE Mechanism y APPLICATION : Diagnosis of syphilis

y specimen appears light against a dark background y used to observe living, unstained preparations

BRIGHT-FIELD MICROSCOPE
y Employs VERTICAL LIGHT

y IN-PHASE Mechanism

y produces a dark image against a brighter background y has several objective lenses y parfocal microscopes remain in focus when objectives are changed y Total magnification y product of the magnifications of the ocular lens and the objective lens

UV MICROSCOPE
y has higher resolving power than LM

y ADVANTAGE : provides natural contrast

y UNIQUE FEATURES : UV light Reflector lens : QUARTZ

ELECTRON MICROSCOPE
y Ultrastructure and subcellular structures

y UNIQUE FEATURES : e- beams (W filaments) fluorescent screen/TV monitor electromagnetic field

ELECTRON MICROSCOPE
y ADVANTAGES: high resolution; high magnification y DISADVANTAGES : Requires vacuum enclosed system high voltage mechanical stability different way of specimen preparation well-trained staff

Two Types of ELECTRON MICROSCOPE