HHISTCG ( Lab ) - Microscopy

8/6/2019 HHISTCG ( Lab ) - Microscopy

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MICROSCOPY



DEFINITION OF TERMS:

yResolving power/Resolution

yMagnification

yNumerical Aperture

yRefractive Index

yRefraction

yWorking distance



Resolving Power

y Capacity of the microscope to SEPARATECLEARLY TWO POINTS

y ability of a lens to separate or distinguishsmall objects that are close together

y wavelength of light used is major factor in

resolutionshorter wavelength greater resolution

y Depends on the objective (NOT EYEPIECE!)



Magnification

yRatio of image size to the actual size

yObjective Magnification X Ocular

Magnification



Numerical Aperture

yMeasure of the size or angle of the

cone of light delivered by the

illuminating condenser lens to theobject plane and of the cone light

emerging from the object

yLight-gathering capacity of the

microscope



Refractive Index

yMeasure of the light-bending ability of the medium



Refraction

y bending of light away from the specimenwhen light passes thru the glass slide



Working Distance

� distance between the front surface of lens and surface of cover glass or specimen



TYPES OF MICROSCOPEy 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



POL ARIZING MICROSCOPE

y Birefringence :

Capacity to change the direction of the axis of light

y Modification : with two filters

POL AROID : between the light source andcondenser

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 constituentsproduce 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 INTERFERENCEy2 LIGHT BEAMS

beam splitting : between the light source andcondenser

: 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 specimeny excellent way to observe living cells



FLUORESCENCE MICROSCOPEy 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

OPTIC AL

SY

STEM :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 betweenthe objective and ocular

y DICHROICMIRROR : near the

objective (NOT INSIDE THE

DRAW TUBE)

y MECHANISMS OF ACTIONS

OF THE PARTS

y APPLIC ATION : malaria and

protein structures



DARK-FIELD MICROSCOPE

y With OPAQUE DISC :eliminates scattered light

-uses a specialcondenser with an opaque

disc that blocks light fromentering the objective lensdirectly

y Employs STRONGOBLIQUE LIGHT

y OUT-OF-PHASEMechanism

y APPLIC ATION : Diagnosisof syphilis



y specimen appears light against a

dark background

y used to observe living, unstained

preparations



BRIGHT-FIELD MICROSCOPE

y Employs

VERTIC AL

LIGHT

y IN-PHASE

Mechanism



y produces a dark image against a brighter background

yhas several objective lensesy 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



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; highmagnification

y DISADVANTAGES : Requires

vacuum enclosed system

high voltage

mechanical stability

different way of specimen preparation

well-trained staff

HHISTCG ( Lab ) - Microscopy

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