AND
ITS APPLICATION
BY
DR.K.SRIPAD
Course : Advances in Diagnostic Pathology
Course Teacher : Dr. Sathyanarayana
MICROSCOPE
IT IS AN INSTRUMENT WHICH ENABLES TO SEE
MICROORGANISMS AND THEIR STRUCTURE
OTHERWISE INVISIBLE TO THE NAKED EYE
MICROSCOPY : STUDY OF MICROSCOPES
TYPES OF MICROSCOPES
Based on the principle of magnfication:
1.LIGHT MICROSCOPE : OPTICAL LENSES USING
LIGHT RAYS
- Bright field
- Dark field
- Flouresence
- Phase contrast
2. ELECTRON MICROSCOPE : USES A BEAM OF
ELECTRONS IN PLACE OF LIGHT RAYS
FUNDAMENTALS OF LIGHT
MICROSCOPY
Magnification
Resolution
Contrast
MAGNIFICATION
Amount of enlargement of the specimen
- magnification is finite
- could be calculated
- Empty magnification
1X
5X
20X
100X
RESOLUTION
It is the ability to distinguish two adjacent
points (or particles) as distinct and
separate from each other.
Factors affecting resolution :
Wave length of the light
Numerical aperture of the lens system
Distance between and lens and specimen
Contrast
CONTRAST
Difference in the maximum intensity between the
darkest and lightest points in an image
too much or too little - you loose information
Several methods to add contrast
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Why Darkfield?
Biological specimens are often watery materials
held together by transparent gels and
suspended in water. It is like the grade school
joke about seeing a polar bear in a snow storm,
nothing stands out.
However some specimens show up well with
darkfield illumination. Arguably, removing the
blinding glare of the brightfield light probably
allows such contrast as there is to be seen.
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PROCEDURE
ADVANTAGES
ADVANTAGES (contd.)
VERY SIMPLE AND EFFECTIVE TECHNIQUE
MORE SUITABLE FOR EXAMINATION OF THE
- UNSTAINED BIOLOGICAL
SAMPLES
- MICROORGANISMS SUSPENDED
IN FLUID
- TISSUE CULTURES
- WATER BORNE SINGLE CELLED
ORGANISMS
Types of DFM
1. Dark field scanning microscope
2. Computer enhanced DFM
3. Dark field electron microscope
Applications
1.Examination of live blood cells
2.Examination of leptospires
3.Examination of unstained bacterial flagellae salmonella,
chlamadomonas and pseudomonas
4.To study the cell division of anaerobic thermphilles
pyrodictium abyssi & Thermoproteales tenax
5.Examination of bio film, microcolonies and attached
bacterial cells
6.Analysis of microtubule related motility
7.Study of chromatin architecture
Applications (contd.)
8.Immunology :
- immunolabelling technique
- labelling of nuclear lamina
- Fibroblasts
9.Surgery :
Diagnosis of dislocation and other defects
Hard X-ray DF imaging for better contrast
10.Cancer Therapy:
Cancer cell imaging-Nanoshell enabled photonics
based imaging
Laeser photothermal therapy of epithelial carcinoma
and breast cancer
Applications (contd.)
11.Metallurgy :
- Study of microstructure and characterization of carbon
-Study of shape effects in colloidal silver particles and
gold beeds
12. Molecular cell biology :
- Visualization of single heavy atom or molecule
- enumeration of cells
- study of intrinsic optical signals in rat neocortical cells
- growth rate
- colonization kinetics
- plasmolysis
Applications (contd.)
- behaviour and interaction of micro
organisms on surface
-determination of rate of motility and number
of motility events
13. Neurobiology
14. Accoustic microscopy
Limitations of DFM
1. More care in sample preparation need to be
excercised
2. Sample material need to be spread thinly
3. Sample media to be filtered to exclude
contaminants agar, water, saline,etc.,
4. Microslides need to be thoroughly cleaned for
extraneous dust and dirt
5. Dust and other particles also are observed
along with the specimen
6. Colour is lacking or minimal
7. Actual size of the specimen is impacted
usually the width of the object is exaggerated
Coagulated blood
Crustacean larva
17 June 2006
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Thank you