DARK FIELD MICROSCOPY

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

Antan van leewenhoek

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

BRIGHT FIELD MICROSCOPY

Pathway of light :
light source
condenser
specimen
objective
eyepiece

Pathway of Light in Bright Field

Microscope
Virtual Image formed by ocular lens

Magnified primary image

This lens not for magnificationconcentrates light

11

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.

Dark Field Microscopy

Principle :
To view a specimen in dark field, an opaque
disc is placed underneath the condenser lens,
so that only light that is scattered by objects
on the slide can reach the eye.
Instead of coming up through the specimen,
the light is reflected by particles on the slide.
Everything is visible regardless of color,
usually bright white against a dark
background.

PATHWAY OF LIGHT IN DARK FIELD MICROSCOPE

14

AUGUST KOHLER 1903

PROCEDURE

Light intensity should be increased to maximum

Open the diaphragm
Open the condenser aperture
Remove any colour or other filters
Use correct microslide 1mm
Focus the sample in the lower magnification 10 X
Insert the stop or occult disc
Raise the condenser to the highest position
Observe the sample by lowering the condenser slowly

ADVANTAGES

IT GIVES GOOD VIEWING IN THE NATURAL

ENVIRONMENT
VISUALIZING THE SPECIMENS WITH LOW
CONTRAST
PRODUCES AN IMAGE WITH A DARK
BACKGROUND
GIVES HIGH RESOLUTION 0.2
MICROMETERS

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

Metallurgical Bright & Dark Field Images

17 June 2006

38

Aids TH1 - McDonald

Thank you