2. Dark Ground Illumanation
• Drak gruond illumination sometimes is called
Darkfield microscopy.
• Utilizes scattered or oblique light.
• Light rays that ordinarily pass through or around
the specimen are blocked and allowing onlyoblique
rays to illuminate the specimen.
• In dark field, light seems to radiate from the
specimen while all the rest of the field is black.
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3. Dark Ground Illumination…
• Sections are supposed to be thin and free from
extraneous refractile materials e.g. air bubbles, oil,
red blood cells, sandy crystals, etc.
• DGI is used for;
a] Initial examination of suspensions of cells such as
yeast, bacteria, small protists, or cell and tissue
fractions including cheek epithelial cells, chloroplasts,
mitochondria, even blood cells
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4. Dark Ground Illumination…
b. Initial survey and observation at low powers of
pond water samples, hay or soil infusions,
purchased protist.
c.Examination of lightly stained prepared slides.
d] Determination of motility in cultures.
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5. History
• In the 1840’s,the ultimate test objects for light
microscopes were diatoms, in particular the species
then known as Navicula Spencerii.
• This diatom was named after Charles Spencer, the
New York lens maker whose lenses could resolve
this diatom’s striae.
• It was found that the use of oblique light was
required to resolve the striae.
6. History…
• Oblique illumination was the first step toward
dark field microscopy.
• In dark field, light seems to radiate from the
specimen while all the rest of the field is black.
Resolution is as good as that in bright field while
contrast is enhanced.
• In 1905dark field popular after Schaudinn
discovered Treponema pallidum the causative agent
of Syphilis using dark field microscopy.
7.
8. Details revealed under darkfield illumination;
Fig. “a”the body of the deer tick (Ixodes demmini),Fig. “b”
the heavily stained helminth trematode (Echinostoma
revolutum,and Fig. “c” the silkworm trachea andspiracle
9. Darkfield (darkground) microscopy
• Is a simple and popular method for making
unstainedtransparent specimens clearly visible.
• Such objects often have refractive indices very close in
value to that of their surroundings and are difficult to
image in conventional brightfield microscopy.
• E.g., many small aquatic organisms have a refractive
index ranging from 1.2to 1.4,resulting in a negligible
optical difference from the surrounding aqueous
medium. These are ideal candidates for darkfield
illumination.
10. Dark field microscopy…
• In darkfield illumination the central light blocked
out, allowing only oblique rays to "strike" the
specimen mounted on the microscope slide.
• The top lens of a simple Abbe darkfield condenseris
spherically concave, allowing light rays emerging
from the surface to form an inverted hollow cone of
light with an apex centered in the specimenplane.
11. Dark field microscopy…
• If no specimen is present and the numerical
aperture of the condenser is greater than that ofthe
objective, the oblique rays cross and all such rays
will miss entering the objective because of their
obliquity.
• The field of view will appeardark.
• To obtain a dark field a special condenser with a
blacked-out centre is used.
12. Dark field microscopy…
• If the condenser is not available it is possible to
obtain a dark field under the x10and x40objectives
by inserting an opaque disc or stop in the filter
holder below the condenser, so that the direct lightis
blocked out, but indirect light gets through.
• The stops must be of the correct size for theobjective
in use.
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13. Dark field microscopy…
• If the stop is too small, too much light will pass into
the objective and a dark field will not beobtained.
• If the stop is too large, insufficient light will be
available to illuminate the specimen.
• Make sure the condenser iris is WIDE OPEN.
15. Principle of dark field Microscopy
• 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.
• Everything is visible regardless of color, usually
bright white against a dark background. Pigmented
objects are often seen in "false colors," Better
resolution can be obtained using dark field as
opposed to bright field viewing.
15
16. Principle of dark field Microscopy…
• Oblique rays from the darkfield condenser strike
the specimen in and are diffracted, reflected, and
refracted into the microscope objective.
• Then, the object will appear self-luminous (bright)
against a dark or black background.
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17.
18. Dark field condenser
• The dark field condenser produces a circle of light.
• The light is at an extremely oblique angle tothe
surface of the slide.
• This oblique light comes to a focus onthe
specimen.
• It then diverges so strongly that no direct lightfrom
the condenser enters the objectivelens.
• Only light that is reflected, refracted ordiffracted
by the specimen enters theobjective.
19. Low Magnification DarkField
Condensers
• A low magnification dark field condenser can be
nothing more than an ordinary bright field condenser
with an opaque disk of the proper diameter placed in
its front focal plane.
• The diameter of the opaque disk must be just large
enough to prevent any direct light from enteringthe
objective.
• Many microscope manufacturers produce a“universal”
condenser that has one or more dark field disks that
match objectives of differentNA.
20. Low Magnification DarkField
Condensers…
• The apparent size of an opaque disk can bemade
larger or smaller by raising or lowering the
condenser.
• One disk may thus be useful for a small rangeof
objective NAs.
• If the condenser has a NA greater than 0.95, better
results at low magnification can be achieved if the
condenser is oiled to the slide even though the
objective is used dry.
21. 21
Changing the opaque stop size affects light coneformation
and numerical aperture in a simple Abbe condenser
22. The light stop illustrated in Figure “b” is an ingenious device that expands and
contracts a "reverse iris" diaphragm to increase or decrease the size of the stop
using a lever control arm.
23. High Numerical ApertureDark
Field Condensers
• The need for keeping the objective NA lower than the
condenser NA lead to the production of higher and
higher NA condensers and to the introduction of the
“funnelstop” for the objectivelens.
• The funnel stop is a funnel shaped cone that isinserted
into the objective lens to limit itsaperture.
• Modern objective lenses do not use funnel stops,
instead, some high NA oil objectives have an iris
diaphragm built into them.
• This iris is used to reduce the NA of the objectivelens.
24. High Numerical ApertureDark
Field Condensers…
• High magnification dark field condensers must
always be oiled to the specimenslide.
• This is because the angle of incidence of light
leaving the top of the condenser is muchgreater
than the critical angle for glass to air; thus, no
light emerges from the condenser until it has
immersion oil applied to its surface.
25. High Numerical ApertureDark
Field Condensers…
• Critical angle is important in darkfield.
• The dark field condenser produces a veryoblique
angle of light.
• If this angle is greater than the critical angle at any
interface, the illumination will be totally internally
reflected. For this reason, the specimen’s
immersion medium is important.
• The critical angle for glass to air is 41degrees and
from glass to water it is 61degrees
26. High Numerical Aperture Dark Field
Condensers…
• Low power dark field condensers work fine for
specimens in water.
• A high power dark field condenser may not be
useful with a water immersed specimen for the
above reason.
• It is always best to immerse the dark field
condenser to the slide with oil even for low power
dark field condensers as long as the critical angle is
not exceeded.
32. Properties of Dark Field Condensers
TYPE Hollow
cone NA
range
Limit NA
of
objective
s
Number
of
reflecting
surfaces
correction
Paraboloid 1.00-1.40 0.85 1 parabolic partial
Cardioid
Bispheri
c
bicentric
1.20-1.33 1.05 1 spherical
and
1 cardioidal or
2 spherical
aplanatic
Cassegrain 1.40-1.50 1.30 1 Aspherical
1 spherical
aplanatic
Spot ring
bicentric
1.40-1.50 1.30 2 spherical aplanatic
33. Thedarkfield microscope
• The microscope is used to examine live
microorganisms that are invisible in the ordinary
light microscope, can not be stained by standard
methods or are so distorted by staining thattheir
characteristics they can not be identified.
34. Dark field Microscopy Applications
• In optical microscopy, dark-field describes
an illumination technique used to enhance
the contrast in samples that are unstained,
transparent and absorb little or no light.
• It works by illuminating the sample with light that
will not be collected by the objective lens and thus
will not form part of the image.
• This produces the classic appearance of a dark,
almost black, background with bright objects on it.
35. Dark field Microscopy Applications…
• The dark field microscope can be used to study
marine organisms such as algae, plankton, diatoms,
atoms, fibers, yeasts, protozoa as well as some
minerals and crystals and thin polymers.
36. Properties of specimen that are used
in dark field microscope
• A dark field microscopy is used to examine live
microorganisms that
Are either invisible in the ordinary lightmicroscopy.
Specimen that can not be stained bystandard
method.
Specimen that are distorted by staining ,that their
characteristics can not be identified.
37. Types of specimen that can be used
in dark field microscopy.
• Very thin histological sections can be used if unstained or if
only certain components are stained, as in silverstains.
• Biological fluids from animals and plants,
• Cell cultures.
• Microbes.
• Foods
• Fibers
• Crystals
• Colloids, and sub-microscopic particles are all suitable for
dark field microscopy.
38. ADVANTAGESOFDARKFIELDMICROSCPE
i. It is ideal for viewing objects that areunstained,
transparent and absorb little or nolight.
ii. Can be used to study marine organisms suchas algae
and plankton
iii. Use in the research of live bacterium as wellas
mounted cells and tissues.
iv. Useful in examining external details, such as outlines,
edges and surface defect than internalstructures.
v. Used to view specimens that have similarreflective
index as their surrounding.
39. DISADVANTAGES
i. Dark field images are prone to degradationand
distortion
ii. Selective to specimen
iii. A specimen that is not thin enough or its density
differs across the slide, may appear to haveartifacts
through out the image.
iv. The preparation and quality of slides, grossly may
affect the contrast and accuracy of a dark fieldimage.
v. Require regular maintenance.
vi. Air bubbles when using oil or water on condenser or
slide may cause distortion or degradation ofimage.
