Dark ground microscopy

1. DR.YOGITA MISTRY
GMC,Surat

2.  Historical aspect
 Principle
 How to convert other microscope into dark
field microscope
 Uses
 Advantages
 Disadvantages

3. Dark – field microscopy is an optical
microscopy illumination technique used to
enhance the contrast in unstained samples
.
 In this the specimens is illuminated by
inclined rays and the field of vision is
rendered dark by preventing the axial rays
with use of a circular stopper in the light
path .
 The result is a bright image of the object
against a dark background.

4.  In this the specimens is illuminated by inclined
rays and the field of vision is rendered dark by
preventing the axial rays with use of a circular
stopper in the light path .
 The result is a bright image of the object against
a dark background.
 This produces the classic appearance of a
dark, almost black, background with bright
objects on it.

5.  In 1830, J.J. Lister (the father of Joseph Lister)
invented the darkfield microscope, in which the
standard brightfield (Abbe) condenser is replaced
with a single or double-reflecting dark field
condenser.
 In 1906 in Vienna, Karl Landsteiner and Viktor
Mucha were the first to use darkfield microscopy
to visualize T pallidum from syphilis lesions.

7.  Light reaches the object
mounted on the stage through
the condenser .
 The condenser lens focuses
the light towards the sample.
 The patch stop provided in the
condenser lens blocks light
around the central region
allowing light to pass only the
periphery of the lens
 Of the light that enters the
specimen , most is directly
transmitted , while some is
scattered from the sample .

8.  The scattered light alone
enters the objective lens
and produce the image .
 The directly transmitted
light is not collected and is
omitted .
 Thus the field of vision is
rendered dark and the
image created is by the
scattered light and
therefore , cell component
that reflect light are clear
in the image .

9.  Light enters the microscope for illumination of
the sample.
 A specially sized disc, the patch stop (see
figure) blocks some light from the light source,
leaving an outer ring of illumination. A wide
phase annulus can also be reasonably
substituted at low magnification.
 The condenser lens focuses the light towards
the sample.
 The light enters the sample. Most is directly
transmitted, while some is scattered from the
sample.
 The scattered light enters the objective lens,
while the directly transmitted light simply
misses the lens and is not collected due to
a direct illumination block (see figure).
 Only the scattered light goes on to produce
the image, while the directly transmitted light is
omitted.

11. BRIGHT FIELD MICROSCOPY DARK FIELD MICROSCOPY
1.Light from a plane-wave
source is focused through an
object by a condenser.
2. Some light is blocked
(absorbed) by opaque
parts of the object, or
reflected away at
boundaries between
components/materials of
different refractive indices.
3. The remainder passes
through the objective lens, to
the observer.
1.An opaque disc is put
between the source and
the condenser, blocking
out the middle of the beam.
2. The condenser focuses the
beam onto the sample.
3. No light enters the objective
directly from the source.
Light from the beam is
scattered by the sample –
some scattered into the
objective.

12. 4.This produces a bright
background, with object
details appearing darker in
the image than their
surroundings. The
brightness of the brightest
parts of the image is
determined by the source
brightness and block size.
5. This results in poorer
contrast compared to
darkfield, as the dark areas
are generally grey rather
than black.
4.Only light scattered by the
object enters the objective.
This produces a dark
background, with sample
details appearing brighter
than surroundings. The
brightness of the brightest
parts of the image is
determined by the amount
of light scattered by the
object.
5. This results in superior
contrast to bright-field, as
dark areas may be
completely black, while
increasing the brightness of
the light source brightens
the bright areas

13.  When viewing a specimen with the
10x objective while slowly raising
and lowering the condenser, a point
will be reached where a bright spot
will appear in the field of view as
illustrated in Figure 2(a).
 As the condenser is slightly raised
or lowered, a dark spot similar to the
one shown in Figure 2(b) will
appear, if the condenser is properly
centered.
 In cases where the condenser is not
properly aligned and centered, a
typical field of view might look like
that shown in Figure 2(c), and if the
microscope is not properly adjusted
for , the viewfield might appear as it
does in Figure 2(d).

14. Rheinberg illumination is a special variant
of dark – field illumination.
 It is named after its inventor , Julis
Rheinberg .
 In this variant , transparent coloured filters
are inserted just before the condenser so
that light rays at high aperature are
differently coloured than those at low
aperature .

16.  There is no special microscope model as a
dark-field microscope.
 The light passing through the central part of
the condenser lens system is to be screened
off allowing only the light in the periphery of
the lens to pass .
 This ensures illumination of the object by
inclined rays only.
 This can be achieved by placing a circular
disc called patch stop in between the iris
diaphragm and the lens in the condenser.
 This along with low-power objectives will
suffice for ordinary dark-field observation.

17.  A specimen is placed on the microscope stage as
usual, and the illumination should be made as
uniform as possible.
 An aperture diaphragm in the condenser
(contrast lever), it should be opened up wide.
 After focusing at low power, the slide with
occulting disks is placed in the light path between
source and condenser, bringing it as close to the
bottom of the condenser as it will go.

18.  In reflected darkfield
microscopy, an opaque
occluding disk is placed in
the path of the light
traveling through the
vertical illuminator so that
only the peripheral rays of
light reach the deflecting
mirror. These rays are
reflected by the mirror and
pass through a hollow
collar surrounding the
objective to illuminate the
specimen at highly oblique
angles.

19.  Mechanism involved :
 Each of these reflecting devices (housed
in mirror blocks or cubes) is tilted at a 45
degree angle facing the light traveling
along the vertical illuminator and,
simultaneously, at a 45 degree angle to
the optical axis of the microscope.
 Both of the respective mirrors direct the
light downward at 90 degrees toward the
specimen and also permit the upward-
traveling reflected light to pass through to
the viewing tubes and eyepieces for
observation.
 The best-designed vertical illuminators
include condensing lenses to gather and
control the light, an aperture iris
diaphragm and a pre-focused, centerable
field iris diaphragm to permit the desirable
Köhler illumination.

20.  Affixed to the back end of the vertical
illuminator is a lamp house containing the
light bulb, usually a high-performance
tungsten-halogen lamp. For very faint
darkfield samples, the lamp house can be
replaced with one containing a mercury
burner.
 The burner lamp may be powered by the
electronics built into the microscope
stand, or (in simpler models) by means of
an external transformer.

21.  Within the vertical illuminator, light
emitted by a 50 or 100-watt low
voltage-high intensity tungsten-
halogen lamp passes through a
collector lens and then through
the aperture and field diaphragms
before striking the opaque stop in
the opening port of the darkfield
mirror block located above the
objective at the front of the
illuminator.
 The opaque stop blocks the
central portion of the light beam
allowing only a hollow cylinder of
light to pass into the mirror block,
as illustrated in Figure .
 The field and aperture
diaphragms are opened to their
maximum positions to avoid
blocking peripheral rays of light
from the source.

22.  Dark-field microscopy produce a clear image in a
dark back background.
 It is ideal for viewing objects that are unstained,
transparent and absorb little or no light.
 It is very simple but effective technique for
observing live and unstained biological samples .
 It clearly shows even transparent objects .
 It is very simple to set up dark – field microscope
with only basic equipments.
 No sample preparation is required . Hence
allows viewing of the live cells.

25.  Not only the specimen, but dust and other
particles scatter the light and are easily
observed.
 Glass slides need to be thoroughly cleaned
of extraneous dust and dirt.
 It may be necessary to filter sample media
(agar, water, saline) to exclude confusing
contaminants.
 The sample has to be adequately
illuminated. sometimes strong illumination
may become necessary and care must be
exercised , so that the specimen is not
damaged by strong illumination

26.  Sample materials need to be spread thinly; too much
material on the slide creates many overlapping layers
and edges making it difficult to interpret structures.
 To create the image, this technique relies on scattered
light from specimens. Color is lacking or minimal; this
can be disappointing to the viewer. The actual size of
specimens is also impacted; the width of objects
becomes exaggerated.
 Numerous problems can arise when adapting and
using a dark field microscope. The amount and
intensity of light, the position, size and placement of
the condenser and stop need to be correct to avoid
any aberrations.

27.  The internal structure of organisms cannot be
studied as the light passes around, rather than
through the organism.
 Risk of HIV transmission from infectious
specimens.
 Sometimes strong illumination is required for
visualization– may damage specimen.
 Images can be difficult to interpret to those
unfamiliar with dark field microscopy.
 A specimen that is not thin enough or its density
differs across the slide, may appear to have
artifacts throughout the image.
 The preparation and quality of the slides can
grossly affect the contrast and accuracy of a dark
field image.

28.  Determination of motility in cultures.
 Useful in diagnosis of Treponema, borrelia and leptospira
infection.
 Demonstration of live fresh blood and its components-
ability to for function and the cellular resistance of the
leucocytes--- value for immune disorders and tumours.
 Therapeutic tests can also be carried out by adding the
medicine directly to the blood sample and observing the
reaction. The examination is extremely motivating for the
patient since he can witness the diagnostic findings directly
alongside the physician.
 You can be useful in study marine organisms such as
algae and plankton, diatoms, insects, fibers, hairs, yeast
and protozoa as well as some minerals and crystals, thin
polymers and some ceramics.
 It is more useful in examining external details, such as
outlines, edges, grain boundaries and surface defects than
internal structure.

29. The major companies are:
 Nikon
 Olympus
 Ziess
 Leica
 Meiji

31. How to turn any light microscope into a dark field scope.mp4

32.  Microscopy and microtechnique.
R.Marimuthu. MJP publishers.
 Koneman: Textbook of Diagnostic
Microbiology
 Dark field microscopy.
www.ruf.rice.edu/~bioslabs/methods/microscopy/d