This Presentation shows working and basics of Dark Ground Microscope

1. DARK GROUND
MICROSCOPY
Presented by: Dr.Himali Gopal Pachpande
MDS-II
Dept. Of Oral Pathology & Microbiology

2. CONTENT
 Introduction
 History
 Principle
 Comparison of Bright Phase Microscopy & Dark Phase Microscopy
 Objectives and Condensers
 Setting up of Dark Phase Microscopy
 Errors Causing Difficulty in setting of dark phase microscopy

3. CONTENT
Variant of Dark phase microscopy – Rheinberg illumination
How to convert Light Microscope to Dark ground microscope
Uses of dark ground microscopy
Advantages of dark ground microscopy
Disadvantages of dark ground microscopy
Specimens seen through Dark ground microscope
References

4. INTRODUCTION
• Dark-field microscopy is an optical microscopy illumination technique used
to enhance the contrast in unstained samples
• For an object to be examined microscopically, it must first be visible.
• Visibility is dependent on contrast, as is illustrated by the fact that a spider's
web is difficult to see against the sky, yet stands out clearly when viewed
against a dark background
• The result is a bright image of the object against a dark background.

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

6. PRINCIPLE
1. Light reaches the object mounted on the stage through the condenser
2. The condenser lens focuses the light towards the sample
3. The patch stop provided in the condenser lens blocks light around the
central region allowing light to pass only to the periphery of the lens
4. Light that enters the specimen, most is directly transmitted, while some
is scattered from the sample
5. The scattered light alone enters the objective lens and produces the
image
6. The directly transmitted light is not collected it simply misses the lens
and is not collected due to a direct illumination block

8. SUMMARY OF PRINCIPLE
• 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

10. COMPARISON BETWEEN
BRIGHT FIELD AND DARK FIELD MICROSCOPY
• BRIGHT 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
• DARK FIELD MICROSCOPY
1. An opaque disc is put between the source and
the condenser, blocking out the middle of the
beam.
2. The condenser focuses the parts of the object or
reflected away at the beam onto the sample

11. 3. The remainder passes through the objective
lens, to the observer.
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. Results in poorer Contrast
3. No light enters the objective directly from the
source. Light from the beam is scattered by the
sample - some scattered into the Objective
4. Only light scattered by the object enters the
objective. This produces a dark background, with
sample details appearing brighter than the
surroundings. The brightness of the brightest
parts of the image is determined by the amount
of light scattered by the object
5. Results in superior contrast
COMPARISON BETWEEN
BRIGHT FIELD AND DARK FIELD MICROSCOPY

12. OBJECTIVES AND CONDENSERS
• LOW POWER OBJECTIVES
a. Work at some distance from the object, therefore dark ground illumination is obtained simply by
inserting a small circle of black paper pasted on glass in the filter carrier
b. The central rays which normally pass through the object and into the objective is cut off
c. Only light entering the objective will be the scattered light
• HIGH POWER OBJECTIVES
a. Much shorter working distance require a special condenser which will focus a hollow cone of light at
an acute angle.
b. The angle is acute if oil is not used – Total internal reflection will occur.
c. To get best results, the condenser must be accurately centred and focussed to get the maximum
amount of light on the object without its entering the objective

13. OBJECTIVES AND CONDENSERS
• Because of the very acute angle of light required, very few dark ground condensers can be used with a
numerical aperture (NA) in excess of 1.0
• A 2mm objective having NA 1.3 can be used if a funnel stop (small metal tube) is inserted at the back
which reduces the working aperture to less than 1.0
• CONDENSERS-
a. Fixed focus type of dark ground condenser – used only with extra thin glass slides and cover slips
b. Focussing dark ground condensers – allow a variety of slides and coverslips to be used

14. SETTING UP THE DARK-GROUND MICROSCOPE
• Make a thin preparation, using a thoroughly clean thin slide and coverslip, and take care
not to have air bubbles in the preparation
• Direct, or adjust, light through the condenser so that it is evenly distributed.
• Rack the condenser down, and place a drop of immersion oil on the top lens of the
condenser and on the lower side of the slide. Place the slide on the microscope stage and
slowly rack up the condenser until the two surfaces of the immersion oil without forming
air bubbles; such bubbles would reflect light in all directions
• Focus on the object with a low-power objective such as the at height of 16mm.

15. • With the centring screws, adjust the condenser until the point of light is in the centre of the
field.
• Place a drop of immersion oil on the coverslip and focus the object with the high-power oil
immersion objective. Perfect darkground illumination should result if a funnel-stop
objective is used; If an iris diaphragm is incorporated in the objective it is adjusted to give
maximum performance.
• After the use the oil should be carefully cleaned off both the condenser and objective
SETTING UP THE DARK-GROUND MICROSCOPE

16. ERRORS CAUSING DIFFICULTY IN SETTING UP THE
MICROSCOPE
• The slides or coverslips are too thick
• The preparation has too many air bubbles present
• Condenser is not properly focused or centred
• Lighting is not sufficiently intense

17. VARIANT IN DARKGROUND MICROSCOPY
• Rheinberg illumination is a special variant of dark
ground 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
aperture are differently coloured than those at low
aperture

18. HOW TO CONVERT LIGHT MICROSCOPE
TO A DARK GROUND MICROSCOPE
• 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 the illumination of the object by inclined rays only
• This can be achieved by placing a circular disc called a 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.
• A specimen is placed on the microscope stage as usual, and the illumination should be made as
uniform as possible

19. • 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
HOW TO CONVERT LIGHT MICROSCOPE
TO A DARK GROUND MICROSCOPE

20. USES
• Determination of motility in cultures
• Useful in the diagnosis of Treponema, borrelia and leptospira infection
• Demonstration of live fresh blood and its component’s 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.
• Can be useful in the study marine organisms such as algae and plankton, diatoms, insects, fibres,
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.

21. T. denticola :
wet mount + Fluroscent
staining
Spirochetes :
wet mount

22. ADVANTAGES
• Dark-field microscopy produces 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 a 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
equipment
• No sample preparation is required Hence allows viewing of the live cells

23. DISADVANTAGES
• Not only the specimen, but dust and other particles scatter the light and are easily observed
• Glass slides need to be thoroughly cleaned
• The internal structure of organisms cannot be studied as the light passes around, rather than
through the organism
• Sometimes strong illumination is required for visualization, which may damage the specimen
• Images can be difficult to interpret for those unfamiliar with dark-field microscopy
• The preparation and quality of the slides can grossly affect the contrast and accuracy of a
dark field image

24. Inflammatory cells in Human Blood sample & Poikilocyte

25. Uses in Hematology:
1. To study RBCs Morphology
- Anisocytosis
- Poikilocytosis

27. REFERENCES
1. Bancroft J. and Gamble M. Theory and Practice of Histological Techniques. 5th
Edition.
2. Culling C. Handbook of Histopathological & Histological Techniques. 3rd
Edition.