Dark field Microscope

4. “It is a technique used to observe unstained
sample causing them to appear brightly against a
dark, purely black background”.
It is one of the simplest and cheapest contrast enhancing
technique and works well for the specimen that have refractive
index which is different from its surrounding medium and
difficult to see.
It is alternative to phase contrast microscope.
In dark field microscope, light seems to radiate from the
specimen while all rest of the field is dark.

5.  Only the light scattered by the object enters the
objective that produces a dark background with
sample details appear bright
 Specimen suitable for dark field microscope

6. Discovery
 Lord Faraday was the first to scientifically
examine the color of small gold particles
 Richard Zsigmondy also extensively studied
the properties of nano-particles to developed
ultra microscopy, which is called dark field
microscopy.

7. The dark field microscope creates a contrast between the
object and the surrounding field. The objective and ocular
lenses are used in dark field microscope are same as light
microscope but special condenser is used.
Fallowing steps are Involves:
o First light enters dark field microscope.
o Then a specialized disc_ Light stop, blocks some
light from light source, leaving an outer ring of illumination.
o Condenser Lens focuses the light towards sample.

8. o The light enters the sample, mostly is directly transmitted
while some is scattered from sample.
o The scattered light enters objective lens, while directly
transmitted light simply misses the lens and not collected due
to light stop.
o Only scattered light goes on to produce image while directly
transmitted
light is omitted.

9. Dark field microscope is consists of fallowing setup:

10.  The first condenser made
specifically for dark field was
produced by Francis H.
 Parabolic glass reflector to create
a hollow cone of light.
 Mounted below the microscope
stage.
 Made up of two uncorrected lenses.
The top lens of an Abbe dark field
condenser is concave lens.
 You can adjust your condenser
for optimal brightness, contrast,
depth of field, etc.

11. “Stops are opaque discs located just under the
bottom lens of the sub-stage condenser”.
 When using stops, the aperture need to be opened.
 Simplest is a “spider stop” consists of central opaque stop
blocks out the central rays.
 This device works fairly well, even with the Abbe condenser
 The diameter of the opaque stop should be approximately
16-18 millimeters for a 10x objective of numerical aperture
0.25

12. Individual stops can be interchanged simply by removing
the screw in the bottom of the support spider and replacing
the stop with a new size

13.  To achieve a dark field image, it is essential to place a dark
field filter –Patch Stop, into the filter holder of condenser.
 The filter prevents light of lamp to directly enter the
objective. The specimen will be illuminated from side and
will scatter some of light to enter the objective so specimen
appears bright on dark background
 There are two possibilities to get dark field image.
Using Condenser
Using Dark field filter_ Patch Stop

15.  Dark field microscope use in different ways to view variety of
specimens that is hard to see in light field as live bacteria
 Dark field microscope often used when specimen is clear or
translucent. Examples are:
Pollen samples.
Live blood samples.
Aquatic environment samples
 View everything in liquid sample, debris & all, dark field
microscope is best.
 Finding cells in suspension.
 For initial examination of suspension of cells
 Determination of motility in cultures.

16. Total magnification of dark field
microscope range from 10X to 1000X

17. Resolution is important to quality of information in an image.
“It is the ability to distinguish two points separate apart
from each other.”
 Resolution power of dark field microscope depends on the
number of factors in its construction.
 The high resolution of dark field microscope is 0.02μ allows
for easy detection of thin & extremely fragile bacteria as
compared to ordinary light microscope

18.  Dark field microscope is a technique creates
contrast in transparent unstained specimens as
living cells.
 It depends on controlling specimen
illuminations
 Dark field illumination uses a carefully aligned
light source to minimize the quantity of directly
transmitted light entering the image plan,
collecting only light scattered
 Dark field can improve image contrast_
particularly transparent objects while
requiring little equipment setup.
 Diatom under Rheinberg illumination

19. Fallowing are important applications of dark field microscope:
o Study insects, hairs, fibers, yeast, protozoa as well as some
minerals and crystal etc.
o Research study of live bacterium, as well as mounted cells and
tissues.
o Examining external details, such as outlines, edges, grain
boundaries.
o Viewing blood cells, different types of algae_ Biological Dark
Field Microscope.
o Viewing hairline metal fractures_ Metallurgical dark field
microscope.
o Viewing shrimp or invertebrates_ Stereo Dark field microscope.
o It is specifically use in haematology for examination fresh blood.

20.  Used to view transparent, unstained specimens clearly
& examine external of specimen with detail.
 High resolution of dark field microscope is allows for
easy detection of thin and fragile specimen.
 It provides good results especially through
examination of red blood samples.
 It can yield high magnification of living bacteria &
low magnification of tissues, cells of certain
organisms.
 Use dark field to study marine organisms
 They have simple and economic setup

22.  Air bubbles in slide can cause problem & dust and other particles
are readily visible.
 Need intense amount of light which can hurt eyes and cause glare.
 The internal structure of organisms can’t be studied as light passes
around rather than through organism.
 Risk of HIV transmission from infectious specimen.
 Lowest resolution in final image & poor depth of field.
 Dark field images are prone to degradation, distortion and
inaccuracies.
 It is not a reliable tool to obtain accurate measurements of
specimens.

23.  J. James,Light Microscopic Techniques in Biology
andMedicine(1976).
 M. G. L. Gustafsson, Proc. Natl. Acad. Sci. USA 102,13081
(2005).
 V. Poher, H. Zhang, G. Kennedy, C. Griffin, S. Oddos, E. Gu,D.
Elson, M. Girkin, P. French, and M. Dawson, Opt. Express15,
11196 (2007).