Dark Field
Microscopy
Presented by:
Ananya Rajappa B R (23SM1017)
Aniket Sharma (23SM1019)
Ann Theress (23SM1020)
Fig: A colony of green planktonic algae Anna Thomas (23SM1021)
Volvox under the dark field microscope. Anoushka Singh (23SM1022)
Image Credit: D. Kucharski K. Anusha Ananda (23SM1023)
History
In 1830, J.J. Lister (the father of Joseph Lister) invented the
dark field microscope, in which the standard bright field
(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 dark field microscope to visualise T pallidum
from syphilis lesions.
What is Dark Field Microscopy?
Dark Field Microscopy is used to examine live micro-
organisms that are either invisible in the ordinary light
microscope, cannot be stained by standard methods, or are
so distorted by staining that their characteristics then
cannot be identified.
In dark field microscopy, the object appears bright against a
dark background.
Principle
In Dark Field (or dark ground) Microscope, the object
appears bright against a dark background. This is made
possible by use of a special dark field condenser.
• The dark field condenser has a central opaque area that
blocks light from entering the objective lens directly and
has a peripheral annular hollow area which allows the
light to pass through and focus on the specimen
obliquely.
• Only the light which is reflected by the specimen enters
the objective lens whereas the unreflected light does not
enter the objective. As a result, the specimen is brightly
illuminated; but the background appears dark.
Applications
Enhanced contrast for transparent structures
Visualisation of thin bacteria
Rapid detection of Treponema pallidum
Observation of Bacterial and Protozoan motility
Study of marine organisms and materials like crystals and
polymers
Examination of cells and tissues
Surface and edge detail examination
Advantages
Simple and cost-effective setup
Ideal for Unstained, Transparent Specimens
Higher resolution and contrast
No sample preparation needed
Suitable for observing live organisms
Detailed view of external features
Effective for water-borne organisms
Disadvantages
Images formed are prone to degradation, distortion and
inaccuracies
Since the sample has to be adequately illuminated,
sometimes strong illumination may damage the specimen
Not only the specimen but dust and other particles
scatter the light and are easily observed
Low Level of light and limited colours
Advancements
Integration with Fluorescence Techniques
Digital imaging and Real-time analysis
Label-free and non-invasive imaging
Portability and Accessibility
Enhanced contrast for organisms without staining,
preserving their natural state
Super Resolution Techniques extending visibility to
nanoscale structures
Thank you
