|Author: Dr. Robert Berdan|
Oblique illumination was the first step toward dark-field microscopy sometimes referred to as dark-ground microscopy. Dark-field microscopy illuminates specimens with oblique light in the form of a hollow cone. Oblique light from the cone is focused on the specimen but only light that is reflected, refracted or diffracted enters the objective. The specimens are generally highly refractile and must be spaced apart. Dark-field microscopy works primarily by increasing the contrast of the specimen. It does not work well with objects that are crowded or too thick and it can be used to study biological sections if unstained or if covered with silver or gold particles. Other good specimens for dark-field include: cell cultures, microbes, plankton, foods, fibers, crystals, colloids, arthropods, autoradiographs or tissues labelled with metal particles. The subjects appear bright against a black background and can produce striking images. The addition of coloured filters or Rheinberg filters can also be used with the technique though the colours do not provide additional information (R. Berdan 2017). Dark-field microscopy today is also used to examine pathogenic bacteria and in live blood analysis (see article on blood on this web site).
Syphilis is a sexually transmitted disease caused by Treponema pallidum, a bacterium classified under the Spirochaetes phylum. Schaudinn and Hoffmann discovered the bacteria Treponema pallidum in tissue of patients with syphilis in 1905. In 1906, Landsteiner (he also discovered different AB0 blood types) introduced the use of dark-field microscopy for the detection of the spirochete causing syphilis which in turn increased the popularity of dark-field microscopy (Tampa et al. 2014).
Above is a comparison of different light microscopy techniques on Volvox aureus - a fresh water algae. a) bright-field b) dark-field c) phase contrast d) differential interference contrast e) dark-field and a Rheinberg filter f) fluorescence microscopy using green light excitation to reveal autofluorescence – all 100X.
The ultra-microscope was the first dark-field microscope. Richard Zsigmondy studied nanoparticles and developed the first ultra-microscope with Siedenkopf. Zsigmondy received the Nobel Prize in 1925 for his work on nanoparticles. Today there is a renewed interest in nanoparticles and dark-field microscopy has regained importance and popularity. I use dark-field microscopy to study and photograph mainly aquatic micro-organisms and find that it complements other forms of microscopy.
Dark-field can be added to almost any light microscope for less than many other techniques. A simple method uses spider-stops (shown above) to create a cone of light by placing the spider-stops in a filter tray below the condenser or on top of the light source below the condenser. These metal filters can be purchased for a few dollars. Early on I used small coins like a penny, dime, nickel or quarter and placed them in the center of a light blue filter or on the light source under the condenser to achieve dark-field lighting. You will need different sized coins for different objectives and moving the condenser up or down also varies the light cone size. Spider-stops and coins generally only work with low power objectives but are easy to try though results will vary. Another method to add dark-field requires a desk lamp or fiber optic lamp and lighting the specimen from above and removing the condenser (R. Vossen, 2004). I have used this “top-lighting” method successfully with larger aquatic arthropods and low magnification objectives 2.5X and 5X. Some phase condensers have a dark-field option that can be used with low power objectives (10 to 40X) and if there isn’t a dark-field option sometimes you can achieve good dark-field by using the wrong phase ring with some objectives.
Dark-field photograph of Chaoborus sp (midge fly larvae) and its prey Ceriodaphnia a genus of the Daphniidae 100X.
Amphipod Gammarus sp using dark-field microscopy 25X.
Desmid Micrasterias sp – a unicellular algae viewed by dark-field illumination. 400X
An aquatic flat worm with fertilized red eggs and a pair of eyes (dark spots on the right) 25X.
Above left is a Motic dark-field slider and on the right is a Motic phase-contrast condenser with a dark-field option which can be used with 10, 20 and 40X objectives. Note the spider-stop in the slider only fits into some Motic condensers.
Diagram comparing dark-field illumination and bright-field illumination. Dark-field uses a cone of light and only the refracted, reflected or diffracted light enters the objective producing a dark background and revealing refractile subjects. Diagram modified from Washington University website (https://public.wsu.edu/~omoto/papers/Fig1.html).
Several images taken with dark-field microscopy A) ciliate Nassula ornata 200X b) Ciliate Stentor sp 100X c) Water mite 40X d) Copepod 50X e) Algae Pleodorina sp 200X f) Diatoms 200X g) tissue section of monkey retina 100X h) woody stem 100X i) arthropod head - Chironomid sp. 100X.
High magnification dark-field microscopy requires special dark-field condensers and the objectives for use with these possess an iris diaphragm which can be used to reduce the numerical aperture of the objective. Numerical aperture (NA) is commonly used in microscopy to describe the light acceptance cone of an objective. Objectives with larger numerical apertures offer more light gathering power and higher resolving power. The NA of the objective is written on each objective. More expensive objectives have higher numerical apertures and generally higher magnification objectives have greater NA’s then lower power objectives. For high resolution dark-field the NA of the condenser must be larger than the NA of the objective lens in order to prevent direct light from entering the objective. A 100X objective with NA = 1.25 requires a condenser with NA 1.4.
Most high magnification dark-field condensers require oil immersion fluid between the condenser and microscope slide because the angle of incidence of light leaving the top of condenser is greater than the critical angle for glass to air; thus no light emerges from the condenser until it has the immersion oil applied to the condenser surface (Bagnell 2012). High magnification objectives designed for dark-field microscopy also have a built in iris diaphragm that permits the NA of the objective to be reduced.
To summarize some of the main requirements for high magnification dark-field microscopy:
- Dark-field requires the use of a cone of light, no direct light enters the objective only reflected, refracted and diffracted light and high magnification objectives require special condensers.
- The condenser numerical aperture (NA) should be larger than that of the objective.
- Dark-field requires a bright light source because only the refracted and diffracted light enters the objective.
- Glass slides used for dark-field should be very clean and between 1.1 and 1.2 mm thick.
- The condenser should be perfectly centered.
- Oil immersion fluid is required for use of high magnification objectives (60X and 100X).
- High magnification objectives with a NA above 1.2 require an iris diaphragm in the objective to reduce its’ numerical aperture (NA).
Dark-field microscopy is ideal for specimens with smooth reflective surfaces. Specimens with a different refractive index or refractive index gradients from their surrounding solution bend the light into the objective. Some of this is light is also diffracted entering the objective and can undergo interference. Under appropriate conditions dark-field microscopy can detect particles or fibers significantly smaller than the resolution limit of a normal light microscope (0.2 microns or 200 nm). This is possible due to diffraction disks provided the distance between the particles or fibers is greater than the resolving power of the objective. For this reason a dark-field microscope can detect suspended particles down to 40 nm in size and even bacteria flagella which is approximately 20 nm in width.
The downsides of dark-field microscopy are that it is not useful with thick specimens and it is less useful in identifying internal details. Also dirt, dust and particles in water show up as bright spots. I often need to clean background spots in images using image editing software like Photoshop.
Dark-field is a technique that can be added to almost any light microscope and is economical in cost. Sometimes spider-disks or even coins can be used with low magnification objectives. The use of sliders or multipurpose condensers can produce better results for low power objectives (10, 20 and 40X). High magnification dark-field requires special condensers that have a numerical aperture greater than the high magnification objectives which have an iris diaphragms to reduce the objectives NA below that of the condenser. Dark-field has the ability to detect specimens below the limit of a normal light microscope resolution (200 nm) so that particles and fibers (20-40 nm) can be detected. This makes dark-field useful in the study of nanoparticles and microbes but it can also create beautiful images of aquatic-microorganisms.
Tampa, I Sarbu, C. Matei, V. Benea and S. R. Georgescu (2014) Brief History of Syphilis. J. Med Life 15: 4-10. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3956094/
R. Bagnell (2012) Dark Field Microscopy – Chapter 9 Pathology 464 – Light Microscopy. https://www.chem.uci.edu/~dmitryf/manuals/Fundamentals/Dark%20Field%20microscopy.pdf
Abramowitz and M.W. Davidson Darkfield Illumination – Specialized Microscopy Techniques. https://www.olympus-lifescience.com/en/microscope-resource/primer/techniques/darkfield/
E. Hayden (2002) BioImaging Adventures on the Dark Side: An Introduction to Darkfield Microscopy BioTechniques 32: 156-760. https://pubmed.ncbi.nlm.nih.gov/11962596/
Berdan (2017) Rheinberg Filters for Photomicrography. https://www.canadiannaturephotographer.com/rberdan_Rheinberg_filters2017.htmlR. Vossen (2009) Darkfield illumination with a desk LED lamp. Micscape Magazine. http://www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/artsep09/rv-darkfield.html