The history of dark-field optical microscopy

Figure 1 Among the oldest published schematics of oblique microscopy[2].
Joseph Jackson Lister (1786-1869), father of Joseph Lister, is often accredited with the development of the first dark-field microscopy technique, often overshadowed by his pioneering work in achromatic lenses. In the book Micrographia (1837) by Goring and Pritchard [1], appears to be the first published use of dark-field illumination techniques. Oblique illumination, as seen in Figure 1, of samples with substage illumination Rev. Joseph Bancroft Reade discusses a specific sample – “for it scarcely shewed(sic) them at all; indeed, as the reader well knows, they require oblique, not direct light” (see page 40 of Micrographia). This encapsulates common usage of dark-field microscopy, where many samples appear translucent under direct bright-field illumination but can be seen clearly with the enhanced contrast of dark-field microscopy. This quote also suggests that oblique lighting was a known technique for contrast enhancement among microscopists at the time.

Figure 2 Amici Lenticular Illuminator antique [3].
A telescopic prism illuminator called the Amici Lenticular Illuminator was most often used for oblique illumination throughout the 1800s [4]. This illuminator, as seen in Figure 2, could be mounted independently to the stage, thus allowing the microscopists to vary the angle of illumination for optimum contrast, Figure 4. It was then imagined that illuminating the sample with uniform oblique light from all sides would enhance the contrast. A parabolic reflector was developed in 1855 by Francis H. Wenham and George Shadbolt. But, as it was made with a mirrored metal interior; it was intrinsically achromatic[5].



Figure 3 The Wenham parabolic illuminator used throughout the 1800s.

This evolved into the Wenham Parabolic Illuminator, Figure 3, which used a parabolic glass ring and adjustable central stop which accommodated collection objectives with various apertures. This piece remained popular for many years. At this point, “darkground” microscopy began to truly resemble modern-day dark-field microscopy with radial illumination rather than from a single azimuth. Oblique lighting is still used today as the technique can increase the optical resolution as both the zeroth and some higher orders of the diffracted light are contributing to the image formation. However, with oblique illumination, the image often has directional shadows dependent on the incident angle of illumination, variations in refractive index/optical path differences leading to obtained images having a pseudo-3D look. Such shadows should not exist in dark-field, as illumination is from all azimuths.

Figure 4 How change in angle of illumination results in change of contrast between sample and background [5].

With Abbe’s work in both resolving power and its relationship with the refractive index of the medium between the objective and glass slide came the dawn of immersion lenses. To adopt these new findings, immersion paraboloids were first developed by Dr James Edmunds and built by Dr John Barker and published in the proceedings of the Royal Irish Academy in 1870 [6]. Such condensers are still used today, however, most incorporate either aplanatic or achromatic correction or both.

These 1800’s dark-field microscopes were involved in many exciting discoveries specifically in medicine as many biological samples appear translucent in brightfield illumination and at the time, many biological samples were considerably difficult to resolve using 1800’s optics due to their size. Most noteworthy was the discovery of Treponema pallidum in 1905 by Schaudinn and Hoffmann; this is a spirochaete bacterium with various subspecies which leads to treponemal diseases including syphilis and yaws, Figure 5. Due to the bacterium translucent nature, it appears invisible under bright field illumination.

Figure 5 Dark-field photomicrograph of Treponema pallidum bacteria [7].

Colloidal metallic solutions were used, likely unbeknownst, throughout history, such as the roman Lycurgus cup seen in Figure 6 which is embedded with gold-silver nanoparticles to achieve a dichroic effect. Even with the dawn of optical microscopy, these nanometre-sized metallic nanoparticles were unresolvable as a result of diffraction limiting.

Figure 6 The Lycurgus Cup, a fourth-century A.D. Roman artefact where gold-silver nanoparticles were used to give the dichroic effect. In direct light it has a jade greenish colour, whereas the transmission of light through the glass results in a ruby red colour [8].

Richard Adolf Zsigmondy, an Austrian-Hungarian chemist (1865-1929) had a fascination with colloidal solutions. Zsigmondy wondered why different colloidal solutions of ‘fine gold’ had such a radiant colour when painted onto porcelain and why different solutions of these fine gold colloidal solutions had different colours, Figure 7. Alchemist Johann Kunckel in the quest of transmuting base metals into gold rediscovered how to produce glass with the deep red lustre as seen in historical artefacts. Zsigmondy developed the “ultramicroscope” in 1902 with Zeiss which he used to show that the ruby glass developed by Kunckle was, in fact, colloidal gold-glass mixture rather than a chemical. The ultramicroscope, Figure 8, was an early iteration of a dark-field microscope, where ‘ultra’ referred to the microscopes capability to observe objects which were smaller than the wavelength of the ultraviolet-visible illumination light. Zsigmondy developed a second iteration called the immersion ultramicroscope where colloidal solutions could be observed. In 1925 Zsigmondy was awarded a Nobel Prize in Chemistry for his research into colloids and for the development of the ultramicroscope [9].

Figure 7 Artistic depiction of light scattering in a gold colloidal solution, published by Zsigmondy in 1907 in Jena [10].
The ultramicroscope developed in the early 1920s was essentially a light-sheet microscopy method. The concept involved using illumination at a right angle to detection hence avoiding white light collection and collecting the light scattered off the sample. Which is the means by which dark-field microscopy operates. The ultramicroscope was later patented and adopted by other microscopy companies such as Leitz.

“The ultramicroscope, the chief feature of which is that by means of a special contrivance the sun’s rays are concentrated so as to produce a very powerful light upon the material to be examined under a compound microscope, has enabled investigators to see minute particles hitherto invisible.”

Figure 8 The ultramicroscope depicted in 1902 developed by Heinrich Siedentopf and Richard Zsigmondy[13].
Such microscopes developed further in the coming years once scientists realised that through using hollow illumination cones were implemented of a suitable angle, superior contrast could be achieved. To form the hollow cone of light, central stops were used – much like modern dark-field microscopes. A paper titled “Modern Dark-Field Microscopy and the History of Its Development” published in April 1920 [12], the author discussed how Zsigmondy’s ultramicroscope concept was not truly new, that physicists and chemists were informally well acquainted with the oblique lighting method. Zsigmondy’s work is accredited with convincing any sceptics still unconvinced of the existence of atoms and molecules.C. STEVENS, University of Washington Science Volume 30 issue 762 1909 [11]

In the past few decades, dark-field microscopy has evolved to become a spectroscopic technique by incorporating a monochromator and spectrometer. This setup allows the Rayleigh scattering spectra to be collected for single metal nanoparticles. Dark-field microspectroscopy was first demonstrated by Yguerabide and Yguerabide in 1998 [14]. This paper used Rayleigh and Mie light scattering theory to theoretically predict the light scattering by gold nanoparticles of various sizes in solution and experimental spectral data was in good agreement with these calculations. At around the same time, near-field dark-field microspectroscopy was demonstrated by Feldmann’s group [15]. In these setups, rather than using dark-field style objectives, large incident angle (oblique) light is used.

Figure 9 Typical transmission dark-field microspectroscope setup, adapted from [16].

In most modern table-top optical microscopes, there is an option to use dark-field illumination, either using particular dark-field objectives or dark-field condensers when in transmission, Figure 9. The output scattered light can be sent to a spectrometer for spectral analysis. Alternatively, in homebuilt microscopes, a dark-field objective or condenser can be used to illuminate the sample, while light collected by the collection objective in transmission can be directed toward a spectrometer.




[1]      C. R. Goring and A. Pritchard, Micrographia: Containing Practical Essays on Reflecting, Solar, Oxy-hydrogen … – C. R. Goring, Andrew Pritchard. 1837.

[2]      J. Quekett, Practical treatise on the use of the microscope, including the different methods of preparing and examining animal, vegetable and mineral substances : Quekett, John : Free Download & Streaming : Internet Archive. 1848.

[3]      “History of Dark Ground (Dark Field) Microscope Illumination.” [Online]. Available: [Accessed: 12-Aug-2019].

[4], “History of Dark Ground (Dark Field) Microscope Illumination,”, 2013. [Online]. Available: [Accessed: 07-Mar-2018].

[5]      W. Chambers, T. J. Fellers, and M. W. Davidson, “Oblique Illumination | MicroscopyU,” NIKON INSTRUMENTS INC. [Online]. Available: [Accessed: 06-Mar-2018].

[6]      J. EDMUNDS, “Microscopy. The Immersion Paraboloid,” Nature, vol. 18, no. 454, pp. 278–278, Jul. 1878, doi: 10.1038/018278d0.

[7]      C. Hubbard and CDC, “Dark field photomicrograph of Treponema pallidum bacteria.,” 1971. [Online]. Available: [Accessed: 07-Mar-2018].

[8]      I. Freestone, N. Meeks, M. Sax, and C. Higgitt, “The Lycurgus Cup – A Roman Nanotechnology.”

[9]      what-when-how, “Ultramicroscope (Inventions).” [Online]. Available: [Accessed: 06-Mar-2018].

[10]    Nanobiotechnology group Johannes Gutenberg University Mainz, “Plasmon Spectroscopy History,” Johannes Gutenberg University Mainz. [Online]. Available: [Accessed: 06-Mar-2018].

[11]    L. Kahlenberg, “Colloids and the Ultramicroscope,” Science (80-. )., vol. 30, no. 762, pp. 184–184, Aug. 1909, doi: 10.1126/science.30.762.184.

[12]    S. H. Gage, “Modern Dark-Field Microscopy and the History of Its Development,” Trans. Am. Microsc. Soc., vol. 39, no. 2, p. 95, Apr. 1920, doi: 10.2307/3221838.

[13]    H. Siedentopf and R. Zsigmondy, “Uber Sichtbarmachung und Größenbestimmung ultramikoskopischer Teilchen, mit besonderer Anwendung auf Goldrubingläser,” Ann. Phys., vol. 315, no. 1, pp. 1–39, Jan. 1902, doi: 10.1002/andp.19023150102.

[14]    J. Yguerabide and E. E. Yguerabide, “Light-Scattering Submicroscopic Particles as Highly Fluorescent Analogs and Their Use as Tracer Labels in Clinical and Biological Applications II. Experimental Characterization Measurement of Light-Scattering Intensity and Spectra of Particle Suspensions,” 1998.

[15]    T. Klar, M. Perner, S. Grosse, G. Von Plessen, W. Spirkl, and J. Feldmann, “Surface-Plasmon Resonances in Single Metallic Nanoparticles,” 1998.

[16]    S. Patskovsky, E. Bergeron, D. Rioux, M. Simard, and M. Meunier, “Hyperspectral reflected light microscopy of plasmonic Au/Ag alloy nanoparticles incubated as multiplex chromatic biomarkers with cancer cells,” Analyst, vol. 139, no. 20, pp. 5247–5253, Sep. 2014, doi: 10.1039/C4AN01063A.

[17]    “AMICI-TYPE ‘LENTICULAR ILLUMINATOR’ ON STAND’.” [Online]. Available: [Accessed: 07-Mar-2018].


Written by Grace Brennan @ the Department of Physics, University of Limerick, Ireland


How Geckos could help us become more like Spiderman. (Interaction forces @ the nanoscale)

There are a number of interaction forces which have a significant role in the interesting mechanical properties that arise at the nanoscale. Nanoscale forces (mechanical not chemical) have been attributed to allowing Geckos to hang on glass surfaces by a single toe! On a geckos footpad, there are hair-like setae which have nanoscale β-keratin spatulae (~200nm in width) at the ends.

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Source Nanoview shows the spatulae. Geckos – Van der Waal forces

The action of a gecko adhering to a surface is not chemical, it’s completely mechanical and due to the unique design of the setae. If a gecko is not in contact with a climbing surface, the setae are curved toward the body and the spatulae are misaligned. But, when the gecko is planted on a surface, the setae bend outwards – flattening out the setae onto the surface. With this action, the spatulae tend to align with the material. If every setae (6.5million) and spatulae aligns – a single gecko could lift 113kg! 

Of course, this is amazing. But the icing on the cake is that geckos can detach their feet in 0.015 seconds. So, it is an easily reversible adhesion. Additionally, geckos adhesion/detachment is not hugely dependant on the surface. Experiments have been carried out where scientists analyse how geckos walk on both hydrophobic and hydrophilic surfaces – only a 2% deviation in adhesion/detachment was seen. We know that gecko Setae are highly hydrophobic (repel water), since van der Waals force is the only mechanism which allows two hydrophobic materials to adhere in air, we can deduce the reason for adhesion to be van der Waals.  Van der Waals forces are a weak interaction distance dependent force which encompasses three contributions – the permanent dipole-dipole attraction Keesom force, the dipole-induced dipole interaction Debye force, and the induced dipole-induced dipole London force. Another amazing feature of Gecko adhesion is the self-cleaning abilities. Geckos actually get cleaner with repeated adhesion and detachment because the dirt/pollen/dust is energetically inclined to stick to the climbing surface rather than the geckos toe!

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Spiderman figurine hanging by gecko-inspired adhesion tape.  Source


After scientists saw the incredible adhesion properties Geckos have, they got to thinking – how can we replicate this synthetically? We could have adhesives that adhere strongly, detach easily and even self-clean! We all know about issues with adhesives, they are typically adherent only (one way) or saw with something like velcro tape, it gets unusable after repeated use due to dirt build-up. How can we use this Gecko design knowledge to make special glues or to carefully pick up and put down small/delicate items or even climb walls like Spiderman? Scientists have made Gecko tape and have actually incorporated this technology into robotics, by making an artificial mechanical gecko. See the fantastic video below! Maybe we could translate this technology to help humans climb walls!

For more details on Gecko adhesion, please see this fantastic paper: You have access Gecko adhesion: evolutionary nanotechnology Autumn et. al.

Nanomaterials bridge the gap between the state of matter transition of molecules to bulk solid materials. There are distinctive size-dependent properties of nanomaterials which arise mostly as a result of the large surface area to volume ratio, which I discussed in my  Nanoscale Phenomena post. At this small size, often times the material length is less than the de Broglie wavelength of the materials charge carrier (electrons and holes) or less than the wavelength of light. In this instance, the periodic boundary conditions (which are theoretical boundaries around a single unit cell – which could be extrapolated to form a full lattice) break down or there may be a change in the density of atoms at the non-crystalline surface of the nanoparticle. This leads to interesting nanoscale forces, like van der Waals forces and others.

Nanoparticles dispersed in a high dielectric constant solution typically develop an induced surface charge. As like charges repel – nanoparticles of the same surface charge also repel thus preventing nanoparticles from clustering together. These surface charges can arise in a few different ways – ionization or a dissociation of surface charge groups, for two dissimilarly charged surfaces in close proximity – charges can “hop” between the surfaces, and an uncharged surface could adsorb or bind to ions in solution. An electrostatic double layer may form to neutralise a charged surface which results in a zeta potential, which is the electric potential between the particle surface and the edge of the double layer – this potential is in the order of milliVolts. A higher zeta potential is a consequence of a larger number of surface charges and results in a higher stability.

Capillary forces are as a result of the formation of a liquid meniscus. This force must be considered for powder and granular samples, multi-particle systems interacting with each other or with a surface, nanoparticle assembly/self-assembly and static friction or stiction forces in nano/microelectromechanical systems.  Concave shaped capillary bridges are typically attractive whereas convex bridges are repulsive.

Image result for capillary bridge
Capillary Bridge source

At the nanoscales other than van der Waal and electrostatic forces, other forces like hydration, solvation, and structural forces are relevant. These forces can, of course, be positive or negative, but can also oscillatory or much stronger than van der Waal or electrostatic forces at short distances. Each of these nanoscale mechanisms and interactions are vital for progressing all sorts of technologies from defense to medical.

Now, because the main focus of this website is on nanomedicine – let’s talk medical applications. The first medical adhesive I think of are plasters. There are so many issues with plasters that I can think of – they either don’t stick very well or they are a nightmare to take off (ouch) and they seem a little unhygienic. In theory, the properties of Gecko adhesion could really help here. Research has been carried out to make gecko-inspired tissue adhesive by mimicking the nanotopography seen in gecko feet. These researchers created a biocompatible and biodegradable elastomar which with further research could be invaluable for the medical field. This is of course not the only bioinspired material – in fact there is a whole area of research dedicated to it and this link is a great way to stay up-to-date with the most exciting research!

Examples of Artifical Setae – 1, Gecko Tape 2 and Geckel (which also incorporates mussle technology) 3