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


Nanomaterials used in Nanomedicine

∑Materials which have at least one dimension less than 100nm are classified as nanomaterials. These materials can be may shapes and sizes like spheres, rods, wires, cubes, plates, stars, cages, pyramids among some funny named shapes like nanohedgehogs, nanocandles and nanocakes! See the paper Morphology-Controlled Growth of ZnO Nanostructures Using Microwave Irradiation: from Basic to Complex Structures for some really inventive names for various shaped nanomaterials!

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Nanocube Source
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Nanotriangles Source
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Nanostar Source


Aside – scientists are pretty terrible at naming things, for example, the “creative” names given to optical telescopes – the Extremely Large Telescope , Large Binocular TelescopeOverwhelmingly Large TelescopeVery Large Optical Telescope.

These nanoparticle shapes come in different sizes and different materials too. Broadly we can categorize nanomaterials into two groups – organic or inorganic (but it is possible to have a hybrid inorganic-organic nanoparticle too). Organic nanoparticles aren’t nanoparticles from your local farmers market – they are nanoparticles which contain carbon (and often hydrogen too which forms hydrocarbons) whereas most inorganic nanoparticles don’t contain carbon atoms. Organic nanomaterials include carbon (except fullerenes) , polymeric and lipid-based nanocarriers. Inorganic nanoparticles include metallic/plasmonic, magnetic, upconversion, semiconductor and silica based nanoparticles.

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Example of Organic Nanomaterials – Carbon Nanotubes Source
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Example of Inorganic nanomaterial – Cadmium Selenium Quantum Dots, Metallic nanoparticles which fluoresce in different colors dependent on the size of the particle – Source

Organic Nanomaterials

The main groups of organic nanocarriers are liposomes, micelles, protein/peptide based and dendrimers. Protein/peptide based nanocarriers are amorphous (non-crystalline) materials generally conjugated to the therapeutic agent and is often further functionalised with other molecules. Micelles and liposomes are formed by amphiphilic (both hydrophilic and hydrophobic parts), micelles form monolayers whereas liposomes form bilayers.  Lastly, dendrimer nanocarriers are tree-like structures which have a starting atom core (eg. nitrogen) and other elements are added through a series of chemical reactions resulting in a spherical branching structure. This final structure is not unlike blood hemoglobin and albumin macromolecules.

These vesicular nanocarriers can be used to trap both hydrophobic and hydrophilic drugs and even small nanoparticles inside the aqueous/lipid core. This provides protection for drugs and facilitates significant drug loading – minimising toxicity and increasing blood circulation time (increasing possibility that the drug will reach the therapeutic target from avoiding opsonisation).

Inorganic Nanomaterials

inorganic nanomaterials are stable, robust, resistant, highly functional. and are quite easily cleared from the body. Furthermore, inorganic material exhibit truly exciting mechanical, optical, physical and electrical phenomena at the nanoscale which can be tailored through changes in material, phase, shape, size and surface characteristics. Oftentimes, it is necessary to add a biocompatible surface to inorganic nanoparticles to avoid toxicity,  especially for heavy metals.

Semiconductor Nanomaterials

Quantum dots are the most well-known semiconductor nanoemitter. These are typically very small in size ~5nm, which is smaller or equal to the exciton Bohr radius giving quantum confinement. Electrons are subatomic particles with a negative elementary electric charge, electron holes is an empty position in an atom or lattice that an electron could occupy. An exciton is a  bound state where an electron and electron hole are electrostatically attracted to each other through Coulombic forces. An exciton bohr radius is the separation distance between the hole and electron. Due to 3 dimensional confinement effects, quantised energy levels are produced in the filled low energy valence band and in the empty conduction band of the quantum dots which is very unlike bulk semiconductors. The energy gap between the conduction and valance band varies with the size of the quantum dot which explains the tunable emissions (colour) when excited. Additionally, alloyed quantum dots can be further tuned because the bandgap is approximately equal to the weighted average of the composite semiconductor material. Quantum dots excited in the near-infrared are expected to be revolutionary in biomedical imaging. There has been concerns about the stability and toxicity, as many quantum dots lose luminescence intensity when exposed to light/air/oxygen/water and they are generally composed of heavy metal materials.

Upconversion Nanomaterials

Upconversion nanomaterials consist of two parts, first – the host dielectric lattice (e.g., NaYF4) with one or more guest trivalent lanthanide (atomic numbers 57–71) ions (e.g., Er3+, Yb3+). Upconversion is an anti-stokes process, two or more lower energy photons are absorbed (either simultaneously or stepwise) via long-lived real electronic states of the lanthanide dopant and a higher energy photon is emitted. The lanthanide element has a specific electronic configuration with energy levels which is usually independent of the host material type, the nanoparticle shape and its size.

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Electrons are arranged in shells around an atom’s nucleus, where the closest electrons to the nucleus have the lowest energy. Each shell can hold a certain number of electrons (principal quantum number) – the first shell (1) can hold 2 electrons, the second (2) 8 and the third (3) 18. Within these shells are subshells (defined by the  azimuthal quantum number) and are labelled s,p,d or f which can hold 2,6,10 or 14 electrons respectively.

In the case of upconversion, the 5s and 5p shells are full whereas the 4f-4f shells are not. But, because 5s and 5p are full – they shield the 4f-4f shells which allows sharp line-like luminescence, i.e. the luminescence peak is not broad. This luminescence is also resistant to photobleaching, high photostability and are nonblinking, which of course is beneficial over fluorescent molecules which experience high levels of degradation. Through careful design, upconversion nanomaterials can display a variety of emission and excitation wavelengths from UV to NIR.

These upconversion nanoparticles can be incorporated with photosensitizers to produce reactive oxygen species which generally require activation by UV light. This therapy procedure is called Photodynamic therapy and can be used for treating a wide range of medical conditions including malignant cancers and acne. Upconverison nanomaterials also have applications in multimodal imaging through the use of specific dopants – high atomic number dopants for computed tomography (CT) imaging, radioisotopes for  single-photon emission tomography (SPECT) imaging or positron emission tomography
(PET) imaging.

Magnetic Nanomaterials

At the nanoscale, certain magnetic materials below a specific size exhibit a special form of magnetism called superparamagnetism. Superparamagnetic nanoparticles behave as single domain paramagnets when under an external magnetic field but once the field is removed – there is no residual magnetisation. Typically, these materials are Iron oxide nanoparticles. Additionally, these nanomaterials tend to be non-toxic and can be readily coated with molecules for further functionalization. These nanoparticles are commonly used as MRI contrast agents in magnetic resonance imaging (MRI). Furthermore, magnetic nanoparticles can be used in nanotherapy either through magnetic-field-directed drug delivery or through magnetic hyperthermia which involves localized heating of diseased tissues and therefore, cell death.

Silica Nanoparticles

Silica is a highly biocompatible biomaterial which is often used in nanomedicine.

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Mesoporous Silica Nanoparticles Source

Mesoporous silica nanoparticles are silica nanoparticles which have been template-patterned to have pores throughout the particle. This is done through the use of surfactants like Cetrimonium bromide (CTAB), which is extracted after synthesis leaving holes where the CTAB once was. In these pores, water insoluble materials can be added, such as drugs for chemotherapy, dyes for imaging or even small nanoparticles. These pore sizes can be controlled to encapsulate various sizes of biomolecules. Silica is often used to coat nanoparticles to achieve biocompatibility and to simplify further functionalisation.



Plasmonic Nanomaterials

Now, saving the best for last – plasmonic nanoparticles.

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Localised surface plasmon of Plasmonic nanoparticles Source

Plasmonic nanoparticles consist of noble metals like gold, silver, copper and aluminium. At the nanoscale, these materials can support Localized surface plasmons, which is a collective oscillation of the free surface electrons at the interface between the nanomaterial and the surrounding dielectric medium when resonance occurs between the natural resonant frequency of the surface electrons and the frequency of the incident light photons. The LSPR can be tuned with the material, size and shape of the nanoparticle.

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Plasmonic nanoparticles plasmon resonance varying with change of shape of gold nanoparticles. Aspect ratio = the ratio of the width of a nanorod to its length. Source

Plasmonic nanoparticles can scatter and absorb light, for example, for smaller nanoparticles absorption tends to dominate (more light is absorbed – which is generally converted to heat energy) and for larger nanoparticles scattering tends to dominate (which is exploited in bioimaging). For this reason, smaller nanoparticles are often used in photothermal therapy. In Photothermal therapy, plasmonic nanoparticles accumulate in diseased tissues then are irradiated with resonant light, the nanoparticles absorb this light energy and convert it to heat energy, resulting in localised heating of the damaged tissue. This localised heating causes cell death, thus this therapy can be used for cancerous tumors. This heating can be visualised using thermographical measurements or using a dark field microspectroscope, plasmon scattering can be used in medical imaging. Please give Biomedical applications of plasmon resonant metal nanoparticles, Liao et. al. a read for additional information.