The history of dark-field optical microscopy

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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.

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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].

 

 

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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.

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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.

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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.

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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].

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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.”

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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.

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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.

 

 

REFERENCES

[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: https://www.microscope-antiques.com/hxdarkfield.html. [Accessed: 12-Aug-2019].

[4]      microscope-antiques.com, “History of Dark Ground (Dark Field) Microscope Illumination,” microscope-antiques.com, 2013. [Online]. Available: http://www.microscope-antiques.com/hxdarkfield.html. [Accessed: 07-Mar-2018].

[5]      W. Chambers, T. J. Fellers, and M. W. Davidson, “Oblique Illumination | MicroscopyU,” NIKON INSTRUMENTS INC. [Online]. Available: https://www.microscopyu.com/techniques/stereomicroscopy/oblique-illumination. [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: https://phil.cdc.gov/details.aspx?pid=2335. [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: http://what-when-how.com/inventions/ultramicroscope-inventions/. [Accessed: 06-Mar-2018].

[10]    Nanobiotechnology group Johannes Gutenberg University Mainz, “Plasmon Spectroscopy History,” Johannes Gutenberg University Mainz. [Online]. Available: http://www.nanobiotech.uni-mainz.de/87_ENG_HTML.php. [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: http://www.microscope-antiques.com/lentic.html. [Accessed: 07-Mar-2018].

 

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