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Light or optical microscopy has considerably advanced since the publication of L’occhio Della Mosca (The Fly’s Eye) by Giambattista Odierna in 1644 CE. In that sensational book, the Italian naturalist provided the first detailed account of organic tissue – enabled by studying organic matter under the lens of newly invented microscopes. Now, the latest optical microscope technology is capable of imaging viruses that live in organisms.
Conventional Light Microscopes: Widefield and Confocal
Conventional light microscopes – widefield and confocal optical microscopes – can accurately depict image samples at resolutions of around 230 nm (widefield) or 180 nm (confocal) in the XY axes. On the Z-axis (analogous to focal length in photography), these traditional microscopy methods work at resolutions of around 1000 nm (widefield) or 500 nm (confocal).
This is only possible due to continual improvements in microscopy techniques. To get to these resolutions, conventional light microscopes need to be specially calibrated and samples prepared and lit in laboratory conditions. In other words, even these resolutions are not attainable with the microscope equipment that is sold to schools and hobbyists.
The largest viruses can be up to 1000 nm, and so these specially operated conventional light microscopes are capable of visualizing some virus organisms. However, the smallest viruses are only 17 nm in diameter and would be too small for widefield or confocal resolutions to detect.
Reaching the Theoretical Resolutions Limits of Light Microscopes
Modern microscopy techniques can break the theoretical limits of resolution for optical microscopes so that higher resolutions become possible and viruses can be visualized.
This was a gradual process beginning in the late 19th century when the concept of condenser lens systems to concentrate light on the specimen – improving the microscope’s performance – was first understood. New techniques sought to capture light reflected from the specimen to form an image.
Although some scientists were aware of this principle in the 19th century, it was not possible to effectively concentrate and capture light until electric lamps became available as light sources for microscopes.
This growing understanding of how methods of illumination could impact optimum performance in light microscopy was formalized in 1893 by German physicist August Köhler. Köhler illumination was an essential step in the effort to achieve the theoretical limits of light microscopy in the late 19th century and early 20th century.
The technique leads to even sample illumination while making sure that the resulting microscope image does not include the light source as well. Köhler illumination is still used by advanced light microscopes today and is necessary for microscopes to reach the theoretical limits of resolution.
Köhler illumination helps light microscopes to overcome the contrast and resolution challenges faced by unmodified light microscopes. Sample illumination was further improved when Dutch physicist Frits Zernike discovered phase contrast in 1953, and then Polish-French physicist Georges Nomarski discovered differential interference contrast in 1955.
Do Researchers Use Light Microscopes to Study Viruses Today?
Although light microscopy was succeeded in terms of maximum resolution by electron microscopy in the middle of the 20th century, there are still benefits to using the lower-resolution optical microscopes for studying viruses today.
Light microscopy does not require samples to be stable. Stabilizing samples for electron microscopy – or other advanced methods – often has undesired effects on the sample’s chemical or mechanical makeup. Light microscopes, however, are less destructive (although illuminating light can still react with samples in various ways).
Light microscopes – even those modified for maximum performance – are considerably cheaper and more accessible than electron microscopes, not to mention the other more advanced scanning probe microscopy instruments developed in the 1980s.
This means that more researchers worldwide can study viruses. This is important, as it means that researchers can identify and describe viruses earlier on in their infection and begin to work on tests, vaccinations, and a better understanding of the new virus.
References and Further Reading
Hampton, Cheri et al. (2016) “Correlated Fluorescence Microscopy and Cryo-electron Tomography of Virus-infected or Transfected Mammalian Cells.” Nature Protocols. [Online] https://www.doi.org/10.1038/nprot.2016.168.
Heintzmann, Rainer and Gabriella Ficz (2006) “Breaking the Resolution Limit in Light Microscopy.” Briefings in Functional Genomics. [Online] https://doi.org/10.1093/bfgp/ell036.
Logan, Liz (2016) “Early Microscopes Revealed a New World of Tiny Living Things.” Smithsonian Magazine. [Online] https://www.smithsonianmag.com/science-nature/early-microscopes-revealed-new-world-tiny-living-things-180958912/.
Murphy, Douglas B. and Michael W. Davidson (2011) Fundamentals of Light Microscopy and Electronic Imaging. Oxford: Wiley-Blackwell. ISBN 978-0-471-69214-0.
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