Through the looking glass

The microscope is the mainstay of cutting edge research in many fields of biology today. When was it invented? What did the initial versions look like? What are some of the latest versions, and what can we use them for? This article provides glimpses into the history of microscopes before recounting some of its more recent and exciting developments.
 
“Where the telescope ends, the microscope begins. Which of the two has the grander view?” —Victor Hugo, Book 3 Chap. 3 of Les Miserables.
 
Few of us can forget the first time we managed to make brick-like rows of cells, peppered with dots of cytoplasm, appear from a flimsy piece of stained onion peel after twiddling the wheels of a school microscope. An indispensable tool in many labs, the microscope is an instrument that helps us examine objects that are too small to be seen by our naked eye. This powerful invention has opened up the previously invisible world of cells and microorganisms to us. Even today, microscopes form the spine of many major areas of life science research, like cell biology.
 
Figure 1. Skin of a tomato as seen through a microscope.
Source: Umberto Salvagnin. License: CC-BY. URL:
https://www.flickr.com/photos/kaibara/7781208904/.
 
A brief history
 
The first microscopes date back to the early 1600s. While it is not clear who the original inventor was, it is believed that the term ‘microscope’ was coined in 1625 by a friend of Galileo Galilei, a German doctor and botanist named Giovanni Faber1. In the years that followed, the microscope was increasingly used to examine and record biological structures. The most memorable contributions to the field of microscopy came about 50 years later, by Antonie van Leeuwenhoek, celebrated today as ‘the father of microbiology1.
 
van Leeuwenhoek was originally a trader of drapes and linens. He became fascinated with lenses, which were commonly used as magnifying glasses to count threads, and soon mastered the art of lens-making. He made several hundred lenses and many different types of simple microscopes - small lenses mounted on brass plates - whose magnification far exceeded that of even the compound microscopes his contemporaries could craft. These state-of-the-art microscopes allowed van  Leeuwenhoek to make ground-breaking observations. He was among the first people to observe cells; discover bacteria and protozoa; and study animal and plant tissues, as well as mineral crystals3.
Today’s researchers are trying to zoom much further into the cell. They seek to probe and photograph the wheels and cogs of all living systems – the biomolecules. One invaluable technique that has made this possible is fluorescence microscopy. Biochemists have discovered a whole host of fluorescing molecules - proteins that spontaneously emit light of one colour after being excited by light of another colour. By chemically coupling these labels or markers to other proteins of interest that do not emit light of their own, researchers are able to ‘see’ proteins as they move and interact with each other.
 
Optical microscopes are limited by what is known as the ‘diffraction limit’, which only allows them to discern details larger than half the wavelength of light, almost of the order of microns. So, two spots, which are less than a micron apart, will appear as one spot. However, a full understanding of many key bimolecular functions, processes and diseases requires a nano-scale picture. Researchers have developed ingenious ways to overcome this constraint and capture the nano-world. These  ‘superresolution fluorescence microscopy’ methods work by controlling the fluorescent labels in different ways. In fact, the 2014 Nobel Prize in Chemistry was awarded to Eric Betzig, W.E. Moerner and Stefan Hell for the development of these techniques.
 
 
Electron microscopes
All the microscopes discussed in this article are optical microscopes - they use light to generate an image. There are also other types of microscopes, including scanning probe microscopes, ultra microscopes and electron microscopes. 
 
Ernst Ruska and Max Knoll are credited with creating the first electron microscope in 1932. As its name suggests, electron microscopes use electrons instead of light to form an image. Glass lenses are replaced by electromagnets, but the working principle remains the same as an optical microscope. Electrons have a much shorter wavelength than light. This means that the electron microscope can resolve, or reveal details on a much smaller scale than an optical microscope. In fact, the best electron microscopes can tell two atoms apart! Electron microscopes can also achieve significantly higher magnifications – almost a thousand times better than a compound microscope.
 
Studying biological samples in an electron microscope, however, has one major problem. Samples are studied in a vacuum, and need to be prepared or ‘fixed’ by one of many methods. This means that live cells cannot be viewed or photographed.
 
Making better microscopes - an ongoing quest
 
Even after three centuries of pioneering work that have made microscopes tremendously powerful and sophisticated, there is room for improvement. “As a physicist, I think there is a huge amount of physics left to be explored and exploited in microscopy,” says G. V. Pavan Kumar, Assistant Professor in the Department of Physics at IISER, Pune. His work is a continuation of the legacy of the many physicists before him whose efforts, through the ages, have helped overcome the limitations of microscopes. 
 
Biological samples are largely transparent. Staining these samples with contrast agents is one way to make them visible under the microscope. But this means that the specimens need to be killed and fixed before staining. Is there a way we can look at living cells instead? Transparent samples, that don’t affect the amplitude of light rays, diffract it. But they also modify another parameter imperceptible to our eye, and called its phase. The Dutch mathematician and physicist, Frits Zernike, discovered a method to convert these phase changes into contrasts in intensity. Using a special disk and a phase plate, he separated and increased the phase difference between direct light and the light diffracted by a specimen. The subsequent interference of the separated light waves resulted in an amplitude contrast that is visible to the human eye.
 
Until recently, phase-contrast was a qualitative method to see cells and tissues non-invasively. Efforts are now focused on extracting quantitative information from the phase change, with scientists trying out several experimental approaches to achieve this. Pavan Kumar and his colleagues are currently dabbling with one such state-of-the-art technique, which could benefit both material science and biology. They have technology that can tailor a phase pattern into the light shone on the sample. The diffracted light, whose phase is modified by the sample, is compared with a reference beam by interference. The phase difference information between the two beams of light is then extracted. A very accurate image of the sample can be constructed using this information, with details of cellular structures and motions on a nanometer scale. “Among other things, this is a method of labelfree imaging, which has great advantages in biology,” says Kumar.
 
Foldscope
Microscopes don’t come cheap. Or they didn’t, until now. A paper microscope called the Foldscope (https://indiabioscience.org/columns/indiabioscience-blog/foldscopeevents...), developed by Manu Prakash, a biophysicist at Stanford University, is set to change the status quo. This origami-based microscope, which can be printed and assembled from a sheet of paper, costs about a hundred rupees. Yet, it provides a magnification of over 2000X, weighs less than a 1 rupee coin, and doesn’t need an external power source to work6.
 
 
The on-going quest for nano-scale clarity has many takers - maybe not among drapers anymore; but, certainly from chemists, physicists and engineers, among others. Their efforts will go a long way in furthering our understanding of the wonderful mechanisms of biology, and hopefully, give us ways to correct those that go wrong. Biology, however, is not merely a muse for innovators. After all, it has been experimenting with the nano-world eons longer than us and has learnt a trick or two, which we can only hope to mimic.
 
Additional readings/resources
  1. Microscopy Society of America. Web. 10 January 2016. http://www.microscopy.org/.
  2. Chapter 10.11: Microscopes. “Fundamentals of Optics”, Jenkins & White. McGraw-Hill International Edition.
  3. Cells Alive! Web. 10 January 2016. http://www.cellsalive. com/
  4. Microscopedia. Web. 10 January 2016. http://www.microscopedia.com/Resource/Application/22?ccgid=4
  5. AAAS ScienceNetLinks “Pond 2: Life in a drop of pond water”. Web. 10 January 2016. http://sciencenetlinks.com/lessons/pond-2-life-in-a-drop-of-pond-water/
References
 
  1. Wikipedia: Microscopy. Web. 10 January 2016. https://en.wikipedia.org/wiki/Microscope
  2. Microscopes for schools. Web. 10 January 2016. http://www2.mrc-lmb.cam.ac.uk/microscopes4schools/yoghurt.php
  3. Wikipedia: Antoine van Leeuwenhoek. Web. 10 January 2016. https://en.wikipedia.org/wiki/Antonie_van_Leeuwenhoek
  4. Gizmodo “How 17th Century Artists Helped Make the Microscopic World Visible”. Web. 10 January 2016. http://gizmodo.com/how-17th-century-artists-helped-make-themicroscopic-w...
  5. NIH National Institute of General Medical Sciences “Life: Magnified” Online. Web. 10 January 2016. http://irp.nih. gov/catalyst/v22i4/nih-microscopy-lights-up-dullesairport
  6. Foldscope: Microscopy for everyone. Web. 10 January 2016. http://www.foldscope.com/

Harini Barath is a science writer based in Bangalore. She writes and curates content for IndiaBioscience. Her background and research training are in the field of condensed matter physics. She can be reached at harini@indiabioscience.org
 

 

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