Introduction to Light Microscopy: Tips and Tricks for Beginners

By Dee Lawlor

This book offers a beginner’s guide to using light microscopes. It begins with a brief introduction to the physics of optics, which will give the reader a basic grasp of the behaviors of light. In turn, each part of the microscope is explained using clear and simple English, together withdetailed photographs and diagrams. The reader will learn the function, care and correct use of each part. A troubleshooting section also helps resolve some of the most common issues encountered in light microscopy.

Most people have a general idea of how to use a microscope, but many never get the full benefit, because they receive no training. With easy-to-follow steps and detailed images, this guide will help everyone achieve the best results, and be confident using their microscope.

This book is intended for anyone using a light microscope, such as university students, people in lab environments, hobbyists, educators who teach science to young children, and anyonewith a general interest in these valuable tools.

 

• Language: English
• Publisher: Springer
• Release date: May 31, 2019
• ISBN: 9783030053932

Book preview

© Springer Nature Switzerland AG 2019

Dee LawlorIntroduction to Light Microscopyhttps://doi.org/10.1007/978-3-030-05393-2_1

1. Introduction

Dee Lawlor¹  

(1)

Aberdeen, Aberdeenshire, UK

Dee Lawlor

The Microscope not only assists studies, and develops objects of profound interest, but it also opens up innumerable sources of entertainment and amusement, in the ordinary conventional acceptance of these terms; -it discloses to us peculiarities and attractions in abundance; -it impresses us with the wonderful and beautifully-skilful adaptation of all parts of creation, and fills our minds with additional reverence and admiration for the beneficent and Almighty Creator

Jabez Hogg [1], The Microscope.

Abstract

The discoveries, advancements, and technologies that we enjoy today are the cumulative effort of millions of curious people over thousands of years. From the people who invented the wheel, the Greek philosophers, Renaissance explorers, instigators of the eighteenth-century enlightenment, Victorian taxonomists … as a contemporary scientist, you are the latest link in a very long chain.

The history of science—and how we have arrived where we are today—is a fascinating topic. I have started this book with a brief history of the microscope. I have also included a brief history of glass, as this has had a direct impact on the development of microscopy.

While the history of science is not essential knowledge, I encourage all scientists to learn about the evolution of their field and to explore the path that has brought us to where we are today.

Nothing is a greater symbol of science and discovery than the microscope. Microscopes are an easily accessible technology and they have the power and potential to show us the world from a whole new point of view. Edward Tufte said, ‘the commonality between science and art is in trying to see profoundly—to develop strategies of seeing and showing’. The microscope has been, and continues to be, a huge contributor to education and our understanding of the world.

1.1 History

Optics—the study of light—is one of the oldest branches of science. The word ‘optics’ was coined by the Greeks and comes from the Latin ‘ta optika’ which means ‘look’ or ‘appearance’. Light has been a source of great fascination for many of the great minds throuhgout history. In 300 BC, Euclid wrote one of the earliest known works on optics, in which he described his theories on the geometry of vision. In 350 BC Aristotle wrote in Problemata about the magnification of light and images. In 1021, Ibn al-Haytham, the Arab mathematician, astronomer and physicist, wrote the Kitāb al-Manāẓir (the Book of Optics). Many of the theories and ideas suggested by these great minds have today been proven to be correct. Interestingly, any the mistakes were mostly made on the biology side, but much of the physics was spot on.

The history of the microscope stretches back to 750 BC Assyria, where we find the earliest known examples of manufactured lenses. The Egyptians and Mesopotamians are credited with inventing glass; however, many cultures developed the technology independently for themselves. The Chinese, for example, are known to have been making glass from before 200 BC [2]. The lenses are the most important part of the microscope. Their quality, purity, precision, and alignment are essential for creating a high-quality image. The oldest known lens in the world is the Nimrud lens, which currently resides in the British Museum. Also known as the Layard lens, it is a 1.5 inch diameter piece of rock crystal, originating from modern-day Iraq. Research on the lens suggests that it has a focal length of 12 cm and a magnification of approximately 3×. There are several theories as to its original use—it was possibly used as a magnifying glass, used for concentrating sun light to start fire, or possibly even part of an ancient telescope, but it could also just have been for decorative purposes. It is made from rock crystal and thus the quality of the lens has not deteriorated over time. Early glass was full of impurities and imperfections and lenses made from crystal were considered to be superior for much of history. Quartz crystal was a popular choice as it is renowned for its clarity and strength, which makes it ideal for polishing and grinding. In fact, quartz crystal is still used to make the prisms in many microscopes manufactured today, and crystal is known to not degrade the way glass can over time. Around 100 AD, Egyptians glassworkers in Alexandria made a significant leap in glassmaking technology. They made improvements in furnace technology and this allowed them to reach higher temperatures and to have better control over the heat. This improved the quality of the glass by ensuring a more even and thorough melting and mixing of the components. They added manganese oxide to the glass mixture and created the first clear glass. The knowledge and skills travelled west into Europe and eventually we would see every country develop it’s own trade and techniques, experimenting with different temperatures and chemical compositions. Glassmaking occurred all over Europe but it was Venice that became the glassmaking capital of the Western world. The world famous Murano glass was first produced in Venice in the thirteenth century and they would also become the finest mirror-makers in the world—in fact, it was Venetians who made the mirrors for the Hall of Mirrors in the Palace of Versailles (they worked in France though).

Italy is credited with inventing the first reading glasses in the thirteenth century. Large lenses with a gentle curvature give a low level of magnification. This low level of magnification was found to allow a person to read a book more comfortably or to see an object in the distance with better clarity. Over time, different lenses were made to correct different visual conditions such as short-sightedness, long-sightedness, and astigmatism (when the shape of the eyeball is more ovoid than spherical).

The magnifying glass was a common instrument by the sixteenth century and was used by many naturalists to examine samples. The large, gently curving lens of the magnifying glass were of low to medium strength magnification. For higher levels of magnification, the loupe was used—the smaller lens, with a stronger curve, gave a higher level of magnification. The loupe is best known as the instrument used by jewellers to examine stones and settings. 

The invention of the first microscope is credited to Hans and Zacharias Jansen in the late sixteenth century. The Jansens were a Dutch father and son duo who worked as opticians. Using their extensive knowledge of lenses, the Jansens found that when they aligned several lenses in a tube, they could view objects at much greater magnifications than what could be achieved using the single lens of a magnifying glass or loupe. Their invention looked like a small telescope and it was the first compound microscope.

In the seventeenth century, the Flea Glass was developed. The Flea Glass was composed of two lenses—one convex and one flat—that were mounted in a circular frame, on a wood or ivory handle. It looked very much like a small magnifying glass and was used in much the same way—the user would hold it close to the eye and use the ambient light for illumination. The Flea Glass differs to the magnifying glass in that the lenses were smaller. The Flea Glass was available in a range of magnifications, but was mostly low to medium magnification. 

Leeuwenhoek’s microscope—also invented in the seventeenth century—was an advancement on the Flea Glass. It was a simple design where the sample was placed on the point of a pin. The pin was attached to a small board, with a small and highly curved lens set into it. The user held the lens up to their eye while facing a light source and the sample was focused by moving the pin nearer to, or further away from, the lens. Leeuwenhoek himself described how samples looked best when viewed at moderate magnification, as this gave the best balance of light and resolution. Technically, the Leeuwenhoek should be classed as a loupe, as it is a small, single, medium magnification lens. To posterity, however, it is considered a microscope.

Throughout the seventeenth century, the microscope saw some of the greatest scientific discoveries come to light. A well-known example to biologists all around the world is Robert Hookes ground-breaking book, Micrographia. It was first published in 1665 and was one of the world’s first public introductions to the cell and other microscopic wonders. It quickly became a best seller and is still in publication today. Hooke is also believed to have been the first microscoper to accurately calculate magnification. By using a glass scale which had been accurately divided into portions of an inch and then seeing how many sections were visible under different objective lenses, he concluded that ‘for if one division, as seen with one eye through the microscope, extends to thirty divisions on the rule, which is seen by the naked eye, it is evident that the diameter of the object is increased or magnified thirty times’.

An interesting fact about Hooke is that he was acutely aware that he was limited by the technology of his time, and in Micrographia he admits ‘so ill and imperfect are our Microscopes’. Hooke’s microscope was a tube model microscope, typical of the seventeenth century contemporary models. This style of microscope was more along the lines of the design developed by the Jansens (a tube with multiple lenses). As the name would suggest, the microscope constitutes a wooden or paper tube, with lenses at the bottom end and an eye cup at the top. In the latter eighteenth century, they would start to be made of brass. There were several interchangeable lenses of different magnifications and these were the first objectives as we would recognise them today. The light was provided either by a mirror reflecting the ambient light or by an oil lamp, whose light was focused through a water-filled glass globe onto the sample.

The microscope did not escape the elaborate aesthetic of the eighteenth century and we see these machines become miniature works of art. The Baroque style in the first half of the eighteenth century and then the Rococo style in the latter half, were applied liberally to microscopes. Tubular style microscopes in the later eighteenth century stood on three legs, which made them sensitive to vibrations. The wood and paper bodies were also subject to warping. A significant advancement in microscopy that was made in the 1730’s was the development of the achromatic lens. Chromatic aberrations result in the different colours of light being spilt and a rainbow effect being seen in the image. Charles Hall discovered that by adding an extra lens, he could realign the colours of light and thereby resolve the issue of chromatic aberrations. See Sect. 5.​11 for more on chromatic aberrations and how they are resolved. Similar to the Leeuwenhoek model, in 1740 the German physician Johann Lieberkuhn developed a model which was a lens positioned in the centre of a silvered speculum, and this helped add more light for the user. Lieberkuhn would go on to create several different styles of microscope, including the solar microscope which debuted in 1738. The solar microscope was similar in function the camera obscura, as it could be used to project the image of the sample onto a wall, thereby displaying it for all to see.

Throughout much of the sixteenth, seventeenth and eighteenth century, the microscope was seen as a novelty item. Of course, it was also being used to make scientific discoveries, but the technology was still limited. Achievements such as Robert Hookes Micrographia are a beautiful example of what the imaging technology at the time could achieve, but we still did not have the magnification, clarity, and resolution to delve inside the cell. It was in the nineteenth century when the industrial era arrived and work left the home in favour of the factory, office, or lab, that the microscope became a real working machine. The microscope as a toy has endured, but the designs for work and the designs for play, diverged significantly during the industrial revolution.

To meet its new role as a working machine, the microscope became sturdier and more robust. Microscopes were now made out of brass or cast iron. Decoration was done away with and the characteristic U-shaped base came along. The heavy base helped resolve the vibration issues that the eighteenth century tripod suffered. The heavy base also shifted everything upwards—the light source could now sit directly underneath the stage, as opposed to being shone in from the side. In 1830, Joseph Lister resolved the issue of the spherical aberration by optimising the distance between lenses. See Sect. 5.​11 for more on spherical aberrations. The new reclining model of microscope (known as short-stand, as opposed to the eighteenth century high-stand) allowed for the user to sit instead of needing to stand while viewing the sample, further improving stability of the microscope and the image.

Throughout the nineteenth century, mass production of microscope began. Microscopes were being produced all around Europe—with England, France, and Germany being the main contributors. European microscopes were openly traded all around Europe and exported to North America. North America did, of course, start manufacturing its own range of microscopes, which were reported as being enormously superior to the European models [3]. In 1859, it is reported that approximately 2000 large microscopes were manufactured in Europe in a year; by 1869 it was closer to 4000. This brought down the price which made them more accessible for scientists and non-scientists alike.

Up until the late nineteenth and into the early twentieth century, the illumination of the sample was done either with focused lamp light or with light reflected by a mirror. Poor lighting had been a limiting factor in the quality of the image and the next leap in image quality came with the addition of the electric light. This gave us brighter illumination, which increased the sharpness and resolution of the image. By seeing brighter, we could see more detail and at higher magnifications.

Today the microscope has become a highly sophisticated piece of machinery. Advancements in optics, lighting, and newer fields such as digital imaging are constantly improving and refining the technology. At its core, however, it is still just a tube with lenses. I don’t say this to be disparaging, but to be reassuring to the user that no matter what system they are faced with, a strong knowledge of the basics will get them through.

The future of microscopy is in the ability to see smaller and clearer. We have already imaged a single atom and scientists are continually breaking the barriers of what we thought