A GLASS-SPHERE MICROSCOPE
By Giorgio Carboni, January 1996
Translated by: Sabrina Gemelli, Giacomo Giuli, Karen Coleman
Acknowledgments to David W. Walker for his helping in the revision of the English text
The Leeuwenhoek microscope
From Leeuwenhoek's microscope to our model
The construction of the microscope
Preparing the objective
The focusing mechanism
The illuminating system
Mounting the objective
Use of the microscope
Travelling in the microcosm!
Textile fibers examination
Events happen in nature in many dimensions. Most of them pass unnoticed by observation because their scale is too big or too small. In fact, we would have to be giants, to follow the path of clouds over continents! And who knows how big we should be, to be able to contemplate the majestic rotations of our galaxy! On the contrary, if we were as small as an ant, we could see the amazing miniature world where bizarre creatures like protists live. The microscope is an instrument that allows us to leave our dimension and explore the microcosm. A snowfall, a flower, a puddle seem normal things, without surprises. Yet, if you could see the beauty of a snowflake, the hidden shapes of flowers, the variety and the strangeness of tiny creatures that live in a puddle, you would surely be amazed. You will notice that you are surrounded by a fascinating and unknown world. The microscope is the right vehicle to conduct you in this amazing world. Using this instrument you will be able to journey in the microcosm.
Usually, while attempting to observe very small objects, we realize the impossibility of distinguishing details smaller than a tenth of millimeter by naked eye. Hopefully, man created instruments, like the microscope, that allowed him to overcome his natural limits. It is not really necessary to be a professional to use a simple but efficient instrument. As people in the past did, with passion and patience, we can try to penetrate into the microcosm, in search of what cannot be seen with only our eyes.
The following article contains the instructions to build a little microscope. It is an instrument similar to the one built by Anton van Leeuwenhoek in the XVIIth century, one of the first microscopes built. Like its illustrious ancestor, our microscope is based on a single but powerful lens.
THE LEEUWENHOEK'S MICROSCOPE
A lot of important scientific discoveries were made by amateurs. Leeuwenhoek was a simple fabric merchant. In his job, little "glass pearls" were regularly used to examine the textiles in detail. None of Leeuwenhoek's colleagues had the idea of observing anything different to textiles, maybe because they did not think there was anything else worth looking at. Leeuwenhoek, however, sparked by a natural and insatiable curiosity, began to observe everything around him. He examined saliva and blood, pond water, vinegar, beer and innumerable other things. Potentially every subject was good, but pond water or even water from a simple puddle (the dirtier the better) was the subject of most interest to examine. He discovered and described many microorganisms. He sent reports to the prestigious English Academy of Science, the Royal Society of London, that widely distributed these documents.
Hence the founder of modern microbiology was a mere amateur, but the scientific community perceived the importance of his discoveries only after many decades. Leeuwenhoek's first advance was to move his attention from textiles to natural objects. To obtain ever-increasing magnifications, he worked on smaller and smaller lenses, finally reaching 1-2 mm diameter lenses. Such small and powerful lenses are difficult to handle and focus. To overcome these difficulties, Leeuwenhoek fixed them between two pierced brass sheets. He arranged the samples to be observed on the tip of a screw, so that he could regulate precisely the distance between them and the objective. The observer had to keep the instrument very close to his eye and look through the lens.
Essentially this instrument was composed of just one lens. Given the high curvature of its surfaces, this lens was very powerful and allowed magnifications of up to 300X, almost one third of the magnification of a modern compound microscope. In optics, this microscope is defined as simple, because it is formed by just one lens. In the same period of Leeuwenhoek's studies, the English physicist Robert Hooke had already built a compound microscope, made up of two groups of lenses: objective and eyepiece. However, the fabrication techniques of lenses were not developed enough and so this kind of instrument had serious optical defects. This rendered it less effective than a simple microscope. Only in the first half of the 1800's were compound microscopes perfected. Leeuwenhoek built hundreds of microscopes. Some of these are still exist today and are conserved in museums (fig. 1). Essentially, this instrument was not easy to use and lacked an efficient illumination system.
FROM LEEUWENHOEK'S MICROSCOPE TO OUR MODEL
During the 50's, in the "Scientific American" magazine, D.L. Stong rediscovered the old Leeuwenhoek's microscope and improved it a great deal. He adapted it to use microscope glass slides and introduced a moveable mirror to direct light through the slides. Another innovation of Stong's is the method of preparing the objective. Leeuwenhoek was able to produce very little lenses by polishing them manually, using abrasive powders. It seems that he also obtained these lenses from the bottom of high temperature blown-glass bulbs. Probably, he exploited the surface tension of the fused glass to obtain high quality spherical droplets. Stong proposed a simpler method to obtain these spheres. He melted the central part of a glass rod on a Bunsen burner in order to obtain a thin glass wire, then he brought this wire near the flame to produce little glass spheres of high quality (see figure 4).
Recently in "Scienza & Vita" magazine of December '93, I presented a model of glass-sphere microscope directly derived from Stong's model, which introduced some other improvements. The first concerns the mechanical structure, which was made easier to use, and the second is a new illuminating system. In place of the mirror, with which it was very difficult to observe objects clearly, in this new model there is a lamp with a circular diffuser which maintains optimal of illumination at all times.
This microscope can reach a magnifications of 200 times or even more, giving surprisingly clear images. Its' construction gives the possibility of enjoying the sensation experienced by scientists three hundred years ago. The microscope opens an amazing field of experiments to amateurs, in preparing samples to observe and in the creation of permanent slides. For teachers this could be an interesting laboratory experience, at the end of which, each student could have a small microscope made with their own hands. In addition, during this experiment, the teacher would have the opportunity to introduce fundamental concepts in Optics and Biology.
THE CONSTRUCTION OF THE MICROSCOPE
The microscope I am going to describe can be divided in four parts:
-The optical part
-The focusing apparatus
-The supporting structure, or stage
-The illuminating system
For a better understanding of the construction methods, the reader is advised to refer to figures 2 and 3. You can modify the project and, if you discover any interesting new solution, tell me and I will examine with interest your proposals.
The optical part is formed by the objective. In our case it is a small glass sphere with a diameter comprised between 1.2 and 2.5 mm, which works as a magnifying glass. Giving its small dimension, it is very powerful and must be kept at a distance of few tenths of a millimeter from the objects to be observed.
THE PREPARATION OF THE OBJECTIVE
To fabricate the objective (fig. 4) you need a glass rod with a diameter of 3 to 5 mm, a Bunsen burner and a pair of tweezers. You can obtain these tools for a low price at a chemistry store. For the Bunsen burner, you will need a small gas tank, a valve, a pressure reducer and a rubber tube. These objects are easy to find in any nearby hardware store. Using a gas burner of a stove takes a lot of patience and it is not easy to obtain satisfactory results: the flame does not heat the glass enough and there is always the danger of burning your fingers. On the other hand, with the Bunsen burner, you have a concentrated and powerful flame, whose intensity can be regulated. This apparatus allows you to work while comfortably seated and this is very important for the fabrication of these delicate objectives.
To reduce the formations of bubbles in the glass sphere you created, wash well the glass rod with soap and water, then avoid touching it in the central part. After having lit the Bunsen burner and adjusted the flame, heat the central part of the rod while rolling it between your fingers. When the glass is sufficiently soft, remove it from the flame and pull firmly on both hands until you get a thread of glass with a diameter of 0.3 mm about. With the tweezers break the thread in the middle, without touching it with your fingers. Hold one of the thread ends on the side of the flame until it begins to melt, forming a little ball. Feed this ball by approaching the thread to the flame until the ball reaches 1.5 to 2 mm of diameter, then remove the thread from the flame and let the ball cool. Now break the thread about 10 mm from the little ball. You will use this tail to glue the objective in its seat. What guarantees the spherical form of the glass ball is the surface tension of the melted glass. However gravitational force tends to deform the sphere, so to obtain objectives of high quality, it is necessary to stay within small dimensions.
You will need to prepare at least a dozen of little glass balls, then with a strong lens, choose one of the correct size without air bubbles and other imperfections. This will be the objective of the microscope. The other good objectives will be kept in reserve. There will be traces of hydrocarbons on the glass sphere you have just fabricated. The sphere must be delicately cleaned with a tissue wet with alcohol or saliva. The magnifying power of the objective is greater the smaller its size. How can you determine the magnifying power? Simply solve the following equation: I=333/d, where I is the magnifying power and d is the diameter of the sphere expressed in mm. For example with a sphere of 1,66 mm of diameter you will obtain about a magnification of 200 X.
THE FOCUSING MECHANISM
To focus the microscope you must move the objective near to or further from the sample. For this reason the lens is fixed on a metal blade connected to two screws. The first one should have a bigger pitch and allows quicker but less precise movements (coarse adjustment). The second one, with a fine pitch allows a more accurate focusing (fine adjustment).
A second metal blade is screwed in, under the slide holding plane and supports the coarse adjustment screw. These two metal blades, with a thickness of one millimeter, can be of brass or steel. The objective is mounted below the upper metal blade over a hole that we will call the seat. In fig 3 are shown the dimensions for making the seat of the objective. The U curve of the two metal blades keeps the screws lined up and this avoids instability of the objective.
As you can see in figures 2 and 3 the objective holder blade is a little curved, otherwise it would slide freely against the slide holding plane, and it would move around. To give it stability it is necessary to bend the coarse adjustment blade slightly up, in this way the objective holder blade bends elastically and stabilizes itself. Complete these operations before mounting the objective, to avoid running the risk of detaching and damaging it. The tip of the micrometric screw must be smoothed to avoid scratching the slide holding plane.
The construction of the supporting structure, or stage, is particularly simple. It is necessary to construct a little box open on two sides. For the base and the two walls you can use wooden boards fasten with nails and glue. For the upper part, where you laid the glass slides, and where the fine focusing screw slides, it is necessary to use a smooth yet hard material for example Formica. On this plane, it is necessary to make a hole of about 10 mm in diameter to permit the passage of the light of the illuminator. You must also make two holes for the screws which hold the coarse adjustment blade. On one of the two lateral walls of the stage you must make a groove to set the blade. The slide holding plane must be fixed to the base with screws so that it can be removed.
THE ILLUMINATING SYSTEM
Besides the objective, the illuminating system is the most critical part of the instrument. If it is well adjusted, it allows objects to be seen with an amazing sharpness for an instrument so simple, otherwise stripes of light will confuse every detail. It is important that the light source has a circular form, a uniform brightness and an adequate dimension. The sun is not a good source. It is too strong and its emitting surface is too small. Using sunlight, the objects appear as clusters of extremely contrasty granules without details. I have tried to use a swinging mirror to collect the light coming from different sources (lamps or windows), according to the aforementioned suggestions of Stong. It is a simple solution, but the adjustment of the mirror is very critical and, moreover, if you move the microscope you will lose the adjustment you have reached. If you collect the light of a neon lamp, due to its lengthened form, the objects you observe will be distinct only in one direction. For similar reasons it is necessary to exclude the use of naked filament lamps.
A very effective and easy solution is a little box containing an electric torch lamp, fed with a flat battery (4.5 V). This solution always gives better lighting conditions and avoids the problems due to the mirror adjustment. You can also give the microscope to another observer without losing the illumination adjustment. The battery can be fixed to the wall of the box by a rubber band. As everyone knows, batteries are spiteful, they exhaust just when you need them! In example, when you want show the microscope to some friend. So, besides the battery, it is better to install a connector to supply energy from an low voltage electric power supply. When the power supply is used, the battery must be disconnected.
The illuminating box can be obtained from a container for 35mm film (24 mm x 36 mm), cutting it in half (to retain the cover, smooth the edge by melting it slightly over a flame). Set up the lamp in the proper lamp-holder inserted in a lateral hole on the box. To increase the efficiency of the illuminator, coat the inside of the box with white paper, or better, with light green paper to raise the color temperature of light. Fix the box to the stage with a screw. The light should pass through a circular opening made on the cover, with a diameter of 8 mm. You must screen the opening with an translucent plastic disk so that the filament will be hidden and there will be a uniformly illuminated circular disc. This screen should not be so transparent either to let the filament be seen, nor so opaque as to absorb too much light. Observing figure 3, notice how the light source, the hole on the slide holding plane, the sample slides, the conical seat with the objective and the eye are all on the same vertical axis.
MOUNTING THE OBJECTIVE
The objective has to be glued under the focusing metal blade in the conical seat (fig. 3). Before doing this, it is necessary to paint opaque black the objective seat and a part of the blade all around. This reduces reflections and interference from light and it must be done on both faces of the blade. This operation can be done easily by a spray-can, but it is also possible to paint with a brush.
To glue the objective, place a drop of nail-polish only on the glass thread that the sphere is connected to (fig. 5). Without touching it with your fingers, the objective must be pressed a little bit against the seat, to eliminate any possible gaps. In fact, if some light should pass between the lenses and the seat, the contrast of the image would be considerably reduced.
USE OF THE MICROSCOPE
This instrument is suitable for observing transparent objects. For this reason it is better to choose very small objects which are transparent and thin. You must put the sample over a glass slide. With a dropper, drip two drops of water on the sample. Then cover this with a coverslip (fig. 6). When you place the slide under the objective, be careful neither to knock it nor to wet it with water. This lens should be only a few tenths of a millimeter away from the coverslip.
Switch on the illuminator. Center the sample by observing the light variation through the objective. Now bring your eye as close as possible to the objective. You will see the observation field widen (at the beginning it is a problem to find a place for your nose!). Now move the focusing screws to make the image distinct. Moving the objective holder blade and the slide (fig. 7) you can easily explore the observation field.
Never touch the objective with your fingers and, if it is necessary clean it, gently use a wet cotton-bud. While doing this, hold the objective underneath to avoid breaking the thin glass thread to which it is attached. After use, store the microscope and all its' accessories in a closed box.
TRAVELING IN THE MICROCOSM!
Required materials: the microscope, a box of glass slides and a box of coverslips, a dropper or a pipette, tweezers with a thin end. These materials can be obtained from chemical and laboratory product shops, usually found near universities.
Collect a sample of water from a pond or from any pool of water. Those with greenish colors or decomposing vegetable matter are very good. In this kind of water, you can observe tiny beings, with the strangest forms, moving in surprising ways (fig. 8). Some of them are unicellular algae and do not be surprised if rather than having roots, you see them rapidly swimming in the water. This group of unicellular organisms belongs to the Protists kingdom. It is made up of only one cell (eukaryotic), often it has chloroplasts and swim by a flagellum or cilia. Among them there are our ... great-great-grand-father!
TEXTILE FIBERS EXAMINATION
Pick up a tuft of thread from a sweater. Put them on the slide, place two drops of water and cover with the coverslip. Through the microscope these fibers appear as transparent rods. Wool threads are recognizable by the presence of thin irregular and transversal lines. Cotton looks like piles of corn leaves. Artificial fibers have longitudinal stripes and sometimes little bubbles. The identification of the nature of this fibers can be achieved observing their behavior near a flame. The material of nylon stockings is very interesting (fig. 9). Put a piece on the slide, show it to a friend and ask him what it is.
Observe a thin cork slice or a piece of elder pith through the microscope. You will see a lot of small cells. The first biologists called them cells, from the Latin cellulae , that is "little cells". Superior animals and plants are constituted by thousands of billions of cells, while bacteria and protists are unicellular. It is amazing that, in a protist organism, all the physiological functions typical of multicellular organisms are carried out by a single cell. Cilia and flagella to swim, introflexions of the membrane to phagocytate particles, vacuoles full of digestive enzymes to break down and assimilate food, other vacuoles to eject the waste and so on ...
The cells of corn and those of pith elder are dead. If you want to observe live cells, take an onion. Cut it into slices and then take a scale. Try to raise the peel that covers the onion scale with tweezers. Draw out a cutting and put it on a slide, add two drops of water and cover. This epithelial tissue is made of a single layer of cells. This is important because it allows us to see the cells without making any difficult thin sections. Through the microscope the tissue looks like a tiled floor (fig. 10). While isolated, cells usually have a spheroidal form, when they are tightly packed one next to the other in a tissue, they take a polygonal form, like soap bubbles and metal crystals.
While observing the onion cells, you can distinguish a cellular wall and a little spheroidal form, the nucleus. The nucleus contains the DNA, the "plan" of the whole onion. If you have methyl blue (you can buy it in a shop of chemical products), prepare a 0.5 % solution in distilled water (you can find this in a pharmacy). Take a peel of a fresh onion or one has been standing in water for a few days, thus biologically active. Dip the peel in the dying solution. The methyl blue will color the nucleus in the cells with a deep blue. If the cells are still alive you will be able to see one or two spherical features of a darker color in the nucleus. These are the nucleolus. This is the place where ribosomes are produced. They are organelles destined to the synthesis of proteins.
A leaf is too thick to be directly observed by the microscope. It is necessary to obtain a thin cross section. The problem is that the leaf bends when you try to cut it. To solve this problem, take a piece of elder pith (you can extract it from a dry branch of that plant), longitudinally cut it and put the leaf inside it, like in a sandwich. With a new razor blade you can now cut thin slices of the leaf without it bending (fig.11). At the place of the elder pith you can use a carrot, or some styrofoam provided it is homogenous and not made up of an agglomerate of little spheres.
With some practice you will be able to cut slices with the thickness of about one cell. In the upper part of a leaf section, you can distinguish a cell-layer lined up in a kind of palisade. In the lower part, you can see a spongy tissue in which the gaseous exchanges take place and, on the epidermis, small openings called stomas. Inside these cells you can see chloroplasts which are organelles where photosynthesis takes place. Here carbon dioxide and water are transformed by the energy of the Sun into sugars and, as a waste product, oxygen.
In a similar way you can prepare and observe other vegetable tissues, for example the stem of herbaceous plants. The section of a violet petal shows epidermal finger-like cells (fig.12). Inside these cells you can see the chromoplasts, organelles which contain the pigments coloring petals.
If you want to see blood red cells (erythrocytes) you have to prepare a blood smear. With a sterilized needle prick a finger-tip. Put a drop of blood on a slide. It is important that the quantity of blood is not excessive, otherwise the red cells could hide the leukocytes. In fact, to make a smear, it is enough to leave a spot of blood of 3 mm about in diameter on the slide. As shown in figure 13, keep the coverslip tilted and bring it near the drop of blood until it touches the slide and adheres to the drop itself. Move the coverslip so as to distribute the blood on the slide underneath. You can observe this slide without adding water and without covering it.
The microcosm is extraordinarily rich with marvels. Strange inhabitants live in unexpected places. Buy some book about using microscopes, they will help you in your search for Vorticella, Rotifera, Diatoms, Paramecia and Amoeba. Who knows, perhaps you could meet a Hydra, a curious being similar to an octopus, colored green because many of its cells possess chloroplasts and carry out photosynthesis. As soon as you say: "What a strange plant!", it will capture a prey with one of its stinging tentacles and ingest it. And maybe you will see it moving doing cut capers or as a caliper "So it is an animal!" The Hydra does not care about this problem it is entirely our own, it settles down on the bottom and stretches its green tentacles at the sun.