1	Using the Microscope Human Structure and Development ANHB 2212 Avinash Bharadwaj
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3	Microscopes and What They Reveal – 1 An ordinary light microscope uses the visible spectrum. It magnifies with the help of glass lenses. Images produced by it can be seen with the eye or photographed. The images show the colours of the tissue as they are on the slide. The main limitation of a light microscope is its resolution – the ability to show two points close to each other as distinct. Up to the limit of resolution, we see a clear, sharp image. If we magnify a light microscopic image beyond 1000 or 1500 times it only appears fuzzy, without revealing further detail : no matter how good the lenses or the mechanism of the microscope. In this unit you will use this type of microscope.
4	Microscopes and What They Reveal – 2 There are other types of microscopes in the same range of resolution, but they use various principles of physics to give images of specific features. Examples of such instruments are : fluorescence microscope and phase contrast microscope (to name just two). A detailed description of these is beyond the scope of this unit.
5	Microscopes and What They Reveal – 3 An electron microscope uses electrons. Magnification is done by means of magnetic coils which act as lenses by broadening the stream of electrons. Electron microscopes can yield vastly greater magnifications at high resolution, compared to a light microscope. Electron microscopes have a vastly better resolution even at lower magnifications. An electron microscope produces monochrome (‘black-and-white’) images which are usually photographed, though they can be seen on a fluorescent screen. In this unit, you do not study electron microscopic pictures as core material except when such a reference is made specifically.
6	Microscopes and What They Reveal – 4 There are in fact two main types of electron microscopes. In a transmission electron microscope (TEM), a beam of electrons is made to pass through a section, just as light passes through a section in a light microscope. In a scanning electron microscope (SEM), a beam of electrons passes over the surface of a tissue and the scattered electrons are sensed by a sensor. This information is just to clarify these terms in case you come across them in textbooks.
7	"This PowerPoint is not an..." This PowerPoint is not an exhaustive resource on the subject of microscopy. It provides familiarity with the microscope and basic instructions adequate for the beginner. These instructions are specific for the Leica student’s microscope used in the histology lab in the School of Anatomy and Human Biology. The descriptions of controls may not be appropriate for other models / makes of microscopes. The photographs are those of the type you will use in the lab.
8	Know Your Microscope Your microscope has a heavy base, a ‘stage’ and the optical system. The base houses a lamp for illumination. The condenser has lenses, which direct a beam of light to the stage. The ‘slide’ (a glass plate which holds the material to be studied) is placed on the ‘stage’. The optical system includes a set of objective lenses and two eyepieces. (There is more to it, but it is not seen and you do not have to do anything with it!) Be careful! The microscope is delicate, with moving / detachable parts. Avoid lifting it! If you must lift it, hold it firmly by the curved column (x) with one hand and support the base with the other hand.
9	Below the Stage… Make sure the main power cord is connected to a power outlet and the switch is on! The Power switch (1) on the microscope is a red wheel on right side of the base. It slides with a click to turn on, and you can rotate it further to control the intensity of light. The small black knob (2) raises or lowers the condenser. You can gently turn the knob to try this.
10	Focusing Knobs
11	Objectives
12	Objectives
13	Objectives
14	Eye pieces
15	The Rotating Head
16	Ready to Use it? This slide onwards, follow the instructions as you work on the microscope. As a prerequisite, you should have studied the earlier slides!
17	Ready to Use it? Turn on the power – check the power cord and the main switch, then turn on the red button at the base. The red button glows when it is ‘on’. As you turn it further, the light intensity increases and you will see a glow through the hole in the stage. Always begin with lowest power (scanner) objective – 4x. Turn it into position if it is not already so. (Which also means that someone was careless! The previous user should have brought the 4x into position at the end of his/her work). Using the coarse adjustment, lower the stage.
18	Further… Check the condenser. We shall not go into the details of adjusting the condenser and centering it. Hopefully these arrangements will not be disturbed. (These adjustments are critical in advanced microscopy). For our purpose : Low power = low condenser, High power = high condenser. Now peep through the eyepieces. Adjust your eye positions until you see a single circle of light when both eyes are open. It is very tempting to close one eye to escape this little exercise, but these bad habits are hard to kill! Adjust the distance between the eyepieces until you succeed with this. Check the iris diaphragm if the light is very dim.
19	Mounting the Slide Select a slide from the box. One side of the slide has labels. This is the ‘upper’ surface. Observe it carefully – you will see a very thin piece of glass (called the coverslip) on this surface. Make sure that the coverslip faces UP. Check the coverslip, do NOT rely on the label. An “upside down” slide can damage itself and the 40x objective. Secure the slide to the stage with the spring clip. On most slides, you will notice the colour of the section in contrast with the clear glass. Bring the section over the hole in the stage. (Use knobs 5).
20	Observation Begin with the scanner (4x) objective. The condenser should be low. Take the stage low down with the coarse focus knob and then raise it slowly until you begin to see an image. Adjust the light intensity (wheel of the switch) and / or iris diaphragm. The ‘field’ of the microscope (the circle of light seen through it) should not be so bright as to cause a glare, it should be light enough to enable you to see some detail. Focus with the coarse adjustment knob and then use the fine focus knob. The visible circular area seen through the microscope is called the ‘field’.
21	10x objective Bring the area of interest close to the centre of the field. Without disturbing the focus, turn the ring of the nosepiece to click the 10x objective in place. Do NOT hold the objective for turning! The image may appear slightly out of focus. Use the fine adjustment knob to sharpen the image. If you need to use the coarse adjustment knob, do so very carefully. Notice that the area of the section seen is smaller now, with the object of interest roughly in the centre.
22	40x objective With the 10x lens note the area of interest and using the stage movement, bring it roughly in the centre of the field. Raise the condenser. You may also need to open the iris diaphragm. High magnification needs more light! Rotate the nose piece so that the 40x objective clicks in place. Be very careful – watch how close the lens is to the slide. Also be aware that someone may have fiddled with the lenses – that would make the lens touch the slide. If the objective does click into place easily : Check if the slide is placed correctly. Check if the objective is screwed-in properly. If in doubt, seek help.
23	40x objective Do NOT touch the coarse adjustment knob! Use only fine adjustment to get a sharp image. If you face difficulty in obtaining a sharp image, call a demonstrator! The rule is : if the slide in focus with the 10x objective, you only need fine adjustment with the 40x objective. If it does not work this way, you need help! When you are done with the 40x lens return the nosepiece to the 10x or 4x objective position. Never take the slide off the stage with a 40x objective in place. This can scratch the slide and / or the objective.
24	Closing down… When you are done : Return the nose piece to the 4x position. Switch power off with the button on the base. Switch power off from the main outlet and remove the plug. Hold the plug, do not pull the wire. Do not leave the power cord dangling. Cover the microscope. Leave your bench tidy.