Microscopy PDF

MICROSCOPY  INTRODUCTION Microscopy it is the science that deals with the study of microscope , microscopy is the technique used to view those objects that we can not see with our naked eyes. There are three main objectives of Microscopy Magnification Resoluation Contrast...

MICROSCOPY  INTRODUCTION Microscopy it is the science that deals with the study of microscope , microscopy is the technique used to view those objects that we can not see with our naked eyes. There are three main objectives of Microscopy Magnification Resoluation Contrast  INTRODUCTION The earliest microscopes used visible light to create images. The various types of light microscopy include bright-field, dark-field, fluorescence, and phase con trast microscopy Many research applications use electron microscopy because of its ability to produce higher quality images of greater magnification Parts of microscope  Bright field Microscopy A microscope that allows light rays to pass directly to the eye without being deflected by an intervening opaque plate in the condenser is called a bright field microscope. it is also the first type to be used in this laboratory. Bright-field microscopy produces an image made from light that is transmitted through a specimen. The specimen restricts light transmission and appears “shadowy” against a bright background Because most bio logical specimens are transparent, the contrast between the specimen and the background can be improved with the application of stains to the specimen  Image formation begins with light coming from an internal or an external light source.It passes through the condenser lens, which concentrates the light and makes illumination of the specimen more uniform. Refraction (bending) of light as it passes through the objective lens from the specimen produces a magnified real image. This image is magnified again as it passes through the ocular lens to produce a virtual image that appears below or within the microscope  The amount of magnification that each lens produces is marked on the lens Total magnification of the specimen can be calculated by using the following formula: limit to magnification with a light microscope is around 1300X , although higher magnifications are possible , image clarity is more difficult to maintain as the magnification increases. Clarity of an image is called resolution  limit of resolution achieved by a light microscope is about 0.2 µm. (That is, at its absolute best, a light microscope cannot distinguish between two points closer together than 0.2 µm.) BRIGHTFIELD MICROSCOPY  Care of the Instrument can be damaged easily if certain precautions are not observed. The following suggestions cover most hazards. Transport 1)Carry your microscope to your workstation using both hands—one hand grasping the microscope’s arm and the other supporting the microscope beneath its base. 2)Gently place the microscope on the table. Clutter Keep your workstation uncluttered while doing microscopy. Keep unnecessary books and other materials away from your work area. A clear work area promotes efficiency and results in fewer accidents. Electric Cord Microscopes have been known to tumble off of tabletops when students have entangled a foot in a dangling electric cord. Don’t let the electric cord on your microscope dangle in such a way as to risk foot entanglement. Lens Care At the beginning of each laboratory period, check the lenses to make sure they are clean. At the end of each lab session, be sure to wipe any immersion oil off the immersion lens if it has been used. Dust Protection In most laboratories dustcovers are used to protect the instruments during storage. If one is available, place it over the microscope at the end of the period.  Components Light Source In the base of most microscopes is positioned some kind of light source. Lens Systems All compound microscopes have three lens systems: the oculars, the objectives, and the condenser. The ocular, or eyepiece, is a complex piece, located at the top of the instrument, that consists of two or more internal lenses and usually has a magnification of 10×. Most modern microscopes have two ocular (binocular) lenses.  Three or more objectives are usually present. Note that they are attached to a rotatable nosepiece, which makes it possible to move them into position over a slide. Objectives on most laboratory microscopes have magnifications of 10×, 40×, and 100×, designated as low-power, high-dry, and oil immersion, respectively Some microscopes will have a fourth objective for rapid scanning of microscopic fields that is only 4×.  The third lens system is the condenser, which is located under the stage. It collects and directs the light from the lamp to the slide being studied. Unlike the ocular and objective lenses, the condenser lens does not affect the magnifying power of the compound microscope. The condenser can be moved up and down by a knob under the stage. A diaphragm within the condenser regulates the amount of light that reaches the slide.  Focusing Knobs The concentrically arranged coarse adjustment and fine adjustment knobs on the side of the microscope are used for bringing objects into focus when studying an object on a slide. Ocular Adjustments On binocular microscopes, one must be able to change the distance between the oculars and to make diopter changes for eye differences.  the interocular distance is changed by simply pulling apart or pushing together the oculars. To make diopter adjustments, one focuses first with the right eye only. Without touching the focusing knobs, diopter adjustments are then made on the left eye by turning the knurled diopter adjustment ring (figure 1.2) on the left ocular until a sharp image is seen. One should now be able to see sharp images with both eyes.  Procedures If your microscope has three objectives, you have three magnification options: (1) low- power, or 100× total magnification, (2) high- dry magnification, which is 400× total with a 40× objective, and (3) 1000× total magnification with a 100× oil immersion objective. it is best to start with the low-power objective and progress to the higher magnifications  Low-Power Examination Use the following steps when exploring a slide with the low-power objective 1. Position the slide on the stage with the material to be studied on the upper surface of the slide. 2. Turn on the light source, If necessary, reposition the slide so that the stained material on the slide is in the exact center of the light source. 3. Check the condenser to see that it has been raised to its highest point. 4. If the low-power objective is not directly over the center of the stage, rotate it into position. Be sure that as you rotate the objective into position it clicks into its locked position 5. Turn the coarse adjustment knob to lower the objective until it stops. A built-in stop will prevent the objective from touching the slide. 6. While looking down through the ocular (or oculars), bring the object into focus by turning the fine adjustment focusing knob. Don’t readjust the coarse adjustment knob. If you are using a binocular microscope, it will also be necessary to adjust the interocular distance and diopter adjustment to match your eyes. 7. For optimal viewing, it is necessary to focus the condenser and adjust it for maximum illumination. This procedure should be performed each time the objective lens is changed. Raise the iris diaphragm to its highest position. Close the iris diaphragm until the edges of the diaphragm image appear fuzzy. Lower the condenser using its adjustment knob until the edges of the diaphragm are brought into sharp focus. You should now clearly see the sides of the diaphragm expand beyond the field of view. Refocus the specimen using the fine adjustment. Note that as you close the iris diaphragm to reduce the light intensity, the contrast improves and the depth of field increases. Depth of field is defined as the range of distance in front of and behind a focused image within which other objects will appear clear and sharply defined. 8. Once an image is visible, move the slide about to search out what you are looking for. The slide is moved by turning the knobs that move the mechanical stage. 9. Check the cleanliness of the ocular, using the procedure outlined earlier. 10. Once you have identified the structures to be studied and wish to increase the magnification, you may proceed to either high-dry or oil immersion magnification. However, before changing objectives, be sure to center the object you wish to observe.  High-Dry Examination To proceed from low-power to high-dry magnification, all that is necessary is to rotate the high-dry objective into position and open up the diaphragm somewhat. It may be necessary to make a minor adjustment with the fine adjustment knob to sharpen up the image, but the coarse adjustment knob should not be touched. Good quality modern microscopes are usually both parfocal and parcentral. This means that the image will remain both centered and in focus when changing from a lower-power to a higher-power objective lens.  Oil Immersion Techniques The oil immersion lens derives its name from the fact that a special mineral oil is interposed between the specimen and the 100× objective lens. this reduces light refraction and maximizes the numerical aperture to improve the resolution. The use of oil in this way enhances the resolving power of the microscope.  Darkfield Microscopy transparent living organisms can be more easily observed with darkfield microscopy than with conventional brightfield microscopy. This effect may be produced by placing a darkfield stop below the regular condenser or by replacing the condenser with a specially constructed one. To achieve the darkfield effect it is necessary to alter the light rays that approach the objective in such a way that only oblique rays strike the objects being viewed. The obliquity of the rays must be so extreme that if no objects are in the field, the background is completely light-free. Objects in the field become brightly illuminated by the rays that are reflected up through the lens system of the microscope. In dark-field microscopy , a special condenser is used so only the light reflected off the specimen enters the objective. The appearance is of a brightly lit specimen against a dark background, and often with better resolution than that of the bright-field microscope.  Although there are several different methods for producing a dark field, only two devices will be described. the star diaphragm and the cardioid condenser. Application Helps in examining movement of motile cell, live organisms that are either invisible in the ordinary light microscope Diagnostic of microorganisms  The Star Diaphragm One of the simplest ways to produce the darkfield effect is to insert a star diaphragm into the filter slot of the condenser housing This device has an opaque disk in the center that blocks the central rays of light. Figure 2.3 reveals the effect of this stop on the light rays passing through the condenser  The Cardioid Condenser The difficulty that results from using the star diaphragm with high-dry and oil immersion objectives is that the oblique rays are not as carefully metered as is necessary for the higher magnifications. Special condensers such as the cardioid or paraboloid types must be used. Since the cardioid type is the most frequently used type.  Phase-Contrast Microscopy A microscope that is able to differentiate the transparent protoplasmic structures and enhance the contrast between a cell and its surroundings, without the necessity of staining, is the phase- contrast microscope. This microscope was developed by the Dutch physicist Frits Zernike in the 1930s. For his discovery of phase-contrast microscopy, he was awarded the Nobel Prize in 1953. Today it is the microscope of choice for viewing living cells and their activities such as motility.  Phase contrast microscope is based on the principle that rays of light move at different speed through material of different refractive index. Advantage Highly transparent material can be seen Intracellular component can be observed e.g endospores Can see living cell there is no need for staining  Two Types of Light Rays Light rays passing through a transparent object emerge as either direct or diffracted rays. Those rays that pass straight through unaffected by the medium are called direct rays. They are unaltered in amplitude and phase. The rays that are bent because they are retarded by the medium (due to density differences) emerge from the object as diffracted rays. Microscopic Measurements MICROMETRY It is a technique used to measure the size of microscopic objects Principle: Calibration of the ocular micrometer using the stage micrometer  MICROMETERY Ocular Micrometer Stage micrometer The ocular micrometer is It is used to calibrate the a glass disc with 100 Ocular micrometer. equal divisions or lines on It looks like a microscope it but with no absolute slid but has a standard value and it is placed in scale etched into it. The the ocular of the smallest divisions are microscope 0.01mm In length. It is just like a tiny ruler. 0.01mm=10 micro m  Micrometry Materials: 1- light microscope 2- ocular micrometer 3- stage micrometer Procedure 1-we place the ocular micrometer in the right ocular lens of the LM. 2-we place the stage micrometer on the stage of the LM. 3- we look into the ocular and focus on the stage micrometer at low power. We move the stage micrometer so that both the ocular and stage micrometer parallel to each other.  CALIBRATION FORMULA One division of ocular=no:of stage micrometer divisions/no:of ocular meter divisions×10