What is the definition of resolution?

What is the definition of resolution?

What is the definition of resolution? 150 150 Nyagu

Microscope Quiz 1. The lens that is within the eyepiece of the light microscope is called the: A) scanning B) low power C) high power D) ocular 2. The wheel under the stage that adjusts the amount of light is called the: A) coarse knob B) body tube C) stage clip D) diaphragm 3. To focus a specimen, it is best to start with which objective: A) high power B) low power C) scanning D) ocular 4. When using the high power objective, you should not adjust the: A) coarse focus B) fine focus C) diaphragm D) stage clips 5. On the image, which letter represents the objective? A B C E 6. On the image, which letter represents the coarse adjustment knob? B C D E 7. On the image, which letter represents the stage? A B AB E 8. On the image, which letter indicates the location of the diaphragm? C D E AC 9. The scanning, low, and high power objectives are mounted on the: A) revolving nosepiece B) stage C) body tube D) eyepiece 10. A microscope has a 4x ocular lens and a 10x objective, what is this microscope’s total magnification? A) 4x B) 14x C) 40x D) 400x Exercise e 5 IIINIINTH Light Microscopy Purpose: To introduce the student to the development, components, and usage of simple and compound light microscopes. those that use some other form of energy as their source of illumination, such as electron microscopes. Their source of illumination is a beam of electrons. A microscope is one of the most important, if not the most important, pieces of equipment used by biologists for research purposes. The microscope allows him or her to see details of a specimen not visible with the naked eye. The human eye allows the brain to see detail by being able to separate characteristics of an object into individual entities or points. This separation is called resolution. This is done all the time with everyday objects such as the letters in a word on the page of a newspaper or faces of people in a crowd. Biologists do this when they observe objects or specimens that are large enough to see with the naked eye. But what if they want to see details of an object or specimen that are below the resolution of the human eye? In other words, what if they want to resolve details that cannot be resolved by the eye? Light microscopes use glass lenses, located near the specimen, to provide the resolution needed to see microscopic details. These lenses or objectives as they are commonly known are constructed according to the principles of optics. They have a numerical aperture (NA) which, with the wavelength of light, quantifies the amount of resolution. The higher the NA, the greater the resolution. The numerical aperture is found next to the objective magnification on the barrel of the objective; it appears as a decimal value with no units. There is a limit of resolution though, due to the wavelength of light used. The resolution of the objective can be no smaller than the wavelength of the light waves traveling through the objective. The theoretical limit of resolution for a light microscope is 0.20 him. The minimum distance between objects that the human eye can resolve is about 0.1 mm at a distance of 25 cm from the eye. If biologists want to view details of a specimen that are closer than 0.1 mm apart, they need to “see” resolution that is much better than 0.1 mm. The human eye cannot do this for them so they turn to the microscope to do this for them. The microscope magnifies the extremely small resolution (detail) so it is visible by the human eye as an image of the actual specimen. Biologists use electron microscopes (EM) to view extremely small details, those within distances less than 0.2 um. The source of illumination in these types of microscopes is a beam of electrons. The wavelength of electrons is much smaller than the smallest wavelength of visible light, therefore greater resolution can be achievable with an electron microscope. The limit of resolution of the most powerful electron microscopes is 0.21 nm, about one thousandth of that of the most powerful light microscopes. The “lenses” of these microscopes are actually series magnets that direct the beam of electrons. one- How does the microscope produce this great resolution? And how does it magnify the resolution so it is visible by the human eye? The answer to the former question will be provided here while the answer to the latter question will be provided in Procedure 5.10, in use There are two types of electron microscopes today: transmission electron microscopes (TEM) and scanning electron microscopes (SEM). In a TEM the beam of electrons passes through a thin section of a specimen and therefore produces only a two dimensional image. In a SEM the beam scans the surface of the specimen producing a three Microscopes can be divided into two classes: Those that use visible light energy as their source of illumination, light microscopes, and 37 dimensional image. As the beam passes over the specimen at an angle, different elevations of the surface topography produce shadows hence the three dimensional image. The one drawback of these high-resolution microscopes is that the specimens must be stained or coated with metal atoms and therefore can not be living at the time of observation. Both types of electron microscopes became generally available to biologists in the late 1940s. Both types of light microscopes require the use of glass lenses. These glass lenses bend rays of light (refract them) to produce magnification. The Arabian scholar Ibn al-Haytham (962-1038) is often credited with developing the earliest ground glass lenses to bend rays of light and produce magnification (1, 3). A more recent development in microscopy is the scanning probe microscope (SPM). These microscopes have extremely small tips or probes that actually trace the surface topology of specimens. The resolution of these new microscopes is similar to that of electron microscopes but because of the different preparation process, they can be used to view specimens in aqueous environments without the use of metal or traditional organic stains (5). Depending on their needs, most biologists use a variety of microscopes. They use simple light microscopes, which are usually on the small side and of limited resolution, for small objects and organisms. These microscopes are especially useful in locations where it is not convenient or practical to carry the larger compound microscope. Compound microscopes are more at home in a quiet and stable location such as a lab or an office. As you will see later, they are capable of much higher resolution than simple microscopes. They will often use electron microscopes for very high resolution when the need arises. as Light microscopes are described as either simple microscopes or compound microscopes depending on the number of lenses that form the image. An example of a simple microscope is a hand lens, sometimes called a magnifying glass. Simple microscopes have a single lens. The larger microscopes visible in the laboratory are compound microscopes; these have two sets of lenses. Antony van Leeuwenhoek (1632-1723), a businessman in Delft Holland, is often credited with using the first simple microscope to magnify natural specimens. He observed many natural samples in the 1670s and 1680s (2). On these simple microscopes, Leeuwenhoek used a primitive mechanical attachment to move the specimen, the forerunner of the mechanical stage (3). Toward the end of this exercise you will be introduced to a type of compound microscope commonly referred to a dissection or dissecting microscope. This microscope allows biologists to observe relatively large objects or specimens with moderate resolution at lower magnifications. Dissection microscopes provide a larger portion of a sample than a simple microscope at the same magnification. Charles Darwin used both types of microscopes (simple and compound) during his studies in natural history and evolution as well as geology. was In this exercise you will become familiar with both simple and compound light microscopes. You will also become familiar with the dissection microscope as well. The first compound microscope developed by Zacharias Janssen in Middleburg Holland around 1600 (1,3). The compound microscope received little popular attention until an English scientist, Robert Hooke (1635-1703), publicized his observations in 1665 in his book, Micrographia (4). (Robert Hooke is also known for the development of a physical law stating that the stretch or compression of a spring will be directly proportional to the force applied, i. e. Hooke’s Law.) Procedure 5.1 A Simple Microscope (Stand Magnifier or hand lens) 38 Exercise 5: Light Microscopy increase in magnification from the lowest to the highest magnification? Questions 1. What is the definition of resolution? Why is this ability important to a biologist or anyone else using a microscope? 2. What is the resolution of the human eye? Compound light microscope? Electron Microscope? What is the difference in magnitude between the resolution of the human eye and the resolution of the compound light microscope? How would you overcome this difference to view small 49 5: Light Microscopy the objects using microscope? compound light a 3. What is the difference between transmission electron microscope (TEM) and a scanning electron microscope (SEM)? When would you use one instead of the other? 4. What is the major drawback with an electron microscope (EM) that doesn’t exist with a compound light microscope? an 5. How is a scanning probe microscope (SPM) different from electron microscope (EM)? 6. List the two general types of light microscopes. What is the difference between them? 7. What physical phenomenon is responsible for the production of magnification? What scholar was thought to be the first to use this phenomenon to magnify images? 1. Obtain a stand magnifier or hand lens, a Syracuse watch glass, and a newsprint slide from the supply counter. 3. Place the microscope on the table with the short side of the base perpendicular to and near the edge of the laboratory table. The distance from the edge will depend on your height. 2. Place the newsprint slide across the top of the Syracuse watch glass. 3. With the stand magnifier or hand lens, observe the letters on the newsprint slide. 4. Completely unwrap the power cord from the base of the microscope. 5. Plug the microscope into the outlet in the center of the laboratory table. 4. Draw the image in your lab notebook. Does the image appear right side up or inverted? Approximately how large is the image compared to the actual letters? (For those of you that feel drawing is not your forte, you are not alone. Leeuwenhoek claimed that an artist drew some of the images he produced with the simple microscope [3].) 6. Locate the power switch on the front left side of the base. On the Leica DME microscopes, the illumination control knob controls the on/off function. It is round and found on the base underneath the stage. Do not turn the microscope on just yet. If the microscope has been left on, turn the switch off. 5. Find the actual magnification of the stand magnifier or hand lens and record this in your lab notebook. Procedure 5.3 6. Is the approximation that you made in step 4 close to the actual magnification? Optical and Mechanical Components of the Compound Light Microscope Procedure 5.2 In this procedure you will become familiar with the optical and mechanical components of your compound light microscope: The Leica ATC 2000 or the Leica DME. Retrieving your Compound Light Microscope from the Microscope Cabinet on 1. Review Figures 5.1 or 5.2 and the available wall charts and identify, your microscope, each labeled component. Be sure to locate components on both sides of your microscope. 1. As per directions from your instructor, obtain a compound light microscope from the cabinet. The majority of the available microscopes are Leica ATC 2000s (ATC). Some students will choose a Leica DME microscope (DME). Although this microscope is the same color as the Leica ATC 2000, it has a triangular base. The DME has several other differences, each of which will be addressed in the appropriate textual sections. If you select or are assigned a DME, use the same microscope for the duration of the semester. 2. Follow the path of light (optical path) through the microscope. Each of the labeled components has a specific function or purpose. The following selection will help you to understand the utility of each component as a part of the microscope system. 2. Be sure to carry the microscope properly by placing one hand under the base of the microscope and the other on the arm of the microscope. The ocular or eyepiece lenses increase the magnification produced by the objectives. They are found at the upper end of the binocular body. 39 Exercise 5: Light Microscopy connect Edition G Figure 5.1 Eyepiece Arm Binocular Body LEICA ATG 2000 Focusing Reverse Nosepiece Infinity Corrected Objectives Fixed Stage Condenser Aperture Diaphragm Control Condenser Alignment Screw Collector Lens with Field Diaphragm Field Diaphragm Lever Rheostat Control Knob Base Illuminator Fine Focus Adjustment Knob Stage Adjustment Knobs Coarse Focus Adjustment Knob 40 Exercise 5: Light Microscopy Figure 5.2 Leica DME Eyepieces Diopter Binocular Body Arm Infinity Corrected Objectives LEICA E Reversed Nosepiece Coarse Focus Adjustment Knob Stage Finger Assembly Mechanical Stage Fine Focus Adjustment Knob Stage Adjustment Knobs Focusing Con- denser Knob Condenser Aperture Diaphragm control Rheostat Control Knob Base leica Condenser Alignment Screw Collector Lens Exercise 5: Light Microscopy
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