Microscope - Chapter 5 - PDF

**Chapter 5** **Microscope** **5-1. Identify the major components of the microscope and their function** The microscope is an important diagnostic tool in laboratories; allowing you to view organisms too small to otherwise be seen. In order to accurately use the microscope, laboratory professiona...

**Chapter 5** **Microscope** **5-1. Identify the major components of the microscope and their function** The microscope is an important diagnostic tool in laboratories; allowing you to view organisms too small to otherwise be seen. In order to accurately use the microscope, laboratory professionals must have a basic understanding of its parts and sections, care, maintenance, and procedures for ensuring optimal illumination and focus. What is Microscopy? - Microscopy is the use of the microscope to view objects that cannot be seen with the naked eye. - It is the first step in the examination of specimens. - Microscopy plays a very important role in helping laboratory professionals view specimens clearly. **The microscope consists of three main parts: the head, the arm, and the base. Each part contains several important sections.** **Microscope Head** The first part of the microscope is the head. The head contains the oculars and the nosepiece**, **which are connected to the objective lens**.** The oculars are the eyepieces of the microscope. They are connected to lenses that have a magnification of 10X. In most microscopes, they are binocular. You can adjust the eyepieces to match the distance between your eyes (interpupillary distance) so that you can see one image. It is important to look at the image under the microscope with both eyes because it improves the field of view. **Microscope Objectives** The objectives are attached to the nosepiece. Microscopes generally have several objectives. The magnification power is designated on the objective, for example: - 10X (low power) - 20X - 40X (high dry) - 100X (oil immersion) Note: Some microscopes may also have a 4X objective. **Magnification** Magnification is the number of times larger the image appears as seen through the microscope than it appears to the eye, at a distance of 10 inches (approximately.25 mm). This ratio, in diameters, may be referred to as \"power\", \"times\" or X. The **ocular (eyepiece)** lens magnification is 10x. Each objective will have the lens magnification designated, for example 100x, 40x, 20x or 10x. To determine the total magnification of an object you are viewing, multiply the power of the ocular lens by the power of the objective lens. Example: 10X ocular lens x 10X objective lens = 100X total magnification **Microscope Arm** The microscope arm contains the stage, stage controls, coarse and fine adjustment knobs, and condenser. The stage, located underneath the objectives, is a platform with clips to hold the slide in place. In order to move the specimen in the field of view, you may need to move the stage right or left, backward or forward using the stage control knobs. These knobs are located at the side and beneath the stage. **Coarse and fine adjustment knobs**, located just above the base, move the stage up and down and are used to bring a specimen into focus. Microscopes use several components under the stage to obtain a clear picture of specimens on the slide. The **condenser** under the stage is typically known as the condenser diaphragm, which is an iris diaphragm.(The field and condenser diaphragms are iris diaphragms. An iris diaphragm is an adjustable assembly of thin metal leaves for varying the size of openings that determine the cross section of the light ray bundle entering the condenser and the objectives). The condenser diaphragm controls the light that illuminates the specimen, passing through the objective to the oculars. By opening and closing the condenser diaphragm, you control the angle of light, thereby controlling the contrast and the depth of field(The distance just above and below the focal plane (area being examined) that can be focused clearly. Depth of field/focus decreases as magnification increases). **Microscope Base** At the bottom of the microscope is the base. The microscope base is used for support. The base of the microscope houses the field diaphragm, illuminator, and the on/off switch for the illuminator. The illuminator may also be called the light source **Field Diaphragm** The field diaphragm, attached to the base, helps to obtain a clear image of the specimen. The leaves of the field diaphragm control the width of the light that reaches the condenser diaphragm. Both the condenser and field diaphragm are essential to obtaining proper illumination or Köhler illumination in your microscope. -Basic Microscope Components Review- The microscope has three major parts: the head, arm, and base. The head contains the oculars and nosepiece, which connects to multiple objective lenses of different magnifications such as 10x, 20x, 40x, 100x. The arm holds the stage, stage controls, coarse and fine adjustment knobs, and condenser diaphragms. Mechanical clips located on the stage hold the slide. The base contains the **field diaphragm**, **illuminator (light source),** and **on/off switch** for illuminator. **5-2. Care and Maintenance of the Microscope** Before you begin routine cleaning of your microscope, make sure that you have the necessary supplies on hand. Your microscope should be cleaned as often as needed; however, yearly maintenance is essential in order to keep the microscope in optimal working order.   All maintenance should be recorded according to your facility's standard operating procedure (SOP) for documenting maintenance.   It is also good practice to cover your microscope after use and cleaning. This will prevent fine dust and particles from filtering into the internal components of the microscope. **Moving the Microscope** If you need to move your microscope to a different location, it is essential to know how to properly carry it to avoid any damage to components. - Unplug and secure the power cord. - If the microscope has a rotatable head, turn the head in before moving. - Grasp the arm with one hand and support the base with your other hand - Always carry the microscope with two hands - Carry the microscope close to your body, or use a cart for long distances. **Procedure to Clean the Microscope:** 1\. When cleaning your microscope, start at the top of the microscope and work down. 2\. Clean the oculars by moistening the tip of a cotton swab or lens paper with lens cleaning solution. 3\. Working from the inside to the outside, gently clean each eyepiece in a circular motion. 4\. With a clean piece of lens paper, use circular motion to polish and remove any cotton fibers. 5\. Using the stage controls, lower the stage to reach the lenses on the condenser diaphragm. 6\. Clean each lens with lens paper. 7\. Clean the stage with a suitable cleaner, as recommended in your facility\'s SOP for maintenance (this helps to prevent the dragging of a slide across the stage if there is oil on the stage). **Replacing the Bulb** -Please refer to the manufacturer's instructions before replacing the bulb- - Unplug the microscope from the electrical outlet. - Locate the bulb, being careful to remove any loose lenses or mirrors if necessary. - Make sure the bulb has cooled down before you attempt to remove it. This usually takes about 15 minutes. - Check the part number of the new bulb (to ensure you have the correct bulb for replacement). - Use ONLY tissue/lens paper or a bulb replacement device to replace the bulb (do not use your fingers because they will leave smudge marks on the bulb that may obscure the light). **Maintenance Best Practices Review** - Ensure that you have the necessary supplies on hand before you begin routine cleaning. - Clean the stage as well as the objectives when you use a new slide.     - Cover your microscope after it is used or cleaned. - Use appropriate practices when moving the microscope to avoid damage. - Follow manufacturer\'s guidelines when changing the bulb. Document any maintenance performed on your microscope according to your facility\'s SOP and consult your supervisor if you have questions. **5-3. Köhler Illumination Introduction** Now that you know the parts of the microscope, it is important that you see a clear image when you look through the oculars. The most important variable in achieving high-quality images in microscopy is illumination of the specimen. Köhler illumination provides optimum specimen illumination by creating the proper light path. Köhler illumination was introduced as a method of providing optimum specimen illumination that would be uniformly bright and free from glare. This would ultimately ensure that an image of the specimen was as clear visually as possible. **Condenser and Field Diaphragms** The correct adjustment of the condenser diaphragm is the most critical step in Köhler illumination. The condenser uses centering screws to center the specimen and control the angle of light that illuminates the field of view. The field diaphragm controls the width of the light. Opening and closing the field diaphragm concentrates the light onto a specific area. Set Up **Köhler illumination:** - Plug in the microscope and turn on the light source. - Rotate the nosepiece so that the 10X objective is locked into place. - Put the specimen slide on the stage and center it under the 10X objective. - Adjust the intensity of the light to a comfortable level with the light source. - Open the field diaphragm all the way and close the condenser diaphragm all the way. - Move the stage to its highest position (this is also called racking up). - Adjust the oculars for interpupillary distance so that when looking with both eyes only one circle of light is seen. - Move the condenser diaphragm as high as possible with the condenser height adjustment knob located under the stage. - Close the field diaphragm halfway and focus on the specimen at 10X using the coarse adjustment knob. - Close the field diaphragm until the diameter of the illuminated image is smaller than the field of view. - Lower the condenser diaphragm with the condenser height adjustment knob until a sharp, focused image of the edges of the field diaphragm is achieved. - Using the centering screws on the side of the condenser diaphragm, center the edges of the image so that the angle of light is centered in the field increasing the resolution of the object on the stage. - Remove the eyepiece and look down the barrel of the eyepiece. - Open the field diaphragm until the illuminated image is just larger than the field of view. - Be careful not to open the field diaphragm too much, as this may result in reduced image quality. If more light is needed to view the specimen, adjust intensity of your light source but not the condenser diaphragm. - Replace the eyepiece in the microscope. Köhler illumination is now set. Köhler Illumination Review A clear view of the specimen is significant to collecting accurate data. Köhler illumination is a best practice used to properly illuminate the specimen before focusing. Without this technique you could run the risk of misinterpreting specimen details.   **5-4. Focusing the Microscope** Specimens are viewed clearly when the microscope is in focus. This process is performed using the fine and coarse adjustment knobs. It is important to proceed carefully to avoid damage to the slide or objective. **Parfocal** is the term used to describe the minimal or non-existent change in the focus as a result of moving from one objective to another. Microscope objectives are parfocal, where focus is achieved by going from a lower to a higher objective. Focusing the Microscope Process: - Put a slide on the microscope stage. - Bring the 10X or 40X objective over the slide. - Using the coarse adjustment knob, raise or lower the stage until you see an object in view. - Use the fine adjustment knob to obtain a clearer view of the object. - Manipulate the light source to obtain the correct amount of light. **Focusing with Oil Immersion** If you are using the 100X (oil immersion) objective to view your specimen, it is a good practice to first focus on the specimen using the 10X or 40X objective, add oil, and then move to the 100X oil immersion objective. You should only need to use the fine adjustment knob, because microscopes are parfocal.  Note: Use oil immersion with 100X objective only and do not drag the 40x objective through the oil. **5-5. Ocular/Stage Micrometer Introduction** An ocular micrometer is used when it is necessary to measure the size of an organism viewed under the microscope. Ensuring proper calibration when using the ocular micrometer is beneficial in order to confirm measurements are accurate. An **ocular micrometer** ( A glass disc inserted between the eyepiece and objectives that contains a measuring scale (in microns) or defined grid; the scale will be visibly superimposed on the field of view). is a ruler embedded in a round glass used for measuring objects under the microscope. In conjunction with the ocular micrometer, you will need a **stage micrometer**(A precisely defined scale on a glass slide used for calculating the dimensional value of an ocular micrometer for each combination of ocular and objective). for the calibration process. **The Ocular Micrometer Calibration Process** The stage and ocular micrometer are used to measure small specimens not easily measured by a traditional ruler. By aligning and calibrating the ocular micrometer under the microscope the measurements can be used to determine the size of the specimen. Add the ocular micrometer to the eyepiece and pre-calibrated stage micrometer to the stage, on top of the specimen to be measured. For this example, we are using a 10X objective. Align the ocular micrometer with the stage micrometer so that both meet at the 0 mark. Identify the farthest point to the right where both the stage and ocular micrometer rulers align. This should look something like the image below. As you can see, the last measurement where both rulers align is at 60mm (millimeters). The larger the number on the ocular micrometer, the more accurate the calculation. For this example, the stage micrometer is 1mm long with 100 divisions, which means that each division is 0.01mm (millimeters). By counting the lines on the stage micrometer up to where it aligns with the ocular micrometer at 60mm, you get 0.4mm (40 divisions x 0.01mm=0.4mm) for the stage micrometer. Take the 0.4mm and divide it by 60mm, which should look like 0.4mm (stage micrometer)/60mm (ocular micrometer) =.00667mm Finally, convert millimeters (mm) into micrometers (μm) by multiplying by 1000. 0.00667mm x 1000 = 6.67µm (micrometers) This means that with the 10X objective each ocular division (OD) is equal to 6.67µm (micrometers). **Measuring Objects Using an Ocular Micrometer** Now that you have calibrated your ocular micrometer, you are ready to measure the size of objects/organisms seen under the microscope using the ocular micrometer as a ruler. - Align the left side of the object with the 0 of the ocular micrometer. - Count the ocular divisions (OD) to the end of the specimen. - Multiply the OD by the calibrated micrometer unit. To calculate the size of the specimen to the right using our previous ocular micrometer calibration, multiply 6.67μm by 11 which is the number of ocular divisions (OD) the specimen covers. 6.67μm x 11 OD = 73.37μm. This means the size of the specimen is 73.37μm. Example: The image of the parasite ova is seen on a microscope slide using the 20X objective. The ocular micrometer in the microscope was calibrated at 11μm (micrometers) for 20X. Count the number of ocular divisions (OD) and multiply by the calibrated micrometer value. This will give you the length of the parasite. 11μm x 11OD = 121μm (micrometers) To calculate the size of the specimen, multiply the ocular micrometer calibration by the number of ocular divisions (OD) the specimen covers. The formula should look like this: Ocular μm (micrometer) x OD (ocular divisions) = size of the specimen. **5-6. Troubleshooting Microscope Issues Introduction** In your daily use of the microscope, you may encounter issues such as not seeing the specimen clearly, dimming, or a burned out bulb. Consult your laboratory\'s standard operating procedures (SOPs), consult your supervisor as needed, and refer to the user manual corresponding to the brand and model of your microscope when troubleshooting microscope issues. **Black Field in the Oculars** If you put the slide on the stage and look through the oculars but all you see is a black field, the possible causes could be: - The microscope is unplugged - Power is not available at outlet - The objective is not clicked into place - The bulb is burned out or not inserted correctly - The condenser is too low and the diaphragm is closed **Blurred Image** If you look into the oculars after attempting to focus and have a blurred image, the possible causes could be: - Dirty objective - Oil has leaked into the objective - Dirty coverslip - Dirty microscope slide - Slide is upside down **Problem Focusing** If you place a slide on the stage and move the objective into place but still have problems focusing, the possible causes could be: - Slide is not seated properly on the stage - Microscope needs to be aligned - Parfocal problems - Jammed focus mechanism - Oil on the stage **Light Flickers** If you turn on the microscope\'s light and it flickers or surges, the possible causes could be: - Wiring could be damaged in the cord - Bulb is getting ready to burn out - Bulb is not inserted correctly - Partial Illumination - If you look through the ocular and you see a field that is partially illuminated, the possible causes could be: - Objective is not clicked into place - Condenser is not centered correctly - Condenser is too low - Field diaphragm is not open enough **All parts of the Microscope**