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MLAB 2360 Clinical I

Microscopy Training

Activity 1: Parts of the Compound Microscope

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but it is helpful when searching for transparent elements in the specimen. 6. Main Switch: On/Of 2. Condenser Lens: Below the stage is the condenser lens.MLAB 2360 Clinical I Microscopy Training 2 Parts of the Microscope Defined: 1. When using the 100X objective. **Note: In Urinalysis and when using hemacytometers. The coarse adjustment is used to first bring the object into APPROXIMATE focus starting first with the stage as close to the objective lens without touching. We will use this “field diaphragm” when setting the microscope for Kohler illumination. the aperture iris diaphragm should be set to 0.9 on the scale. **Note: These microscopes are PARFOCAL microscopes.  Aperture Iris diaphragm: The recommended setting of this aperture iris diaphragm is between 75-85% of the NA of the objective in use. 50X or 100X. . Field Iris Diaphragm: Using the field iris diaphragm ring. 5. it is often necessary to LOWER the condenser to achieve greater contrast. Rheostat: Light intensity knob 3. This focuses light onto the object and is NOT involved in the magnification. Clear images are obtained only when the condenser lens is in proper focus (Kohler illumination). meaning that if you are properly focused on a lower objective (10X). Do NOT use the coarse adjustment when on 40X. Coarse adjustment knob: This knob is used to raise and lower the stage of the microscope. the diameter of the field iris can be adjusted to circumscribe (encircle) the field of view to exclude extraneous light and improve image contrast. This reduces resolution. 4. **Note: The coarse objective will ONLY be used when focusing with the 4X or 10 X objectives to avoid damage to the other objectives. you will be able to switch to a higher objective and maintain relative focus. Then move the coarse adjustment so that the stage moves away from the lens until the object is in relative focus. Fine adjustment knob: This knob is used to focus the image into a sharp. critical focus after achieving relative focus with the coarse adjustment knob. The focusing adjustment is a rack and pinion movement to permit vertical movement of the condenser.

Doing so will cause oil contamination of the 40x objective. Each lens is marked with its Magnification/numerical aperture (NA) and Focal Distance. the eye shades should be extended toward you to prevent . Y-axis: Moves the stage forward and back 9. rotate the coarse and fine adjustment knobs to bring the specimen into focus. X-axis : Moves the stage left to right 8. DO NOT “go back” to the 40x objective. **NOTE: Once you have used the 50x or 100x oil immersion objective to examine a slide. the oil must be removed after used and before you put the microscope away at the end of each class period. Oil is ONLY used on the 50X and I00x oil objectives. Most of our microscopes are equipped with 10x and 40X DRY objectives as well as 50X and 100X OIL objectives. Then look through the left eyepiece with your left eye (cover the right eye) and focus the specimen using the adjustment ring on the left ocular. o When using the microscope WITHOUT eyeglasses. This allows for both eyes to be focused individually.  When first using the microscope. Although the OIL immersion lens is designed to work in oil. while the OIL objectives utilize a SMALL drop of oil between the lens and the slide. Objectives: These are lenses mounted on the revolving nosepiece.  Adjust the Diopter: Looking through the right eyepiece with your right eye. 10.  Using the Eye shades: o When using the microscope with eyeglasses.MLAB 2360 Clinical I  Microscopy Training 3 Condenser centering screws: These screws are used during the Kohler illumination procedure to ensure that the condenser is centered in the Field Iris diaphragm. The objective lenses must be kept clean. Use lens tissue or KIMWIPES to clean the outer. The oculars can be moved back and forth to adjust to the interpupillary distance of the student. 7. Oculars: The ocular lenses usually magnify 10X. The oil will cause the gaskets in the objective to erode and cause permanent and EXPENSIVE damage to the objective. adjust the ocular lenses back and forth until a circular field is viewed with both eyes open. exposed surfaces of the lenses. The DRY objectives use only air between the lens and the slide. rendering it useless until it can be cleaned. The index dot indicates the interpupillary distance on the Interpupillary Scale. Thus the total magnification observed is the multiplication of the power magnification of the ocular times the objective. the eye shades should be in the folded back position.

The "actual" distance between any two marks on the reticle are a function of the objective lenses only. The best way to calibrate your reticle is to use what is called a stage micrometer. In binocular scopes like the ones you are using. one can establish the actual value for each mark on the reticle. By making a comparison of the marks on the stage micrometer to the marks on the reticle. Reticles come in many varieties and in diferent diameters.MLAB 2360 Clinical I Microscopy Training 4 extraneous light from entering between the eyepiece and the eye. . there will be a reticle in ONE of the lenses.  Reticles: (Eyepiece micrometers) They are clear circular glass inserts with a scale inscribed on them. This is a slide that has tiny marks of a known dimensions inscribed on it. The reticle or eyepiece micrometer sits at the focal plane inside the eyepiece lens of the microscope and allows the investigator to make accurate measurements of specimens.

the markings will always be the same but the size of the image superimposed under them will get larger with more magnification. The reticle value. Fingerprints can blur images and mascara can “appear” in the field of view. Let's say each division of the metric stage micrometer above is 0. Dropping eyepieces can damage them beyond repair. So.67µ per reticle division. in this case 6. To calculate the value of one division of the eyepiece reticle we would divide 200µ by 30 and the result would be 6. **Note: Eyepieces should be kept clean. In this example. Calculate the length which corresponds to one division of the eyepiece reticle.MLAB 2360 Clinical I Microscopy Training 5 When you look into your eyepiece lens.67µ. as you change to a higher power objective lens. **Note: 1 micron = 1 micrometer so 1 µ=1 µm   First determine how many divisions of the eyepiece reticle correspond to a certain distance on the stage micrometer by finding the FIRST line of the eyepiece reticle that perfectly lines up with the stage micrometer. **Note: Care should be taken when removing dust covers from microscopes in order to avoid pulling an eyepiece off of the scope and having it drop to the floor or workbench. would apply only to the objective for which the calibration was made.01mm or 10 µm or 10µ (10 micron). 30 divisions of the eyepiece reticle corresponds to 20 divisions of the stage micrometer. the represented value between marks will change proportionately. Rubbing mascara . Each microscope objective must be calibrated independently. The eyepiece reticle is divided into 100 units. Each division of the stage micrometer equals 10µ so 20 divisions of the stage micrometer would equal 200 µ. A fresh piece of lens paper should be used to clean each eyepiece. Gently “fogging” the eyepiece with your breath before wiping can aid in removing fingerprints.

the .be/fNTNZ8nWPd4 Illumination of the specimen is the most important variable in achieving high-quality images in microscopy and critical photomicrography or digital imaging. Activity 2: The Compound Microscope Video http://www.austincc. Place microscope on the workbench as shown in the first video. 3.ht ml Activity 3: Review the parts of the Microscope http://www. therefore. Köhler illumination was first introduced in 1893 by August Köhler of the Carl Zeiss corporation as a method of providing the optimum specimen illumination. Activity 5: Kohler Illumination http://youtu.edu/biocr/1406/laba/microscope/index.austincc. The object of the process is to align the condenser to ensure that the cone of light emitted from the illuminator (light source) is focused directly on the specimen and the apertures are set to maximize contrast.MLAB 2360 Clinical I Microscopy Training 6 when it is on a lens can etch the glass.edu/biocr/1406/labv/microscope/index. 2. it is important to “blow off” as much of the mascara as possible before wiping the lens.edu/biology/ketcham/microscope/scope. in urine samples and when using a hemacytometers. Select a specimen slide from the slides provided by the instructor. As the instructor goes through the Virtual Microscope Demonstration. find the parts of the microscope and perform the tasks in the checklist. The primary advantage of Kohler illumination is even illumination free from glare. however.udel.ht ml Activity 4: Watch Virtual Compound Microscope Demonstration and Practice on Your Microscope http://www.html 1.

Close the right eye. the aperture iris diaphragm knob should be set on the aperture iris diaphragm scale to a setting between 75-85% of the NA of the objective. Kohler illumination is not warranted in these circumstances. When you move the aperture iris diaphragm knob from right to left you will see a hexagon or octagon depending on the . you are to set the aperture diaphragm knob to approximately 0.2 on the scale. 4. Focus is obtained using the diopter ring shown below. Objective Magnificatio n Where video begins N a. The diopter ring is normally located on the right eyepiece and moves both in the counter clockwise and clockwise directions to achieve the necessary focus.25 (as seen below). Adjusting Contrast: 1. 2. and parasitology areas of the lab. As stated earlier. Look through the ocular. microbiology. Close the left eye. 3. therefore. but IS recommended in the hematology. a. Place the 10X objective lens into position and focus the specimen using the coarse adjustment knob. We will perform the Kohler illumination on the 10X objective which has a NA of 0.MLAB 2360 Clinical I Microscopy Training 7 condenser is often lowered to increase contrast then raised to increase resolution. b. Place a specimen slide onto the microscope stage and secure between the specimen holders. c. b. This can be done visually as shown in the video. Use the fine adjustment knob to sharpen the image. Remove the removable eyepiece and place it carefully on the table. Set the interpupillary distance to ensure comfort.

. d. **Note: These slides have had oil used on them before. 4. 2. therefore. Replace the eyepiece carefully. You want to set the aperture iris at approximately 75-85% of the capacity (gray circle) by sliding the aperture iris knob. ii. Continue looking through the oculars and centralize the condenser by twisting the condenser centering screws. Close the right eye. focus the specimen using the coarse and fine adjustment knobs. 5. Open the field iris diaphragm by moving the field iris diaphragm to the far right. i. 10. 9. Activity 6: Focus with an OIL Objective 1. be careful to go from 10X to 100X WITHOUT passing the 40X over the slide. Close the left eye. Use the fine adjustment knob to sharpen the image. Centralizing Field Diaphragm 5. 8. Focus is obtained using the Diopter ring. Select a blood smear that the instructor supplies. This gets the stage in the appropriate position. Once focused. 6.MLAB 2360 Clinical I Microscopy Training 8 model expanding then becoming a circle of light. Place the 10X objective lens into position and focus the specimen using the coarse adjustment knob. While looking through the oculars adjust the condenser knob until the hexagon/octagon of light/specimen is at its sharpest. 7. With the 10X objective in the viewing position. close the diaphragm. Set the interpupillary distance to ensure comfort. You have now achieved Kohler illumination. 3. Place the specimen slide onto the microscope stage and secure between the specimen holders. This can be done by moving the field iris diaphragm knob to the far left. When the image is focused with the 10x objective. carefully hold the nosepiece and move the 100X oil objective into viewing position.

10. 7. and GENTLY remove the excess oil of the slide using a Kimwipe. remove the slide. **Note: These microscopes are PARFOCAL microscopes. you will be able to switch to a higher objective and maintain relative focus. Using a clean Kimwipe and lens cleaner. apply a SMALL drop of oil to the space between the objective and the slide. meaning that if you are properly focused on a lower objective (10X). clean the 100X oil objective. 8. Properly put away your microscope as shown in the first video. 9. lower the stage using the coarse adjustment knob. Use the X and Y axis knobs to move through the slide.MLAB 2360 Clinical I Microscopy Training 9 6. . 11. Once the 100x oil objective is in place. Use ONLY the FINE adjustment knob to focus on the specimen. When you have looked at a minimum of 5 fields (visible area of oculars).