Exercise 2 Microscopy PDF

PRAYER BEFORE CLASS Holy Spirit, Divine Creator, true source of light and fountain of wisdom! Pour forth your brilliance upon my dense intellect, dissipate the darkness which covers me, that of sin and of ignorance. Grant me a penetrating mind to understand, a retentive memory, method and ease of le...

PRAYER BEFORE CLASS Holy Spirit, Divine Creator, true source of light and fountain of wisdom! Pour forth your brilliance upon my dense intellect, dissipate the darkness which covers me, that of sin and of ignorance. Grant me a penetrating mind to understand, a retentive memory, method and ease of learning, the lucidity to comprehend, and abundant grace in expressing myself. Guide the beginning of my work, direct its progress and bring it to successful completion. This I ask through Jesus Christ, true God, and true man, living and reigning with You and the Father, forever and ever. Amen. ANATOMY AND PHYSIOLOGY WITH PATHOPHYSIOLOGY Exercise 2: MICROSCOPY Learning Outcomes At the end of the exercise, the student must be able to Name the parts of the microscope and their functions presented in this exercise; Demonstrate the proper use of a compound light microscope; Place a slide on the microscope and observe the material, in focus, under all magnifications of the microscope The Anatomy Lesson of Dr. Nicolaes Tulp (c. 1632 CE) Oil painting on canvas by Rembrandt van Rijn Leeuwenhoek with his microscope Oil painting by Ernest Board Parts of the Microscope Microscope Parts Eyepieces / Where the viewer looks through; Ocular lens at the opposite end of the system to where the light source is Base flat structure of the microscope that serves as its foundation Stage Where the sample sits; used for holding and navigating the sample Arm connects the base of the microscope to the nosepiece and eyepiece; used to carry the microscope around Microscope Parts Nosepiece Holds the objective lenses & attaches them to the microscope head; may be rotated Objective the primary magnifying lenses lenses Microscope Parts Condenser used for controlling the light as it falls on the sample Light used for illuminating the sample with source transmitted light Diaphragm used for controlling the amount of light entering the condenser Microscope Parts Coarse Used for making larger movements, adjustment such as bringing the objective down to the knob stage (or vice versa) or for lifting the objectives away to make space Fine Used for smaller movements, adjustment such as the final approach to the slide and knob small adjustments during imaging  Care of the Microscope 1. When carrying the microscope, hold it securely with two hands – one under the base, and one on the arm. 2. Keep the microscope upright at all times. 3. Keep microscope lenses clean with lens cleaner and lens paper. Do not use paper towels or your clothes. 4. Use only the fine-focus knob when using the high-power objective lens. 5. Remove slides from the microscope before putting it away. 6. Store the microscope with the low-power objective in place. 7. Lower the stage. 8. Put the microscope away in its proper location. MATERIALS for Exercise 2: Microscopy Please bring: Please request from LESO: 1. Newsprint with letter e 1. Compound light microscope 2. Scissors / razor blades (1 per group) 3. Toothpicks 2. Glass microscope slides 3. Coverslips 4. Lens paper, Cleaning paper, Lens cleaner 5. Small dropper bottle of water 6. 1% Methylene blue solution 7. Histological slides of kidney, stomach, or liver PROCEDURE A. Using the Microscope Familiarize yourself with the parts of the microscope. Plug it in, making sure that the ocular lens or lenses are facing you. Use the low-power objective lens (4x). If it is not in place, turn the nosepiece until LPO clicks into place. Remember to always use LPO when you first look at a microscope slide. Use the knob on the microscope to raise the condenser lens so it is at its highest level. Take a piece of printed material and cut a few letters from the paper. Place the small piece of paper on a microscope slide and add a drop of water. PROCEDURE A. Using the Microscope Place a thin coverslip on the slide by touching one edge of the coverslip to the water and slowly lowering it over the paper specimen. Note: if you drop the coverslip on top of the slide, you will probably trap several air bubbles, which may obscure some of your specimen. Locate the on/off switch on your microscope and turn it on. Place the slide on the microscope stage with the coverslip on top and the letters centered in the circle on the stage of the microscope. Make sure to use the stage clip. Examine the specimen under low power. Use the coarse adjustment knob to bring the specimen into focus. PROCEDURE A. Using the Microscope Adjust the light by adjusting the light from the base or the iris diaphragm lever in the front of the microscope. The condenser lens will also help with the lighting. Draw what the specimen looks like in the activity sheet. Questions: Do the letters appear right side up, or is the image inverted? Is the letter oriented correctly, or is the image flipped horizontally? How much of the letter occupies the field of view? After examining the specimen using LPO, examine the slide under the next higher power. Switch the objective lens, but do not move the stage. Adjust the focus using the fine-focus knob. Questions: Do the letters appear right side up, or is the image inverted? Is the letter oriented correctly, or is the image flipped horizontally? How much of the letter occupies the field of view? Scanner 40x LPO 100x HPO 400x Questions: Do the letters appear right side up, or is the image inverted? Inverted Is the letter oriented correctly, or is the image flipped horizontally? Flipped horizontally How much of the letter occupies the field of view? All of the letter (scanner) Most of the letter (LPO) Only a small part of the letter (HPO) Scanner 40x LPO 100x HPO 400x Compound microscope: two convex lenses (ocular lens, objective lens) Light passes through convex lens = bends Light rays converge at a point = image (inverted) Object = simple arrow pointing up Objective lens = inverted image, arrow pointing down Eyepiece then further magnifies the image, yet remains inverted PROCEDURE B. Preparation of a Wet Mount With a toothpick, gently scrape the inside of your cheek. Smear the cheek material from the toothpick on a clean microscope slide. Place a drop of methylene blue (a stain) on the smear. Place a coverslip on the slide by touching one edge of the coverslip to the side. Then, slowly lower it on the slide until it covers the smear. Examine it under the microscope. Draw a single cell in the activity sheet. From: https://x.com/MsNMinton/status/1631943787768430594  Plasma membrane nucleus cytoplasm https://www.alamy.com/cheek-cells-at-10-times-magnification-under-a-light-microscope-image415209638.html PROCEDURE C. Observation of a Prepared Tissue Slide Examine a prepared slide provided to your class. Examine the entire sample using LPO. Scan the area and look for areas you want to observe more closely. Move to the next higher power and adjust the focus using the fine focus knob only. Draw what you see in the activity sheet. PROCEDURE D. Oil Immersion Lens Once you have examined the specimen using the high-power dry lens, find the spot you want to examine and center it in the field of view. Add a drop of immersion oil on top of the coverslip and carefully move the oil immersion lens into place. Use the fine focus only; otherwise, you might drive the oil immersion lens through the slide and break it. After examining the slide, swing the lens away and remove the slide, carefully wiping away the immersion oil using a clean piece of lens paper. POST-DISCUSSION QUESTIONS 1. If the ocular lens is 10x, what is the total magnification of the following objective lenses? a. 7x = 70x b. 15x = 150x c. 20x = 200x 2. What is the name of the glass plate that is placed on top of a specimen? Cover slip 3. What is the function of the iris diaphragm of the microscope? It controls the light POST-DISCUSSION QUESTIONS 4. When you switch from a low-power objective (for example, 4x) to a higher-power objective lens (for example, 10x), what happens to the working distance between the lens and the coverslip? It decreases 5. When should you use the low-power objective lens on the microscope? - Whenever a new slide is viewed - When using the high-power objective and the specimen is lost in the field of view POST-DISCUSSION QUESTIONS 6. What is the proper way to carry the microscope in the laboratory? Hold it securely with two hands - one on the arm and the other under the base  1. Arm 2. Fine adjustment knob, coarse adjustment knob 3. Base 4. Ocular lenses 5. Nose piece 6. Objective lenses 7. Stage clips 8. Stage 9. Condenser 10. Iris diaphragm 11. Light source References Wise, E., VanPutte, C., Regan, J., & Russo, A. (2023). Laboratory Manual by Wise for Seeley's Anatomy and Physiology. (13th ed.). New York, NY: McGraw-Hill Lawlor, D. (2019). Introduction to light microscopy: tips and tricks for beginners. Springer.