Module 1 Lesson 1 - 4.pdf

Introduction to Basic Microscopy PRPM110L: Pharmaceutical Botany with Taxonomy MODULE 1 (Lesson 1 – 4) Prepared by: Mary Ruth Manansala, RPh Microscope ✓Microscopy ▪ Good, unaided eye can separate objects about 0.10-0.15 mm apart For Example: CHLOROPLAST Microscope ✓M...

Introduction to Basic Microscopy PRPM110L: Pharmaceutical Botany with Taxonomy MODULE 1 (Lesson 1 – 4) Prepared by: Mary Ruth Manansala, RPh Microscope ✓Microscopy ▪ Good, unaided eye can separate objects about 0.10-0.15 mm apart For Example: CHLOROPLAST Microscope ✓Microscopes allow scientists to examine specimens in more detail, whether the specimen is a biological sample, a plant, or a crystal structure. ✓In your advanced courses and research in the pharmacy program further use of microscopes (and cameras) allows you to take pictures of your specimens, make quantitative measurements, and properly document your findings. Learning Outcomes At the end of this modular exercise, you will learn to: 1.Use and handle properly the microscope; 2.Describe the use of each part of microscope in use; 3.Apply techniques in mounting specimens on microscope slides; 4.Focus a sharp and quality image of specimen under study. Lesson 1: Proper Care and Maintenance of Microscope Handle with care Improper handling is a common cause of many problems that occur with microscopes. When carrying a microscope, hold it by the base and the metal support arm. The stage on a microscope is the flat plate where the slides are placed for observation. Avoid picking your microscope up by the stage or the eyepiece holder, as this can cause misalignment. Lesson 1: Proper Care and Maintenance of Microscope Look after lenses When using your microscope, the objective lens is lowered to adjust the focus. However, be careful not to let the lens touch the slide you’re looking at, as this can damage the lens. Furthermore, dirty lenses are notoriously difficult to clean. Lesson 1: Proper Care and Maintenance of Microscope Keep covered Microscopes should always be sold with dust covers. Whether transporting or storing your instrument, make the most of the microscope bag and remember to keep your microscope covered when not in use. The microscope’s eye tubes also need to be kept dust free. If the eyepieces need to be removed, cover the tubes with caps and store them with the microscope Lesson 1: Proper Care and Maintenance of Microscope Store safely Ensure you store your microscope in a clean, dry space with good ventilation. Salt air or damp, for example, can cause damage to equipment over time. Expensive, precision equipment should not be stored next to solutions that may leak. Similarly, keep your microscope away from areas with potentially corrosive chemical fumes. Lesson 1: Proper Care and Maintenance of Microscope Keep clean Oil immersion is a technique used to increase the resolving power of a microscope. Both the objective lens and sample are immersed in a transparent oil of high refractive index so that high magnifications can be achieved while still maintaining good resolution. Lesson 1: Proper Care and Maintenance of Microscope Take care of bulbs After using your microscope, turn off the illuminator and wait for it to cool down before putting it away. Allowing the bulb to cool will extend its life and avoid the unnecessary cost of expensive replacements. Lesson 1: Proper Care and Maintenance of Microscope Clean carefully Microscope lenses are delicate. Treat them carefully to avoid any scratches. Use an aspirator to remove dust. Moisten special lens paper with distilled water or an appropriate cleaning solution. Rubbing gently in a circular motion will remove any sticky residue. Never use anything abrasive on microscope lenses. Lesson 1: Proper Care and Maintenance of Microscope Refer to the user’s manual Your microscope should be sold with a user’s manual and specialist spanners as required. Always refer to the manual when making any adjustments to the microscope and use the supplied spanners. Never use force, inappropriate tools or over-tighten when making adjustments to your microscope, as this will only result in equipment damage. Lesson 1: Proper Care and Maintenance of Microscope Maintain your microscope An annual maintenance check of microscopes is always a good idea. Moving parts should be cleaned and lubricated. Similarly, inspect the power cords and plugs for safety. Lesson 1: Proper Care and Maintenance of Microscope Consider a professional service The company where the microscope was bought can be contacted for a range of products to help keep your laboratory microscopy equipment in tip-top condition. Lesson 2: Parts of the Microscope and Its Uses  Lesson 2: Parts of the Microscope and Its Uses ▪ (1656) from Mod.L. Microscopium, lit. : "an instrument for viewing what is small,“ ▪ from Gk. micro- (q.v.) + -skopion. "means of viewing,“ ▪ "from skopein "look at” ▪ " Microscopic "of minute size" is attested from the 1760s Lesson 2: Parts of the Microscope and Its Uses The Eyepiece Lens Eyepiece contains the ocular lens which you will be looking through to see the magnified specimens with magnification ranging from 5X to 30X, but 10X or 15X is the most common in use. The ocular lens provides a re-magnified image to see when light enters through the objective lens. Lesson 2: Parts of the Microscope and Its Uses The Eyepiece tube It connects the eyepiece and ocular lens to the objective lenses. The Microscope Arm It connects the eyepiece tube to the base where you should hold it when carrying the microscope. The Microscope Base It provides stability and support for the microscope in its upright position. Typically, it holds the source of light or illuminator Lesson 2: Parts of the Microscope and Its Uses The Microscope Illuminator It is a light source that can come in a form of a built-in, low-voltage illuminator light or a mirror that reflects an external light source like sunlight. The Stage and Stage Clips It serves as the platform for slides that hold the specimen in place through a staged clip on either side. Some have a mechanical stage with adjustment knobs that allow the movement of slides to achieve more precise positioning. Lesson 2: Parts of the Microscope and Its Uses The Microscope Nosepiece It contains the objective lenses. You can rotate this part by switching objective lenses and adjusting the magnification power. Lesson 2: Parts of the Microscope and Its Uses The Objective Lenses Objective lenses are combined with the eyepiece lens to increase magnification levels. Objective lenses are the lenses that protrude downward over the specimen. ▪ Scanning lens – 4X ▪ LPO – 10X ▪ HPO – 40X ▪ OIO – 100X Lesson 2: Parts of the Microscope and Its Uses The Rack Stop It prevents users from moving the objectives too close to the slide. Control Focus Knobs Turning the knobs adjusts the distance between the stage and the lens. The coarse adjustment knob is used to bring the specimen into initial focus. The fine adjustment knob is then turned to bring the specimen into sharp focus. Lesson 2: Parts of The Condenser Lens and Diaphragm the Microscope These parts are located under the microscopic stage. The condenser concentrates the light on the specimen and Its Uses whereas the diaphragm with a small movable lever is adjusted that regulates the entry of light. Categories of the Parts of the Microscope Categories of the Parts of the Microscope Mechanical Parts oBase oDiaphragm oPillar oBody Tube oInclination Joint oNose piece oStage oCoarse Adjustment Knob oClip oFine Adjustment Knob  Categories of the Parts of the Microscope Magnifying Parts oEye piece Lens oObjective lenses Eye Piece Lens 10x ocular (eyepiece) and four objectives (4x, 10x, 40x and 100x). To calculate the magnification, simply multiply the ocular lens (10x) by the objective lens. With this microscope you can obtain four different magnifications: 40x, 100x, 400x and 1000x Field of View The field of view when using the 10x objective (100x total magnification) is 2 mm. If 8 plant cells extend across the field of view (2 mm), then each cell is 2/8 or 0.25 mm long. Field of View Remember that the diameter of the field of view changes depending on the power of the objective according to the following table: Field of View If you know the diameter of the fov at one magnification, you can determine the diameter of fov at another magnification with the following formula: For example, if you know the diameter of fov at 100x magnification, the diameter of the fov at 1000x magnification = 1.78mm x 100 divided by 1000 = 0.178 mm or 178 micrometers. Field of View The diameter of the circle that you see is the field of view of the microscope. As you increase the magnification, the field of vision is reduced. The field of view is the area your lens can see, and the magnification is how expanded it is Going to high power on a microscope decreases the area of the field of view  Categories of the Parts of the Microscope Illuminating Parts oMirror oElectric Lamp Lesson 3: Mounting of Specimen on Microscopic Slide  Objects magnified under compound microscopes are mounted onto MICROSCOPE SLIDES. Made of glass or plastic, slides are approximately 1x3 inches and between 1mm- 1.2 mm thick. Cover Slips made of borosilicate or silicate glass hold samples in place and Mounting of Specimen protect them from inadvertent movement and on Microscopic Slide contamination. Preparation Techniques Dry Mount simply position a thinly sliced section on the center of the slide and place a cover slip over the sample. Opaque specimens require very fine slices for adequate illumination. Preparation Techniques Wet Mount Place a drop of fluid in the center of the slide Position sample on liquid, using tweezers At an angle, place one side of the coverslip against the slide making contact with the outer edge of the liquid drop Lower the cover slowly, avoiding air bubbles Remove excess water with the paper towel Preparation Techniques Staining a Slide Staining solutions such as iodine, methylene blue, and crystal violet can be added to wet or dry mounts. Add a drop of staining solution on the edge of one side of the coverslip Position the edge of a paper towel on the opposite end Allow the dye to be pulled across Reference: https://www.microscopemaster.com/microscope-slides.html the specimen  Lesson 4: How to Focus Image Under a Microscope How to Focus Image Under a Microscope 1. Place the microscope on a flat, level, firm table free from vibration seeing to it that the arm is towards you while the stage is going away from you. Do not place it in front of a brightly lit window. How to Focus 2. Turn on the light source and adjust the optimum light setting to ensure the correct Image Under level of brightness by turning or sliding the a Microscope brightness adjustment knob at the base. How to Focus Image Under a Microscope 3. Turn the revolving nosepiece (turret) so that the lowest power objective lens is clicked into position. 4. Place the slide (a dry mount, wet mount, prepared mount) on the mechanical stage secured with clips. How to Focus Image Under a Microscope 5. Look through the eyepiece and move the focus knob until the image comes into focus. 6. Adjust the condenser and light intensity for the greatest amount of light. 7. Move the microscope slide around until the sample is in the center of the field of view. How to Focus Image Under a Microscope 8. Manipulate the focus knob and readjust the condenser and light intensity to obtain the clearest image possible. 9. Once the image is sharp with LPO, move to HPO and do minor adjustments using the fine adjustment knob. (You might need to readjust the sample into focus and/or readjust the condenser and light intensity). How to Focus Image Under a Microscope 10. Complete your drawings (image seen under LPO, HPO). 11. When finished, lower the stage, click the low-power lens into position and remove the slide. 12.Wash and dry the slides and keep them on your storage rack or tray. Be Familiar with the Parts END OF DISCUSSION