Microscopes PDF - Chapter 4

6/26/2024 Microscopes CHAPTER 4 Preamble PowerPoints are a general overview and are provided to help students take notes over the video lecture ONLY. ◦ PowerPoints DO NOT cover the details needed for the Unit exam Each student is responsible for READING the TEXTBOOK for details to answer the UNI...

6/26/2024 Microscopes CHAPTER 4 Preamble PowerPoints are a general overview and are provided to help students take notes over the video lecture ONLY. ◦ PowerPoints DO NOT cover the details needed for the Unit exam Each student is responsible for READING the TEXTBOOK for details to answer the UNIT OBJECTIVES Unit Objectives are your study guide (not this PowerPoint) Test questions cover the details of UNIT OBJECTIVES found only in your Textbook! 1  6/26/2024 Importance of the Microscope  Important for hematology, microbiology, TB, and malaria testing  Compound microscope used in bacteriology, biology, and medicine to examine minute objects such as bacteria, other unicellular organisms, cells and tissue  Advances in fluorochrome stains and monoclonal antibody techniques caused growth in use of fluorescence microscopy in both biomedical analysis and cell biology Basic Components of the Microscope Eyepiece lens Course and fine adjustments Objectives knobs Stage Condenser Stage motion control knobs Power switch Aperture diaphragm Light intensity control Field diaphragm ring 2  6/26/2024 Component Functions  Brightfield microscope - most often used in the clinical laboratory. Consists of three systems: Illumination, magnifying and focusing. I. Illumination System  Light Source – bulb, provides illumination  Intensity control – rheostat (like a dimmer switch)  Condenser – directs and focuses the beam of light onto the material being examined  Aperture Iris Diaphragm – also controls amount of light passing through the material. Interlocking plates (like the iris of the eye)  Field Diaphragm –Controls the area of the circle of light in the filed of view; also used in the alignment of the microscope Component Functions II. Magnification System  Ocular (Eyepiece) – lens that magnifies the image formed by the objective (mag. usually 10 (10x); most microscopes have two oculars called binocular  Objectives – Major part of system, usually three objectives: 10x, 40x and 100x; responsible for primary image formation; determine magnification and resolution of image; Low power, high power and oil immersion Magnification – magnification of objective x the magnification of the ocular equals total magnification. 10x ocular x 10x objective = 100x magnification Resolution – how small and how close individual objects can be and still be recognizable 3  6/26/2024 Component Functions III. Focusing System ◦ Body tube – light passes through to the ocular ◦ Adjustment Controls ◦ Course adjustment – rapid movement over a wide range; give an approximate focus ◦ Fine adjustment – slow movement over a limited range; gives an exact focus AFTER course has been established. NOTE: Use ONLY FINE adjustments with 40x & 100x. Otherwise you will BREAK the slide! Care and Maintenance of the Microscope Good preventive maintenance and care includes: ◦ Regular cleaning of oculars and objectives ◦ Avoid damaging oculars and other optics with eye make-up or other debris ◦ Careful handling to avoid abrupt motions ◦ Protect from direct sunlight, high temperature, humidity, dust and vibration ◦ Use appropriate materials to clean the lenses ◦ Cover when not in use with vinyl or plastic dust cover 4  6/26/2024 Cleaning the Microscope Routine Cleaning Supplies: 1. Commercial lens tissue for optics Caution: Do not use paper towels or rough paper 2. Commercial prepared lens cleaner for the objectives 3. Mild detergent and soft cloth for stage and base of microscope Cleaning Objectives Unplug the microscope Wash hands Remove dust from optical glass surfaces Carefully remove objectives Clean and replace as completed  NOTE: Excessive rubbing can cause damage to iridescent coating on lens Do Not take eyepiece or objectives apart 5  6/26/2024 Replacing Microscope Bulb Unplug microscope and allow bulb to cool Carefully place microscope on its side Open bulb house; use tissue to remove bulb Use tissue (to avoid fingerprints) to pick up new bulb Insert new bulb and close bulb house Setting the Koehler Illumination Plug in microscope and turn on illuminator. Rotate nosepiece to lock 10X objective in place Place smear on stage and center it under the 10X objective Open the field diaphragm all the way and close condenser diaphragm all the way Move up (rack up) stage to its highest position Adjust the oculars for interpupillary distance so that only one circle of light is seen Rack up condenser as high as possible 6  6/26/2024 Setting the Koehler Illumination  Close field diaphragm half way and focus smear at 10X  Close field diaphragm until diameter of illuminated image is smaller than the field of view  Lower condenser with positioning knob until you have a sharp, focused image of the edges of the field diaphragm Setting the Koehler Illumination Adjust condenser using centering screws so that the circle of light is centered in field Open field diaphragm until illuminated image is just larger than the field of view. If more light is needed, use the transformer. Koehler illumination is now set. It is important not to move the condenser up or down or change the field diaphragm. 7  6/26/2024 Operation of the Microscope : Examining Smears  Put smear on stage and center it under the 10X objective  Adjust intensity of the light to a comfortable level with the transformer  Open condenser diaphragm about 70% to achieve a good balance of resolution and contrast  Adjust oculars for interpupillary distance so that when looking with both eyes only one circle of light is seen Examining Smears (continued)  Adjust sharpness of image by moving adjustment ring on adjustable ocular  Once 10X focus is achieved, rotate nosepiece so that the 40X objective is in place  Re-adjust the intensity of light to a comfortable level using the transformer  Use the fine adjustment knob to focus up and down through the different planes of the field 8  6/26/2024 Trouble shooting Microscope Problems – Troubleshooting 1 Problem: Black Field Possible Causes:  Microscope not plugged in  Power not available at outlet  Illuminator not turned on  Bulb burned out  Objective not clicked into place  Condenser too low with diaphragms closed 9  6/26/2024 Microscope Problems – Troubleshooting 2 Problem: Field only partially illuminated Possible Causes:  Objective not clicked into position  Condenser not centered correctly  Condenser too low  Field diaphragms closed too much Microscope Problems – Troubleshooting 3 Problem: Difficulty focusing with 10X objective Possible Causes:  Wrong objective in place  Objective not screwed into place  Not in correct plane of focus 10  6/26/2024 Microscope Problems – Troubleshooting 4 Problem: Difficulty focusing with 40X objective Possible Causes:  Not in correct plane of focus  Not initially focused at 10X Microscope Problems – Troubleshooting 5 & 6 Problem: Blurry image at 10X or 40X Possible Causes:  Dirty objective  Dirty slide  Dirty coverslip Problem: Ground glass appearance Possible Causes:  Condenser too high 11  6/26/2024 Postamble READ the TEXTBOOK for the details to answer the UNIT OBJECTIVES. USE THE UNIT OBJECTIVES AS A STUDY GUIDE All test questions come from detailed material found in the TEXTBOOK (Not this PowerPoint) and relate back to the Unit Objectives 12