Light Microscopy


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Light Microscopy

  1. 1. By: Shefaa Hejazy. Umm Al-Qura University, Makkah. Faculty of Medical Sciences. Hematology Dept. 1st Semester 1433/2012
  2. 2. Introduction Objectives:  Recognize parts of a compound light microscope (LM)  Learn how to focus an object using Objective Lenses  How to adjust the light source by using the condenser and iris diaphragm
  3. 3. Introduction  LM in Haematology laboratory The Microscope can magnify an object 100-1000 times of it’s original size  A microscopic object can be visible by using a system of lenses and illumination source
  4. 4. Principle and function  LM use a system of lenses (objectives and oculars) to manage the path of light beam that travel b/w the object and eyes in order to magnify that object A Light compound microscope have two ocular lenses Objective lenses
  5. 5. Parts of Light Microscope 1. Eye piece (ocular lenses): Magnifying lenses with a magnification power (10x).
  6. 6. 2. Body tube: Contains mirrors and prisms that transmit the image from the objective lens to the ocular lens.
  7. 7. 3. Objective lenses: Primary lenses that magnify specimen a) Low power 10x b) High power 40x c) Oil immersion 100x
  8. 8. 4. Stage: Holds slide in position.
  9. 9. 5. Condenser: Contains lens system that condenses light before it passes through the specimen.
  10. 10. 6. Iris diaphragm: Control the amount of light entering the condenser.
  11. 11. 7. Coarse and fine adjustment knobs: Used for focusing.
  12. 12. 8. Light: Source of illumination, bulb.
  13. 13. Types of Light Microscopy A. Bright field microscopy. B. Dark field microscopy. C. Fluorescence microscopy. D. Phase contrast microscopy.
  14. 14. A. Bright field microscopy Stained preparation/ slides
  15. 15. B. Dark filed microscopy
  16. 16. This method use to examine unstained preparation e.g. Treponema species.
  17. 17. D. Phase contrast microscopy
  18. 18.  Contrast i.e. structures with differing degrees of brightness or darkness  Denser parts of cell appear Bright  While those have density close to water will appear Dark
  19. 19. C. Fluorescence microscopy
  20. 20. Most commonly used fluorescent dyes Acridine orange Auramine/rhodamine
  21. 21. “Cryptococcus neoformans” stained blue with Calcoflour white
  22. 22. Materials  Compound light microscope.  Lens cleaning paper/ cloth.  Immersion oil.  Wet preparation.  Stained preparation.
  23. 23. Wet preparation method
  24. 24. Examination of a Wet Preparation Using 10x and 40x Objectives:  First select the 10x objective and focus the slide using coarse adjustment knob, by moving it up or downward. Then make it clearly visible using the fine adjustment knob.  Turn to 40x objective and again focus using fine adjustment knob until object is clearly visible.
  25. 25. Examination of stained preparation with 100x objective 1. Focus the smear at 10x then rotate to 100x and add a drop of oil on the slide. 2. Upward the condenser and open iris diaphragm at maximum level. 3. Slowly upward the stage by coarse knob until the picture appear and the objective is immersed with oil. 4. Use fine adjustment to gain a clear picture of the slide.
  26. 26. What is the total magnification power?  Power of the objective lens X Magnification of ocular lens 10x Resolving power of the microscope:  It is a measure of its ability to distinguish b/w two adjacent objects.  The absolute limit of resolving power is the light wavelength = (400-800nm).
  27. 27. Maintenance of microscope Microscope is a delicate instrument; thus it should be …  Handled gently.  Keep in a clean environment.  Stage if contaminated should be cleaned immediately with saline to avoid corrosion.
  28. 28. How to clean a microscope A. Optics:  The low and high power should be cleaned after and prior using with lens paper tissue.  The Oil immersion objective lens can be cleaned with lens paper tissue and few drops of Xyelen. B. Non-optics:  Ocular lenses: using a soft camel-hair brush.  Condenser and iris diaphragm: using a soft cloth or tissue moistened with toluene; and the mirror with 5% alcohol.  Other parts cleaned with mild detergent. Grease/oil removed with petroleum ether followed by 45% ethanol in water or 5% alcohol.
  29. 29. Ocular lenses Body Arm Objective Lenses Stage Condenser Coarse adjustment Iris Diaphragm Light source Fine adjustment