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Microscopy

Microscopy. An important means of studying MICROANATOMY . Overview of discovery. Abbe & Zeiss. Campani. George Bass. Chester Hall. Pasteur. Amici. Dollond. Lister. Geroge Adams. From the Bioscope Initiative with permission. Dissecting Microscope.

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Microscopy

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  1. Microscopy An important means of studying MICROANATOMY

  2. Overview of discovery Abbe & Zeiss Campani George Bass Chester Hall Pasteur Amici Dollond Lister Geroge Adams From the Bioscope Initiative with permission

  3. Dissecting Microscope Light is reflected off a specimen into two different objectives and eyepieces (or oculars). Provides a three-dimensional view of object. Usually shows surface features of object. Useful up to about 60 - 80 X magnification.

  4. Compound Light Microscope Light shines through specimen and into a single objective lens and then trough the eyepiece (ocular). Provides two-dimensional view. Specimen must be thin and light must be able to pass through. Useful up to about 1,000 X magnification (w/oil lens)

  5. How Does A Compound Microscope Work? • There are two lenses • Objective lens and Ocular lens

  6. FOCUSING AND MAGNIFICATION • The objective lens (in refractors) or primary mirror (in reflectors) collects lots of light from a distant object and brings that light, or image, to a point or focus. • An eyepiece lens takes the bright light from the focus of the objective lens or primary mirror and "spreads it out" (magnifies it) to take up a large portion of the retina. • This is the same principle that a magnifying glass (lens) uses; it takes a small image on the paper and spreads it out over the retina of your eye so that it looks big.

  7. Eyepiece Lense X Objective Lense = Total Magnification

  8. CHEEK CELLS

  9. Scanning Electron Microscope (SEM) Uses a beam of electrons instead of light. The beam of electrons is passed over the specimen and are scattered. These scattered electrons are detected and processed to form an image on a florescent screen. Useful up to about ???

  10. Scanning Electron Microscope

  11. Scanning Tunneling Electron Microscope (TEM) Uses a beam of electrons instead of light. Uses probe and electrons to determine differences in voltage as probe passes over specimen. Can view objects as small as atoms. SO, a bit strong than scanning but images don’t appear as 3-D …flatter in appearance.

  12. Scanning Tunneling Electron Microscope

  13. TISSUES

  14. Factors that Affect IMAGE QUALITY There are several factors : focusing, brightness but the MOST IMPORTANT ONE IS ……RESOLUTION Resolution : the ability to tell two points apart as separate points. • Resolving power: The ability of an objective to resolve two distinct lines very close together

  15. One Slide Has Better Resolution

  16. What Factors Affect Resolution on Scope? • Aperture Size ( the larger the better) • Wavelength ( the smaller the better)

  17. Compound Light Microscope

  18. Objective  - Infinity corrected PLAN-APO-40X 1.30 N.A. Flat field Apochromat Magnification Numerical aperture Factor

  19. Refraction Short wavelengths are “bent” more than long wavelengths dispersion Light is “bent” and the resultant colors separate (dispersion). Red is least refracted, violet most refracted.

  20. Electromagnetic Spectrum

  21. The light spectrum Wavelength ---- Frequency Blue light 488 nm short wavelength high frequency high energy (2 times the red) Photon as a wave packet of energy Red light 650 nm long wavelength low frequency low energy

  22. Object Resolution • Example: 40 x 1.3 N.A. objective at 530 nm light  .00053 = 0.20 m = 2 x 1.3 2 x NA 40 x 0.65 N.A. objective at 530 nm light  .00053 = 0.405 m = 2 x .65 2 x NA

  23. IS THIS PENCIL REALLY BROKEN? Light passing through two different media ( the water and the air) It bend at differently ..has different REFRACTIVE INDEXES SO WE SEE AN ILLUSION OF A BROKEN PENCIL.

  24. Oil Immersion Lens • Using the Oil Immersion Lens • Place a drop of oil on top of coverslip; oil replaces air. • Oil has an index of refraction (bending) almost identical to glass. AIR DOES NOT! SO by replacing air with oil you eliminate the distraction of the DIFFERENCES OF INDEXES between air and glass! • THIS INCREASES RESOLUTION…….ENOUGH TO GIVE US 1000x up from 4oox …WOW!

  25. FACTORS TO CONSIDER: • FIELD OF VISION- As you increase magnification your field of vision will decrease. • VIRTUAL IMAGE- Your specimen willappearupside down and backwards. SO…remember this when you want to move your slide. • DEPTH OF VISION: This is limited in compound scope as we will see with our cross thread slide observation.

  26. ESTIMATE SIZE OF OBJECTS • PLACE SLIDE ON LOW POWER • Using a clear metric ruler, approximate diameter of field. If 2mm is seen in low power then that field is 2mm! NOW…..look at your slide specimen..cells of tissue, maybe? Estimate the size relative to the field.

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