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Understanding Compound Light Microscopes: Parts, Uses, and Techniques

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virgil
50 diapositives · 676 Views · Sep 29, 2014

This guide covers the key components and functions of compound light microscopes, including how to label parts, use the coarse knob and diaphragm for light adjustment, and calculate total magnification. Learn about resolving power, measuring field diameters, and converting millimeters to micrometers. We also discuss the advantages and disadvantages of light microscopes compared to electron microscopes. Mastering these concepts will enhance your microscopy skills and improve your understanding of cellular structures.

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  1. MICROSCOPES

  2. Microscope Quiz Friday –Jan. 28 • Label parts of microscope • How to use (ex: use coarse knob to find object, adjust diaphragm for light) • Total Magnification (eyepiece X objective) • How to Measure (field diam. • number across) • Convert mm to µm (4 mm = 4000 µm)

  3. Compound Light Microscope Parts

  4. Compound Light • Uses two lenses • ocular • objective • To bend light

  5. Resolving Power • Being able to tell two objects apart • Measure of “clarity”-how clear it is

  6. Resolving Power • smallest separation between two object points that a given lens (or mirror) can still show as two distinct entities, not one • .. .

  7. Pollen Under 1000X LM Over 1000 X SEM

  8. MAGNIFICATION • Increase in the apparent size of an object • MULTIPLY THE OCULAR LENS x THE OBJECTIVE • OCULAR 10x • OBJECTIVE 40 x • WHAT IS THE TOTAL MAGNIFICATION? • 400 x

  9. Leaf 4X Leaf 10 X How do they look different?

  10. ADVANTAGES of CLM: • CAN MAGNIFY UP TO 1000 x • CAN VIEW LIVING THINGS • Resolving power 200 nm or 0.2 µm

  11. Disadvantages of LM • Objects must be thin or transparent so light can go through them • The image is inverted

  12. Pictures of LM microorganisms Can be stained

  13. Dissecting Light Microscope • Image is NOT inverted • Usually 40 X is the limit

  14. Dissecting Scope Viewing Light-colored stage for dark specimens and dark-colored stage for light ones

  15. Dissection Scope View of Insect Wing

  16. ELECTRON MICROSCOPES • USE MAGNETS TO FOCUS A BEAM OF ELECTRONS • TEM (Transmission Electron Microscope) • SEM (Scanning Electron Microscope)

  17. TEM AdvantageCan magnify 1000 X’s more than a light microscope (Uh…1000 X 1000 = 1,000,000 X’s ) • Resolving power 0.2 nm

  18. TEM Disadvantages • Must be in a vacuum (dead) • Sample must be VERY THIN (less than 0.2 nm)

  19. TEM Images

  20. SEM: Scanning ElectronMicroscope Advantages • Electron beam scans the surface • Resolution 10 nm • Magnifies 1,000,000 X’s

  21. SEM Disadvantages • Must be in a vacuum (dead) • Cannot see internal structures

  22. SEM Images • house fly • and its mouth

  23. Choose Critter and Change Image • Molecular Expressions Microscopy Primer: Electron Microscopy Interactive Java Tutorials - Virtual Scanning Electron Microscopy

  24. SPM-Scanning Probe • Scanning Probe Microscope • Viewed ATOMS!!!!!!!!!!! • Does not need sample in a vacuum • Magnifies 10 million times

  25. SPM Images (50 um X 1.4 um) • Steel Surface

  26. SPM Images • DNA

  27. FIRST TO VIEW ATOMS!!!

  28. CHOICES: Diaphragm Objectives Ocular Coarse knob Fine knob Base Revolving nose piece Stage and stage clips Arm condenser Label the parts:

  29. Label the parts: Ocular (eyepiece) Arm Coarse knob Fine knob (little) Revolving nose piece Objectives Stage and stage clips Diaphragm Condenser base

  30. Always begin on lowest lens

  31. That is, as you increase magnification, the actual field of view becomes proportionally smaller. • 4OX 100X 400X

  32. Use REVOLVING NOSEPIECE –don’t grab lenses

  33. EyeSpecimen

  34. Depth of Field = to the thickness of the plane of focus

  35. Viewing “F” with A Light Microscope • Which is an “F” put in a compound light and a dissection light microscope?

  36. Field of View • What is the approximate width in mm? • In µm? (1 mm = 1000 µm) • 4 mm 4000 µm

  37. Why use the letter “e?”

  38. Do the math: • one millimeter (mm.) = 1,000 micrometersµm • So 5.5 mm = ________ µm • 5500 µm

  39. NOTE!!!!!!!!!!!!!!!! • The field diameter at high power is proportional to the ratio of the low to high power objectives. • If 40X is 4000 µm • 400X is 400 µm

  40. FD = field diameter • Low power FD X low magnification high power magnification = high power FD Use when object is between the mm markers

  41. Why do you need to know Field Diameter? • You may wish to estimate the size of the specimens (e.g., cells) you will see in lab.

  42. Field of View • What is the approximate size of this cell? • In mm? • In um? • If 5 fit across… • O.4 mm 400 µm

  43. If the field of view in this question is 2 mm… How long is one cell? 2 mm

  44. If the field of view in this question is 2 mm… If 3 cells fit across then, one cell is: 2mm 3 =.67 mm 2 mm

  45. Wet Mount 1. Add drop of water 3. Add cover slip 2. Place Specimen on 4. Tap out bubbles slide

  46. Making a Wet Mount Slide • Add drop of water • Add cover slip