Tools of the Biologist The Incredible World of Microscopes video

1. Tools of the BiologistThe Incredible World of Microscopes video

2. History • Anton Von Leeuwenhoek Born in Holland 1632 • First to observe living bacteria & drew them. • Also looked at protists, sperm, blood • Used a simple scope • Made over 500 "microscopes"

4. Robert Hooke (1665) • Used compound scope to examine cork. • Coined the term “cell” referring to the many little boxes. Actually saw dead plant cells.

5. Types of Microscopes 1. Simple microscope – Hand lens (magnifying glass) • 3 – 40 times magnification

6. Compound Light Microscope The type we use in our labs • Most commonly used microscope • Uses light and lenses to magnify & view the specimen • Has two sets of lenses – Ocular (eye piece) & Objective (near the object being viewed) • Total magnification on our scopes = 40 – 1000 times • Total magnification = Ocular (10X) x Objective (40X)

7. Ocular – Eye piece Neck – Supports the eyepiece. Objectives – 4 – 100x magnification Arm – Supports neck and objectives. Carry by this Stage and clips – Holds slides in place Adjustments – Coarse & Fine. Focuses image Diaphragm – Controls the amount of light coming through the stage Light – Electric light source Base – Bottom of scope. One hand goes underneath

9. The Diaphragm • Use the Diaphragm to adjust the amount of light • Image of pollen grain under good brightness (left) and poor brightness (right) • Image of pollen grain with good contrast (left) and poor contrast (right)

10. Focusing • Use the Adjustment knobs to focus the image • Coarse adjustment brings the image into near focus • Fine adjustment (smaller knob) brings it into fine focus

11. Resolution • Ability to clearly distinguish two objects that are close together. • Image of pollen grain with good resolution (left) and poor resolution (right) • Resolving power of our scope = 0.2um

12. Rules for using the Microscope • Use only the assigned microscope • Carry & place the scope properly (6cm from edge of table) • Do not let the cords dangle or get into the sinks • Clean lens only with lens paper. NO FINGERS! • Do not reuse the same spot on your lens paper • Start on low (4x) power when you start your observations • Always focus (move the stage) away from the slide • Use the coarse adjustment first then the fine adjustment • Be careful when switching to high (40x) power to se that there is enough clearance between the objective and the slide • Do not use the coarse adjustment knob on high (40x) power • When you are done with the scope, turn off the light switch • Always put scope away with cord wrapped around it, cover on & the low power objective in place • Put scopes away with the numbers facing out into the proper slot • Clean and dry all slides and cover slips before putting them away

13. Wet Mount Add drop of water to specimen Clean slide Put coverslip at a right angle into the side of the water View specimen

14. Making a Wet Mount Slide • 1. Gather a thin slice/piece of whatever your specimen is. If your specimen is too thick, then the coverslip will wobble on top of the sample like a see-saw: • 2. Place ONE drop of water directly over the specimen. If you put too much water over the specimen, then the coverslip will float on top of the water, making it harder to draw the specimens as they float past the field of view! • 3. Place the coverslip at a 45 degree angle (approximately), with one edge touching the water drop, and let go.

15. The Letter “e” 40x 400x

16. Threads 100x

17. Beware of Air Bubbles!!

18. Compound Microscope images Diatom Paramecium Amoeba Vorticella Daphnia Hydra budding

19. 3. Binocular (Has two oculars) Gives a 3D image. • Also called a Dissecting scope or Stereo scope Monocular (1 ocular) Light Microscope • 2D image

20. Since most of the specimens we observe will be clear to near clear, what could be done to enhance the image we view through the scope? • Adjust the diaphragm to allow less light to come through • Use a Stain to make transparent specimens visible. Ie. Iodine, methyl blue • Specimens must be sliced very thin. Use a Microtome to make thin slices

21. Calculating Fields of View Once you have your field of view for Low Power, you will no longer use the ruler: GIVE BACK THE RULER For Medium Power: Low Power Field of View (um) = Medium Power Mag MediumPower Field of View (um)Low Power Mag For High Power: Low Power Field of View (um = High Power Mag High Power Field of View (um)Low Power Mag Medium Power Field of View Low Power Field of View

22. Field of View 1mm 1mm 1mm 1mm 4 cells fit across field Specimen = 3750um 4 Diameter = 3.75 mm or 3750 um Length of Specimen =937.5um

23. Electron Microscopes • Uses electromagnets and streams of electrons to view a specimen • Limit of Resolution is 1000x finer than light microscope • 200,000 – 1,000,000x magnification

24. Two types Transmission Electron Microscope (TEM) 1931 (Germany) Side 1 2286-99 Image is seen on a fluorescent screen • Specimen must be thinly sliced and coated with Au or Ag. • Gives a 2D image of specimen • Specimen must be dead

25. Herpes simplex viruses Staphylococcus aureus These have all had color added to them!!! E. coli bacteria

26. Scanning Electron Microscope (SEM) – 1935 (Germany) • Gives a 3D image • Electrons scan around specimen • Shows only the outside of the specimen • Gives very clear surface details 2256 - 2285

28. Images Diatom Weevil Tick Radiolarian

29. Gold coater - $1,950 used • Transmission Electron Microscopes (TEM):$90,000 - $2,000,000 • UsedScanning Electron Microscopes (SEM):$45,000 - $200,000 Used

31. Limitations of Electron Microscopes • Specimens must be very thin (TEM) • Specimens must be stained or coated • Specimens must be dried out (Mounting chamber is vacuum sealed) • Specimens must be dead • Black and white images only! Any color you may see is added in