How to carry microscope properly One hand holding arm of microscope One hand supporting base of microscope Slide 3
For today only, sign out the microscope later during the lab period rather than now. Slide 4 Please bring one microscope to your work station. Carry it correctly! Each person should have his/her own microscope today. DO NOT PLUG IT IN! Each time you use a microscope, you must sign it out in a notebook on the instructor’s desk. You must indicate your name & microscope number. You are responsible for that ‘scope!
OCULAR LENSESSUBSTAGE LIGHT binocular (2 oculars); never swivel sends light up toward image eyepieces (what you look through); magnifies 10 X controlled by light switch on bottom/side of ‘scope Slide 5
OBJECTIVE LENSESROTATING NOSEPIECE 4X = scanning; 10 X = low power** supports objective lenses 40 X = high power or high dry** 100 X = oil immersion changing objective lenses changes magnification Slide 6 ** = objectives we will use in this course
NEVER clean the lenses of your microscope yourself. Ask your instructor to clean them if they are dirty. Your instructor will use lens paper. Kimwipes scratch and ruin microscope lenses. Slide 7
STAGEMECHANICAL STAGE supports the slidegrips the slide moves up and down during focusing moves the slide using control knobs Slide 8
COARSE ADJUSTMENT KNOB FINE ADJUSTMENT KNOB stage visibly moves up and down stage moves up and down; not visible used for coarse focusing used for fine focusing Slide 9
Never let the microscope cord hang over the side of the desk – somebody might trip and get hurt (and the ‘scope might break)! Now plug your microscope in & turn the light on. Slide 10
CONDENSERIRIS DIAPHRAGM LEVER raises & lowers (keep closest to stage) makes light seen dimmer or brighter focuses light on the specimen (on the slide) controls amount of light going through condenser Slide 11
TOTAL MAGNIFICATION Slide 12 magnification of oculars (10) times magnification of objective lens used cat blood in field of view of microscope
Slide 13 You are observing a slide using the high power objective. What is the total magnification of the image? under low power? under oil immersion? under the scanning objective? QUESTION
Slide 14 As magnification increases, working distance decreases. What is the significance of this? Working Distance
Are working distance and total magnification directly or inversely proportional to each other? QUESTION Slide 15
Resolution Ability to distinguish between 2 closely spaced points. Resolution of human eye: 100 µm (100 µm = 0.0039 inches) If > 100 µm Will see 2 objects If < 100 µm Will see only 1 object Slide 16
Resolution continued … Resolution of compound microscope: 0.2 µm (0.2 µm = 0.0000078 inches) If > 0.2 µm Will see 2 objects If < 0.2 µm Will see only 1 object Slide 17
Limit of Resolution continued … Why can’t you just keep putting more powerful objective lenses on a microscope to keep increasing magnification? Slide 18 can reach a point where you can increase volume, but the sound won’t be clear because you have reached the limit of resolution of the radio
Oil Immersion: Theory After passing through the slide, light must pass through the coverslip (glass) and air before reaching the objective. Slide 19 cover slip slide on stage objective lens
But air and glass have different refractive indexes. This means that they bend light differently (i.e., to different degrees) Slide 20 Air and water also have different refractive indexes, so the spoon appears bent at the air/water interface.
Oil Immersion: Theory continued … Slide 21 With no oil: Not all light goes into objective; Enough light for 4X, 10X & 40X objectives; Not enough light for 100X objective. With oil: Glass & oil have similar refractive indexes; Almost all of the light goes to 100 X objective
Letter “e” Slide and Proper Focusing Activity 2 e Slide 22
1. Obtain letter “e” slide from side counter 2. Move stage as far away from objectives as possible. 3. Put the 10x objective in place. Slide 23 Always use the low power objective to focus your slide – even if you want to view your slide under high power.
4. Look at your letter “e” on the slide with your eyes (i.e., NOT through the ‘scope). Note the orientation of the letter “e” in your labbook. 5. Put the letter e slide on the stage of your microscope; be sure it is clipped into place by the mechanical stage. You can test if your slide is in the mechanical stage correctly by trying to move it around with the appropriate dials. 6. Move the slide so that the “e” is centered in the hole the light comes up through. Slide 24
7.Looking to the side of the microscope (NOT through the oculars), move the stage as close as possible to the objective lens without touching the slide to the objective lens. Why do you have to look to the side of the ‘scope when you are moving the stage closer to the objective lens? ________________________________ Slide 25
8.Adjust the oculars for your eyes. 9.Looking through the oculars, slowly move the course adjustment knob until the image is almost in focus. When you do step 9, are you moving the slide toward or away from the objective lens? ________________________________ Slide 26 When moving stage & objective closer together, you must look at the side of ‘scope rather than through the oculars. When moving the stage & the objective away from each other, you can look through the oculars.
10.Use the fine adjustment knob to finish focusing the slide. NOTE: if the slide doesn’t come into focus, repeat steps 2 – 10. 11.Once the slide is in focus, note the orientation of the letter “e” in your labbook. 12.Compare the orientation of the letter “e” in steps 4 and 11. What changes do you see in the orientation of the letter “e” when looking at it without a microscope (step 4) and with a microscope (step 11)? ________________________________ Slide 27
13.Now move the slide to your right while looking through the ocular. In which direction (left or right) does the letter “e” appear to move? _________________________ 14.Now move the slide away from you while looking through the ocular. In which direction (toward you or away from you) does the letter “e” appear to move? _______________________________ Slide 28
What implications do the answers to the questions in steps 13 and 14 have in terms of looking at tissues under the microscope? QUESTION Slide 29
15.Make sure that the letter “e” is in the center of your field of vision and is in focus. 16. Looking at the side of the microscope, switch from the 10x objective to the 40x objective. In step 16, why do you need to look at the side of the microscope rather than through the oculars? _______________________________________ Slide 30
17. The slide should be in focus because our microscopes are PARFOCAL (i.e., if they are in focus using one objective, they should be in focus using all objectives). Microscopes are not perfectly parfocal, though. If your image is not quite in focus, use the FINE adjustment knob. DO NOT TOUCH the course adjustment knob. Can you see the whole “e” ? _______________________________ Is there enough light? _____________ (If not, move the iris diaphragm lever until there is enough light) Slide 31
As magnification increases, do you see more or less of the “e”? What implications does this have if you are looking at a tissue under the microscope? QUESTION Slide 33 As magnification increases, does the amount of light getting through to your eyes increase or decrease? What implication does this have in terms of looking at something under the microscope? As magnification increases, does the field of view increase or decrease? What implications does this have for looking at cells under the microscope?
Depth of Field (Activity 4) Slide 34 3 colored threads are crossed on a slide. Each thread is 3 dimensional. If you focus on the point at which the threads cross, the threads should not be in focus at the same focal point.
Which thread should come into focus first – top or bottom? objective lens 3 colored threads on slide When focusing correctly, are you moving the threads toward or away from the objective while looking through the oculars? ________________________________________ Therefore, should the focal point be above the orange thread or below the green thread? ____________________ Slide 35
objective lens 3 colored threads on slide Focal Point Therefore, which should come into focus (i.e., reach the focal point) first – the top thread or the bottom thread? __________________________________________ Slide 36
1.Obtain a crossed colored thread slide from the side counter. 1.Focus correctly using the same steps used to focus the letter “e” slide. HINT: once the slide is almost in focus, you have to do this very slowly! 3.Which thread (blue, red or yellow) is on top? __________ on the bottom? __________ Slide 37
3 threads are crossed on a slide – a green thread, a blue thread, and a silver thread. When focusing correctly, first the green thread comes into focus, then the blue thread comes into focus, and, lastly, the silver thread comes into focus. Which thread is on the top? Which thread is on the bottom? QUESTION Slide 38
What was the purpose of looking at a crossed colored thread slide? What implications does this have in terms of looking at tissues through the microscope? Slide 39 What were the purposes of looking at the letter “e” slide?
Preparing/Observing Wet Mount Activity 5 Slide 40 cheek cells = squamous cells flat, plate-like, 6 sided cells; nucleus in center of cell
1. Obtain the following: 1 clean microscope slide 1 clean coverslip 1 bottle methylene blue 1 sterile applicator stick 1 toothpick 1 bottle 0.85% saline 2 small pieces of paper towel Slide 41
2.Put a slide on the paper towel. 3.Put 1 drop of 0.85% saline in the center of the slide. NOTE: 0.85% saline is isotonic saline – i.e., it has the same concentration of salt as most body fluids 4.Using the wooden end of a sterile applicator stick, lightly scrape the inside of your cheek. 5.Put the wooden end of the applicator stick in the saline on the slide and wiggle it around so cells fall off into the saline. Slide 42 throw the used applicator stick in the proper container immediately!
7.Add one drop of methylene blue to the saline – cheek cell suspension on the slide. 8.Your instructor will tell you how to correctly put the coverslip on the slide – do so. Figure 4 NOTE: it’s better to use a toothpick to balance the coverslip than your fingers (because a toothpick is thinner than your fingers) Slide 43 NOTE: you can use a paper towel to soak up excess liquid outside of the coverslip. BE CAREFUL: the paper towel can suck liquid from underneath the coverslip, and you don’t want to do that.
9.Focus on the cheek cell wet mount using the directions given previously in this slide module to focus. 9.Draw what you see under low power in your labbook 11.Draw what you see under high power in your labbook Slide 44 Discard your wet mount and your used toothpick in the containers indicated by your instructor! Neatly return the saline and methylene blue to the set-up area. Throw your used paper towels in the waste basket.
HOW TO PUT YOUR MICROSCOPE AWAY! - Turn microscope light off - Remove slide from microscope - Put low power (10x) objective in place - Unplug microscope (DON’T pull on cord) - Wrap cord tightly around microscope - If microscope has dust cover, put dust cover on microscope - Make sure the number on your microscope corresponds to the number of the cubbyhole you return the microscope to - Put your microscope in the cubbyhole so that the next person taking the microscope will grab the arm, not the objectives - Do NOT cross out your name or microscope number from the microscope sign-out notebook Slide 45