Basic Microscopy

1. Basic Microscopy MAJ Kris Paolino, MD Infectious Disease Staff Chief, Clinical Trials Center Walter Reed Army Institute of Research

2. Disclaimer The views expressed in this presentation are those of the speaker and do not reflect the official policy of the Department of Army, Department of Defense, or U.S. Government

3. Microscopy • Technical field of using microscopes to view samples and objects that cannot be seen with the unaided eye • We will be using light microscopy

5. Calculating the magnification • Identify the magnification of the ocular lens • Identify the magnification of the objective lens in use • Multiply the two together • Example: • Ocular lens = 10x • Objective lens = 40x • Magnification is 400x

6. Oil Immersion lenses • Oil immersion is a technique to increase resolution of a microscope • Use of transparent immersion oils with an oil immersion objective lens • You will need oil to have adequate visualization of the malaria parasites on the high power objectives • The lens will typically state “oil” on the side

7. Correct use of a light microscope • Make sure the scope is plugged in and turned on • Ensure you are starting at the LOWEST power objective • Mount your glass slide using the cover slips or slide holder • Adjust the light source using the diaphragm • Rule of thumb: • Low light intensity for lower magnification • High light intensity for higher magnification

8. Correct use of a light microscope • Adjust the ocular lenses to your eyes as appropriate • Take off your glasses if you wear them • Again, starting at the lowest objective focus using the COARSE adjustment knobs • This is the last time you will use this knob • Some would say to move the lens as close to the slide as possible, and then adjust away DISCLAIMER: Anyone who breaks a slide will automatically be enrolled in our next malaria challenge trial

10. Correct use of a light microscope • Focus in on your target (i.e. red blood cells) at the LOWEST power • Make sure what you want to look at is CENTERED • In the thin smear of the malaria slide you want to try to start at the “feathered edged” of the slide • Cells are more spread out

11. Correct use of a light microscope • Focus in on your slide by switching to the next highest objective lens (it will “CLICK” into position) • Adjust illumination as necessary • Each time you switch up to the next highest objective, re-focus, and re-center in on the slide using the FINE adjustment knob ONLY • i.e. DON’T BREAK THE SLIDE

13. Correct use of a light microscope • When you get to the high power objectives, you will need to use a drop of immersion oil • For malaria diagnostics, you will use the 100x objective • To place the oil, rotate the objective lenses so they are out of the way, and place a single drop of oil directly on the slide • Slowly rotate the oil lens back over the slide so that the drop of oil touches the lens

14. Correct use of a light microscope • Using the oil lens, focus on the slide using the FINE adjustment knob only until the cells are in focus • When you are done, wipe the oil immersion lens and the slide with lens paper only • If you have a camera (smartphones included) try to take a photo of what you are seeing • Include magnification in the teleconsultation

15. Keep your scope clean • USE LENS PAPER TO WIPE THE OCULAR AND OBJECTIVE LENSES AS NECESSARY • DON’T USE OIL ON LOWER POWER OBJECTIVE LENSES • DON’T TOUCH THE LENSES WITH YOUR FINGERS

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19. Basophile