HISTOLOGY - BIOL 0509

LAB INTRODUCTION III

MICROSCOPY

USE OF THE COMPOUND MICROSCOPE

HOW TO HANDLE A MICROSCOPE:

A compound microscope should be treated as a VERY, VERY, VERY fragile piece of equipment.

1. Adjustments should be made gently and with finesse.

2. ALWAYS use both hands when picking the microscope up and moving it from one place to another.

3. When focusing on a slide, ALWAYS start with either the 4X or 10X objective. Once you have the object in focus, then switch to the next higher power objective. Re-focus on the image and then switch to the next highest power. Etc. NEVER advance more than one objective before focusing.

4. Use ONLY the fine focus control when focusing the higher power objectives (20X, 40X, 100X) on a slide. The course focus control is too course for focusing with these objectives. Objectives are fragile and must not be rammed into slides.

5. If an objective or ocular needs to be cleaned use the Q-tips and methanol available on the front desk in the lab. THE FOLLOWING NOTES ON CLEANING ARE VERY IMPORTANT!

a. ONLY CLEAN, UN-USED Q-TIPS MAY BE DIPPED IN THE METHANOL BOTTLE. Even Q-tips that have only been used once may have dirt on them that could contaminate the methanol and cause scratches in subsequent objectives or oculars when they are cleaned.

b. WHEN LENS PAPER IS USED TO CLEAN OBJECTIVES OR OCULARS, USE A NEW, CLEAN SECTION OF THE PAPER EACH TIME YOU WIPE THE LENS SURFACE.

c. NEVER SAVE USED LENS PAPER OR Q-TIPS. THEY SHOULD NEVER BE RE-USED ON ANY OF THE LENSES.

d. IF YOU RUN INTO A PARTICULARLY TENACIOUS BIT OF DIRT ON A LENS, SEE THE CLASS INSTRUCTOR ABOUT REMOVING IT RATHER THAN TRYING TO DO IT YOURSELF.WITH THESE THINGS IN MIND LET'S RUN THROUGH THE USE OF THE MICROSCOPE WITH AN EXAMPLE SLIDE.


INITIAL PROCEDURES:

1. USING BOTH HANDS, REMOVE YOUR MICROSCOPE FROM ITS CABINET AND PLACE IT ON THE LAB BENCH IN FRONT OF YOU.

2. PLUG THE POWER CORD INTO THE BENCH SOCKET AND TURN ON THE LIGHT.

FOR MICROSCOPES THAT DO NOT HAVE AN INTERNAL LIGHT SOURCE:
THE NEXT FEW INSTRUCTIONS ARE GIVEN IN CASE YOU HAVE TO USE ONE OF THE OLDER MONOCULAR MICROSCOPES.
IF YOU HAVE A MICROSCOPE THAT DOES NOT HAVE AN INTERNAL LIGHT SOURCE, YOU WILL HAVE TO USE THE EXTERNAL LIGHT SOURCE THAT IS PROVIDED IN THE CABINET WITH THE MICROSCOPE.
IN THIS CASE, PLACE THE EXTERNAL LIGHT SOURCE IN FRONT OF THE MICROSCOPE, PLUG IT IN, TURN ON THE LIGHT, AND ADJUST THE LIGHT SUCH THAT IT SHINES ON THE MICROSCOPE MIRROR.
NEXT, PUT THE LOWEST POWER OBJECTIVE INTO POSITION OVER THE VIEWING AREA OF THE MICROSCOPE AND LOWER THE OBJECTIVE, USING THE COURSE FOCUS, UNTIL IT IS ABOUT 1/4" ABOVE THE LENS OF THE CONDENSER.
NOW LOOK THROUGH THE MICROSCOPE OCULAR, AND ADJUST THE ILLUMINATION. IT IS POSSIBLE THAT THE EXTERNAL LIGHT SOURCE WILL HAVE TO BE REPOSITIONED TO ACHIEVE THE BEST ILLUMINATION.

3. MAKE SURE THAT THE 10X OBJECTIVE IS IN POSITION OVER THE VIEWING AREA. THE OBJECTIVE SHOULD BE POSITIONED ABOUT 1/4" - 3/8" ABOVE THE SLIDE.

4. PLACE THE SLIDE ON THE MICROSCOPE STAGE SUCH THAT THE PORTION OF THE SLIDE YOU WANT TO VIEW IS UNDER THE OBJECTIVE.

5. FOCUS ON SPECIMEN, FIRST USING THE COARSE AND THEN THE FINE FOCUS CONTROLS. YOU MAY HAVE TO MOVE THE SLIDE AROUND ON THE STAGE OF THE MICROSCOPE TO BRING THE SPECIMEN INTO THE VIEWING AREA.

ADJUST THE POSITION OF THE OCULARS (the interocular distance) SO THAT A SINGLE IMAGE CAN BE SEEN WHEN LOOKING THROUGH BOTH OCULARS AT THE SAME TIME.

If your eyes are too close set or far apart for the intraocular distance to be adjusted properly, you will have to use your microscope as a monocular instrument (i.e. look through one eyepiece with one eye). If you do this, it is important to keep both eyes open in order to avoid eyestrain. With a little practice, you should be able to train yourself to "see" only what is being viewed with the microscope, and ignor whatever the other eye is seeing. If you can't do this, a trick that works is to buy a cheap pair of sunglasses, knock out the dark lenses and put a piece of cardboard in the lenses over the eye that you don't look through the microscope with. This will allow you to "see" only with the eye you are looking through the microscope with even though the other eye is open. In any case, practice keeping both eyes open while looking through the microscope. Eyestrain can give you headaches.

6. VISION DIFFERS BETWEEN PEOPLE AND BETWEEN EYES. IT IS LIKELY THAT WHILE THE IMAGE YOU ARE VIEWING MAY BE IN FOCUS FOR ONE OF YOUR EYES, IT IS NOT IN FOCUS FOR THE OTHER. ONE OF THE OCULARS ON YOUR MICROSCOPE HAS AN ADJUSTABLE FOCUS TO ACCOUNT FOR THIS.

LOOK THROUGH THE NON-ADJUSTABLE RIGHT OCULAR AND COVER YOUR LEFT EYE WITH THE INDEX CARD THAT IS IN THE TOP DRAWER AT YOUR SEAT. DON'T CLOSE THE EYE YOU ARE COVERING! NOW, USING YOUR RIGHT EYE, CAREFULLY FOCUS ON THE IMAGE USING THE COURSE AND THEN THE FINE FOCUS CONTROLS ON THE MICROSCOPE. ONCE THE IMAGE IS IN FOCUS,COVER YOUR RIGHT EYE WITH THE INDEX CARD AND LOOK THROUGH THE OTHER OCULAR WITH YOUR LEFT EYE. ADJUST THE FOCUS OF THE LEFT OCULAR BY TURNING THE KNURLED KNOB ENCIRCLING THE OCULAR TO THE LEFT OR THE RIGHT. ONCE THIS IS COMPLETED YOU WILL HAVE MATCHING FOCUS IN BOTH OCULARS. SINCE SOMEONE ELSE IN ANOTHER LAB SECTION WILL BE USING YOUR MICROSCOPE IT MAY BE NECESSARY TO PERFORM THIS ADJUSTMENT EACH TIME YOU USE THE MICROSCOPE. DOING THIS WILL HELP PREVENT EYESTRAIN.

ONCE THE SPECIMEN IS IN FOCUS, IT IS TIME TO ADJUST THE CONDENSER DIAPHRAGM APERTURE. THIS IS DONE BY ROTATING THE PLASTIC RING ON THE BOTTOM OF THE CONDENSER ASSEMBLY. YOU WILL NOTICE THAT THERE ARE GRADUATIONS ON THE RING FROM 2 TO 30.

7. CONDENSER DIAPHRAGM ADJUSTMENT.

a. USING THE CONDENSER FOCUS ADJUSTMENT KNOB THAT IS LOCATED ON THE LEFT SIDE OF YOUR MICROSCOPE JUST BELOW THE STAGE, MOVE THE CONDENSER UP TOWARD THE STAGE AS FAR AS IT WILL GO.
b. NEXT, WHILE LOOKING THROUGH THE OCULARS OF YOUR MICROSCOPE, ROTATE THE RING SO THAT YOU CAN DETERMINE WHAT SETTING ALLOWS THE LEAST AMOUNT OF LIGHT THROUGH THE APERTURE.
c. NEXT, SLOWLY OPEN THE APERTURE UNTIL YOU REACH THE POINT THAT THE VIEWING FIELD IS AS BRIGHT AS IT WILL GET. THE APERTURE SHOULD BE ADJUSTED TO THIS POINT AND NOT BEYOND IT. YOU NOW HAVE ADJUSTED YOUR CONDENSER DIAPHRAGM FOR MAXIMUM RESOLUTION AT A REASONABLE CONTRAST. IF YOU HAVE COMPLETED THIS ADJUSTMENT CORRECTLY, THE WHITE MARKER LINE ON THE KNURLED PLASTIC RING THAT ADJUSTS THE CONDENSOR DIAPHRAGM WILL BE SET AT A POINT BETWEEN 0 AND 10 DEGREES.

CONTRAST CAN BE INCREASED BY CLOSING DOWN THE CONDENSER DIAPHRAGM TO ALLOW LESS LIGHT THROUGH; HOWEVER, THIS ALSO CAUSES A DECREASE IN RESOLUTION. IN VIEWING SOME SLIDES, AN INCREASE IN CONTRAST MAY BE HELPFUL.
THE APPROPRIATE ADJUSTMENT OF THE CONDENSER APERTURE MAY CHANGE DEPENDING ON WHICH SLIDE YOU'RE VIEWING AND WHAT OBJECTIVE YOU ARE USING. SO BE AWARE, THAT IT MAY BE HELPFUL IN SOME CASES TO CHANGE THE ADJUSTMENT OF THE CONDENSER DIAPHRAGM.

8. AFTER THE SPECIMEN IS IN FOCUS AND THE CONDENSER PROPERLY ADJUSTED, IT IS TIME TO BRING THE FIELD DIAPHRAGM INTO FOCUS AND CENTER IT.

9. FIRST CLOSE THE FIELD DIAPHRAGM TO IT'S SMALLEST OPENING BY TURNING THE KNURLED ADJUSTMENT KNOB LOCATED BEHIND THE FIELD LENS ASSEMBLY IN THE BASE OF YOUR MICROSCOPE.

NEXT, WHILE LOOKING THROUGH THE MICROSCOPE OCULARS, FOCUS ON THE ILLUMINATION FIELD DIAPHRAGM BY TURNING THE CONDENSOR FOCUS ADJUSTMENT KNOB THAT IS LOCATED ON THE LEFT SIDE OF YOUR MICROSCOPE, BELOW THE STAGE. WHEN THE ILLUMINATION FIELD DIAPHRAGM IS IN FOCUS, YOU WILL SEE A SMALL HEXAGON OF LIGHT, SURROUNDED BY A DARK FIELD.

10. NOW, USING THE CHROME-PLATED ADJUSTMENT SCREWS IN FRONT OF THE FIELD LENS ASSEMBLY, CENTER THE DIAPHRAGM IMAGE IN THE VIEWING FIELD. CENTERING IS CRITICAL IF YOU ARE TO OBTAIN THE BEST IMAGE OF THE MATERIAL YOU ARE LOOKING AT. AFTER YOU HAVE CENTERED THE SMALLEST OPENING OF THE FIELD DIAPHRAGM, OPEN THE DIAPHRAGM UNTIL THE OPENING ALMOST FILLS THE FIELD. NOW YOU CAN RE-CENTER THE DIAPHRAGM OPENING FOR EVEN BETTER ALIGNMENT.

11. ONCE YOU HAVE CENTERED THE FIELD DIAPHRAGM, OPEN THE DIAPHRAGM UNTIL THE OPENING JUST FILLS THE FIELD OF VIEW. NO FURTHER!

12. EACH TIME YOU CHANGE OBJECTIVES YOU WILL HAVE TO RE-FOCUS AND RE-CENTER THE FIELD DIAPHRAGM IF YOU WISH TO OBTAIN THE BEST IMAGE POSSIBLE.


WHAT YOU HAVE JUST DONE IS SET YOUR MICROSCOPE UP FOR "PROPER KOHLER ILLUMINATION". THIS WILL GIVE YOU THE BEST RESOLUTION POSSIBLE WITH YOUR MICROSCOPE.

13. ONCE YOU HAVE THE IMAGE OF THE TISSUE YOU ARE VIEWING IN FOCUS WITH A GIVEN OBJECTIVE YOU MAY ADVANCE THE OBJECTIVE TURRET TO THE NEXT HIGHER MAGNIFICATION AND RE-FOCUS AND READJUST YOUR MICROSCOPE AS DESCRIBED ABOVE.

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THE FOLLOWING INSTRUCTION MUST ALWAYS BE ADHERED TO!!!

YOU MUST FIRST FOCUS ON A SLIDE WITH EITHER THE 4X OR 10X OBJECTIVES M BEFORE MOVING ON TO HIGHER MAGNIFICATIONS. BEFORE YOU ADVANCE TO THE NEXT HIGHER MAGNIFICATION OBJECTIVE, YOU MUST BE SURE THAT THE TISSUE IS IN FOCUS WITH THER OBJECTIVE THAT YOU ARE USING BEFORE ADVANCING TO THE NEXT HIGHER MAGNIFICATION!!!
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14. NEVER, I REPEAT NEVER, USE THE COURSE FOCUS WITH THE HIGHER POWER OBJECTIVES (20X, 40X OR 100X objectives).

WHEN YOU ARE FINISHED VIEWING, MOVE THE OBJECTIVE TURRET TO BRING THE 4X OR 10X OBJECTIVE INTO POSITION OVER THE VIEWING AREA. THEN REMOVE THE SLIDE FROM THE STAGE. REMOVING THE SLIDE FROM UNDER HIGHER POWER OBJECTIVES MAY CAUSE THE SLIDE SURFACE TO HIT THE OBJECTIVE LENS SINCE THE LENS IN THESE OBJECTIVES WILL BE VERY CLOSE TO THE SLIDE. THIS COULD RESULT IN DAMAGE TO THE LENS.

OIL IMMERSION

OIL IMMERSION VIEWING IS USED TO

a. DIRECT THE GREATEST AMOUNT OF LIGHT THROUGH THE OBJECTIVE LENS

and

b. TO ACHIEVE THE HIGHEST POSSIBLE RESOLUTION WHEN VIEWING THROUGH THE HIGHEST POWER OBJECTIVES.

FOR YOUR MICROSCOPE, THIS WOULD BE WHEN VIEWING SLIDES WITH THE 100X OBJECTIVE.

IN THIS COURSE, THE 4X, 10X, AND 40X OBJECTIVES WILL BE SUFFICIENT FOR MOST OF YOUR LAB WORK. HOWEVER, IN SOME INSTANCES, THE OIL IMMERSION OBJECTIVE (100X) MAY BE USEFUL. IN ORDER FOR THIS OBJECTIVE TO PROVIDE AN IMAGE THAT IS IN GOOD FOCUS, A DROP OF OIL MUST BE PLACED BETWEEN THE OBJECTIVE LENS AND THE SLIDE.

15. BEFORE PERFORMING OIL IMMERSION MICROSCOPY, YOU MUST FIRST GO THROUGH THE PROCEDURES NECESSARY TO OBTAIN GOOD FOCUS WITH THE 40X OBJECTIVE.

16. THAT ACCOMPLISHED, MOVE THE OBJECTIVE TURRET SUCH THAT THE SPACE BETWEEN THE 40X AND 100X OBJECTIVES IS OVER THE VIEWING AREA.

17. NOW, PLACE A SMALL DROP OF IMMERSION OIL ON THE SLIDE OVER THE POINT WHERE THE OBJECTIVE LENS WILL RESIDE WHEN YOU MOVE IT INTO POSITION OVER THE VIEWING AREA. THIS POINT IS WHERE YOU CAN SEE THE LIGHT BEAM PASSING THROUGH THE SLIDE. THE DROP OF OIL SHOULD BE VERY SMALL, NOT MUCH IS NEEDED AND THE LESS THERE IS, THE EASIER IT WILL BE TO CLEAN THINGS UP WHEN YOU ARE THROUGH.

18. NEXT, ROTATE THE OBJECTIVE TURRET TO BRING THE 100X OBJECTIVE OVER THE VIEWING AREA. DO THIS CAREFULLY, AND WATCH TO MAKE SURE THAT THE OBJECTIVE LENS DOES NOT CONTACT THE SURFACE OF THE SLIDE. IF YOU FOCUSED PROPERLY WITH THE 40X OBJECTIVE IN STEP 10, THERE SHOULD BE NO PROBLEM.

19. ONCE THE 100X OBJECTIVE IS MOVED INTO POSITION, THE LENS WILL BE IMMERSED IN OIL. NOW, USING ONLY THE FINE FOCUS CONTROL, LOOK THROUGH THE OCULARS AND FOCUS ON THE SLIDE. THIS SHOULD REQUIRE ONLY A SMALL ADJUSTMENT OF THE FINE FOCUS KNOB.

WHEN YOU ARE DOING THIS FOCUSING, IT IS IMPORTANT TO BE VERY CAREFUL. IT WILL NOT TAKE MUCH ROTATION OF THE FINE FOCUS KNOB TO CAUSE THE 100X OBJECTIVE TO RAM THROUGH THE SLIDE, DAMAGING BOTH THE OBJECTIVE AND THE SLIDE.

BE CAREFUL!!!

20. ONCE THE SLIDE IS IN FOCUS, IT MAY BE MOVED AROUND ON THE STAGE. AS LONG AS THE DISTANCE MOVED IS NOT TOO LARGE, THE OIL DROPLET WILL REMAIN BETWEEN THE 100X OBJECTIVE AND THE SLIDE. YOU WILL NOTICE THAT WHEN THE SLIDE IS MOVED, IT WILL BE NECESSARY TO RE-FOCUS THE 100X OBJECTIVE USING THE FINE FOCUS CONTROL. AT VERY HIGH MAGNIFICATIONS, VERY SMALL CHANGES IN THE DISTANCE BETWEEN THE SLIDE AND THE OBJECTIVE WILL CAUSE THE IMAGE TO GO OUT OF FOCUS. IRREGULARITIES ON THE SLIDE AND ON THE STAGE OF THE MICROSCOPE ARE LARGE ENOUGH TO CAUSE CHANGES IN FOCUS.

ONCE YOU ARE DONE VIEWING A SLIDE UNDER OIL IMMERSION, IT WILL BE NECESSARY TO CLEAN BOTH THE SLIDE AND THE OBJECTIVE.

21. TURN THE OBJECTIVE TURRET SUCH THAT THE SPACE BETWEEN THE 100X AND LOWEST POWER OBJECTIVE IS OVER THE VIEWING AREA.

22. REMOVE THE SLIDE FROM THE STAGE AND THOROUGHLY WIPE THE OIL FROM ITS SURFACE USING A PIECE OF LENS PAPER. MORE THAN ONE PIECE OF LENS PAPER MAY BE REQUIRED. WHILE SOME PRESSURE MUST BE USED IN ORDER TO CLEAN THE OIL FROM THE SLIDES SURFACE, BE CAREFUL NOT TO PRESS TOO HARD. IT IS POSSIBLE TO CAUSE THE COVERSLIP TO SLIDE OFF THE SLIDE IF TOO MUCH PRESSURE IS USED.

DO A GOOD JOB OF CLEANING THE SLIDE SO THAT YOU WILL NOT HAVE TO CLEAN IT BEFORE VIEWING IT IN THE FUTURE. DRIED OIL IS HARD TO REMOVE!

23. REPLACE THE SLIDE IN YOUR SLIDE BOX.

24. NEXT, THOROUGHLY WIPE THE 100X OBJECTIVE WITH A NEW, CLEAN PIECE OF LENS PAPER. AGAIN, IT MAY BE NECESSARY TO DO THIS MORE THAN ONCE, WITH MORE THAN ONE PIECE OF LENS PAPER.

25. WIPE UP ANY EXCESS OIL THAT IS ON THE MICROSCOPE STAGE.


CONGRATULATIONS!

YOU HAVE JUST FINISHED A SHORT COURSE IN MICROSCOPY. THE INSTRUCTIONS ABOVE ARE TAILORED TO THE MICROSCOPES USED IN THIS COURSE. THEY MAY NOT BE COMPLETELY ADEQUATE FOR MORE SOPHISTICATED MICROSCOPES. IF YOU HAVE THE OPPORTUNITY TO USE A MORE SOPHISTICATED MICROSCOPE IN THE FUTURE, BE SURE TO REVIEW THE INSTRUCTION MANUAL THAT COMES WITH THE MICROSCOPE BEFORE USING IT. FOLLOWING THIS COURSE OF ACTION WILL SAVE YOU TIME, THE HIGH COST OF REPAIRS, AND PROVIDE THE BEST VIEWING.

A FINAL REVIEW OF AN IMPORTANT POINT.

WHENEVER YOU CHANGE SLIDES, OR LOSE FOCUS ON SLIDES YOU ARE VIEWING, ALWAYS START YOUR VIEWING WITH THE 4X OR 10X OBJECTIVES. FOCUS ON SPECIMEN, AND THEN SWITCH TO HIGHER POWER OBJECTIVES.

NOT FOLLOWING THIS PROCEDURE WILL LEAD TO BROKEN SLIDES AND DAMAGED OBJECTIVES. BOTH ARE EXPENSIVE. PARTICULARLY THE OBJECTIVES WHICH, FOR YOUR MICROSCOPES, CAN COST AS MUCH AS $200.00 EACH. ON MORE SOPHISTICATED MICROSCOPES, THE COST OF OBJECTIVES CAN BE $2000.00 - $10,000.00 EACH DEPENDING ON THE TYPE OF OBJECTIVE..

IF YOU DAMAGE ANY OF YOUR OBJECTIVES, IT MAY BE WEEKS BEFORE WE CAN GET A REPLACEMENT. YOU MAY BE WITHOUT THE BENEFIT OF THAT OBJECTIVE ON YOUR MICROSCOPE FOR AS LONG AS IT TAKES.