No doubt you have already used a light microscope in one or more courses to examine cells, tissues, and other specimens.  The type of microscope with which you are familiar is actually only one of many kinds, and its correct name is "brightfield" light microscope.  Although it is used extensively in research laboratories, the brightfield light microscope is not the most useful type for many areas of research.  The reason is that brightfield microscopes require staining of specimens with dyes to achieve contrast sufficient for easy visibility to the human eye.  Such dyes usually kill the cells, and the staining plus associated methods take a good deal of time as well.


     For routine work in bioresearch, a phase contrast light microscope is more useful.  This is a brightfield microscope equipped with special optical devices that enhance contrast in the image without requiring staining.  While "resolution" or "resolving power" refers to the ability of a microscope to reveal structural details of a specimen, it is contrast that enables the human eye to detect these details.  Contrast refers to differences in light intensity in different parts of the image.  In a phase contrast microscope, the naturally-occurring small differences in intensity that are produced by the specimen (barely visible to the eye) are given an optical "boost" that converts them to large differences.


     Phase contrast light microscopes are manufactured by a number of companies (such as Zeiss, Olympus, Bausch and Lomb, Leitz).  Regardless of manufacturer, there are two major designs: upright and inverted.  Upright phase contrast microscopes are typically used for examination of samples mounted on glass slides, while inverted phase contrast microscopes are normally used for viewing cell cultures growing in relative thick vessels such as Petri dishes and bottles.  Inverted phase contrast microscopes make use of special "long working distance" (= LWD) condensers and objective lenses because the lenses cannot get as close to the specimens as usual due to the thickness of Petri dish bottoms or bottle walls.


     The phase contrast microscope in the Tech Facility is an upright instrument manufactured by Zeiss, and is of research quality.  It consists of a Zeiss "Standard 25" microscope body (one of the smaller models) with various major components attached.  From the bottom up, these components are: (a) internal (i.e., built-in) illuminator, (b) condenser, (c) stage, (d) rotating nosepiece with objective lenses, and (e) binocular tube with two eyepieces.  This microscope is an expensive precision instrument, and must be handled with utmost care.



EXERCISE #1:  Major components of the phase contrast microscope


     Ask your supervisor to obtain the Tech Facility phase contrast microscope from its locked storage location.  The instructor will remove its plastic dust cover so that it can be carried properly.  To do so, it is held by its straight upper body in one hand, and supported underneath the base with the other, as indicated by labels on the microscope.  The supervisor should place it on one of the wooden "Microscopy" tables (one with wheels) or on the center lab bench.



     (1) Remove and set aside the plastic dust cover if it is on.

     (2) Locate all of the major components shown in Figure 1 on following page (find corresponding labels on the microscope).





     Microscope controls include (again, starting at the bottom): Field diaphragm (over light source), on/off light switch (at the back of instrument), light intensity control (right side), centering controls for the condenser, height control knob for the condenser, lever for moving the condenser "swing-in" front lens, centering controls for the "phase rings" located in the condenser, coarse and fine focusing knobs, "knurled rings" for rotating condenser and nosepiece (these have a rough surface), and stage movement controls.



EXERCISE #2:  Control components of the phase contrast microscope


     Examine the phase contrast microscope and locate the microscope control components shown in Figure 2 on following page.  OBSERVE ONLY! DO NOT MANIPULATE ANY MICROSCOPE COMPONENTS YET.




     The greatest difference between an ordinary brightfield microscope and a phase contrast microscope is in the condenser.  The condenser not only has the usual function of focusing light

onto the specimen, but also can be rotated into several positions for specific purposes.  The condenser position is denoted by the letter or number that "clicks" into place next to the white marker line at the right-hand side of the condenser.  One position is marked by the letter "J"; this sets the condenser for ordinary brightfield microscopy.  The phase contrast positions are marked as "1", "2", and "3".  In each of these positions there is an optical ring ("condenser annulus") of a particular size, matched to a ring ("objective phase plate") in each objective lens.  For the 10X objective, position #1 is used; for the 40X objective, position #2; and for the 100X objective, position #3.  The position number is marked on each objective lens; for example, the 40X objective has "Ph2" on it.  Every time the objective lens being used is changed, the condenser must be rotated to the proper matching position.


     The "J" position, in which there is no condenser ring, can be used with any objective lens to produce ordinary brightfield microscopy.  Thus, with a phase contrast microscope, one has the option of doing either brightfield or phase contrast light microscopy.


EXERCISE #3:  Rotating the phase contrast microscope condenser



     (1) Locate the white marker line at the right-hand side of the condenser.

     (2) Using only the knurled ring of the condenser (the part just above the "CONDENSER" label, with letter J and numbers on it), rotate the condenser to the "J" position.  Here the letter "J" clicks into place as it lines up with the white marker line.  If the condenser is already in the "J" position, move it slightly away (in either direction) and then back in.

     (3) Note that, in the "J" position only, a control knob (labeled "CD") appears at the front of the condenser.  This controls the opening and closing of the condenser diaphragm.

     (4) Again using the knurled condenser ring only, rotate the condenser into the #1, #2, and #3 positions.

     (5) Rotate once more, into any unmarked position.  Here the condenser is effectively "open"; it has no diaphragm or phase rings, so that this is not a functional position.

     (6) Return the condenser to the "J" position.


EXERCISE #4:  Setting up the phase microscope - basic steps



     (1) Plug the microscope power cord into a nearby socket if not already plugged in, making sure that the cord is out of the way and will not be tripped over.

     (2) Ask your supervisor for the plastic "microscope accessory tray" containing lens paper, slides, coverslips, immersion oil, and other items.  Place it near the microscope (open attached cover).

     (3) Rotate the nosepiece until the 10X objective lens is in place (or check that it is already in place).  Remember to use the knurled nosepiece ring to do this (RESIST the temptation to push on the objective lenses).

     (4) Check that the top lens (also called "front lens") of the condenser is swung IN all the way.  Use the lever labeled SWL, under stage, left, to do so; it pulls outward and pushes inward.  Push it all the way inward.

     (5) Rack the condenser all the way up (until it hits the stop position) using condenser height knob (left side, underneath stage).

     (6) Using the diaphragm control ring over the light source

( = lamp field stop), open the diaphragm all the way by turning the ring clockwise.

     (7) Rotate the condenser (using knurled ring!) to the "J" position ("J" clicked into place, as in EXERCISE #3 above).

     (8) Use the slide marked "TEST SLIDE", found in the "microscope tray".  This contains pen marks covered by a coverslip.

     (9) Place the test slide on the microscope mechanical stage, SPECIMEN UP (i.e., pen marks and coverslip up), and hold it in place with the spring-loaded slide clamp.

     (10) Using the stage movement controls (S-1, S-2), position the slide so that the pen mark area is directly underneath the 10X objective lens (do this without looking through the eyepieces!  It is easier and faster that way).

     (11) Turn on the light power switch, at rear of microscope stand.  The green power indicator lamp, at the right side, rear, will go on.

     (12) Turn up the illumination intensity partway, using the light intensity control knob below the green indicator lamp (rear, right) until some light is visible on the slide.

     (13) Look into the binocular eyepieces.  If you do not obtain a comfortable view with both eyes open, make two adjustments:

    (a) Adjust eyepiece separation distance to match your eyes:

       [a-1] grasp the two knurled rings of the binocular (each labeled "X" on top)

       [a-2] pull and/or push to change separation until you obtain a comfortable view.


    (b) Adjust the left eyepiece focus to match the right:

       [b-1] close the left eye,

       [b-2] focus the specimen through the right eyepiece using microscope focus knobs,

       [b-3] open the left eye

       [b-4] screw the very top part of the left eyepiece either upward or downward until the plane of focus of the left image (appearance of specimen) exactly matches the right one.  To do this, grasp the very top of the eyepiece with one hand (to rotate), and hold the lower part of the ocular steady (at "OC" label) with the other hand.


     (14) While looking into both eyepieces, adjust light intensity with intensity control knob to a comfortable level (if necessary).

     (15) Still looking through the eyepieces, use the coarse focus knob to bring the "specimen" (test slide pen marks) into focus.  If nothing is visible, move the slide back and forth with the stage controls while focusing up and down with the coarse focus knob.


            What happens to this microscope when you use the coarse focus control?  Does the objective lens move up/down?  Does the stage move up/down?  Which turning direction of the focus control (clockwise? counterclockwise?) produces what movement?  Test this by observing the microscope itself briefly (rather than the specimen) during coarse focusing.


     (16) Once the specimen is in approximate focus, use the fine focus knob to improve the focus.


     You will now complete the basic set-up procedure by following additional steps that optimize illumination of the specimen ("Koehler Illumination").




     A procedure for obtaining the most advantageous illumination of a specimen for brightness without glare or reflections, and for evenness (uniformity) of light intensity across the field of view, was first developed and published by E. Koehler (in 1893!).  The procedure involves setting the condenser to a particular height position, centering it, and adjusting the opening of the field stop diaphragm (diaphragm over light source) and the condenser diaphragm.  The condenser diaphragm is only adjustable in the "J", or brightfield, position of the condenser.


     The Koehler illumination procedure is normally followed when work for the day is initiated, and the illumination is then checked periodically or whenever there is a change to a different objective lens.



EXERCISE #5:  Koehler Illumination



     (1) Complete all steps of EXERCISE #4, above (if not continuing directly from EXERCISE #4).

     (2) Make sure that the condenser is racked all the way up, with its top lens swung all the way in.

     (3) Rotate condenser to "J" position (if not already there).

     (4) Focus on a specimen (test slide with pen marks) using the 10X objective and the coarse and fine focus controls.

     (5) While viewing specimen in best focus, close down the diaphragm over the light source (the "lamp field stop") in the base of the microscope, until you can see its edges.

     (6) Lower the condenser very slowly (using condenser height knob) until the edge of the lamp field stop diaphragm is in sharpest focus.  If you are not sure, move it slightly up and down to find optimum focus (of edge of field stop diaphragm, not specimen!).  DO NOT USE THE MICROSCOPE FINE OR COARSE FOCUS KNOBS.

     (7) Center the condenser using the two condenser centering screws (labeled "C") protruding to the left and right just underneath the condenser.  The idea is to position the image of the field stop diaphragm exactly in the center of the field of view.

     (8) Open the field stop diaphragm further, until its image is just touching the edge of the circular field of view.  Does it touch evenly all the way around?  If not, improve condenser centering using centering screws ("C").

     (9) Open the field stop diaphragm further, until it is just outside the field of view.  If necessary, touch up the specimen focus using the fine focus knob.


     Procedure for continuing to use the microscope in brightfield (i.e., without phase contrast):


     (10) Use the diaphragm in the condenser (labeled CD) to adjust the contrast in the image.

     (11) To check for proper illumination, remove one of the eyepieces and hold onto it carefully (or, place it on lens paper, further back on the table where you will not knock it over or onto floor!).  The field of view through the open tube (the visible objective aperture) should be about 3/4 filled with light (i.e., 3/4 its diameter.  If not, adjust the condenser diaphragm a bit (NOT the field stop diaphragm). [NOTE: if illuminated region appears to be off center, ignore this, and make the best 3/4 light-filled estimate you can].

     (12) Put eyepiece back in place, and observe specimen.  If necessary, adjust light intensity using the intensity control below the green indicator light.

     (13) If stopping for the day, or even temporarily, leave the 10X objective in place and turn off the power switch at the rear.  If not stopping, proceed to the next exercise.  In either case, DO NOT CHANGE ANY SETTINGS (EXCEPT FOR THE POWER SWITCH).




     As noted above, for each objective lens there is a matching condenser position and condenser annulus (ring).  These phase contrast condenser rings will be referred to here simply as "phase rings".  In using a phase contrast microscope, it is necessary not only to match the objective with the proper phase ring, but also to align the phase ring with the ring in the objective.  This is accomplished by using a special device, or "telescope", that is inserted temporarily in place of one eyepiece, enabling one to see both rings (condenser and objective).  The condenser phase ring position is then adjusted carefully, using the ring positioning knobs (K-1 and K-2), until the condenser phase ring (i.e., the moving one) is positioned directly over the objective ring (the non-moving one).  The eyepiece is then out back in and the specimen is viewed to verify that it now has maximum contrast.


EXERCISE #6:  Aligning the phase rings with the "telescope"



     (1) Complete steps 1-9 in EXERCISE #5.

     (2) With the 10X objective in place, rotate condenser to position #1.  Note that "Ph1" appears on the 10X objective.

     (3) Replace one of the eyepieces with the "phase telescope", which will be found in its special plastic holder in the microscope accessory tray.  Carefully remove the tape holding the halves of its plastic holder together, and take out the telescope.  Place the removed eyepiece into the holder, tape the halves of the holder back together using the same labeled tape, and place the holder/eyepiece gently into the tray.

     (4) Focus the telescope by grasping its upper black section (black top) and pulling it quite far upward, rotating it slightly as you go, until both of the phase rings are visible clearly.  If you pull too far, push it back downward a bit.

(NOTE: Do not touch lenses at top or bottom of the telescope.  If you get finger prints on them, they must be cleaned off immediately by rubbing with lens paper.)

     (5) Adjust the phase ring position so that both rings are superimposed (one exactly on top of the other and centered).  Do this by using the two condenser adjustment knobs, K-1 and K-2 (Figure 2).  The K-1, K-2 labels are at either side of the "condenser" label.  Knob K-1 must be unscrewed slightly to unlock it, and is then slid back and forth in the open slot at the left side of the condenser.  Knob K-2 will rotate in either direction without requiring unlocking or locking.  Both knobs are used until the rings coincide, then Knob K-1 is re-locked by screwing it clockwise.

     (6) When rings have been aligned, remove the telescope and put the eyepiece back in.  (Put telescope back in its plastic holder, tape the halves together using the original tape, and place it back in the microscopy tray).

     (7) Examine the test slide (pen marks).  The specimen should now have good contrast, with a relatively dark background.  A bright "halo" may be visible at the edges of the lines; this is normal.

     (8) Rotate the condenser to position #2 and rotate the nosepiece so that the 40X objective (Ph2) is in place.  Do this without lowering the stage or changing focus.  The three objective lenses are essentially "parfocal", which means that only a little focusing adjustment is required AFTER switching objectives.


NOTE:  Moving the stage down (via focus controls) to get it out of the way when changing parfocal lenses wastes much time (for re-focusing) and may cause damage to objectives if you accidentally move the stage upward instead of downward.


     (9) If necessary, turn up the illumination intensity.

    (10) Check Koehler illumination for the 40X objective (looking through eyepieces), i.e., as before, get specimen sharply in focus, close down the field stop diaphragm until the edge is visible, and adjust condenser height until edge of diaphragm is sharpest.  Center diaphragm within the field of view using the condenser centering screws, then open diaphragm to just beyond the field of view.

    (11) Repeat steps 3 - 7 above using 40X objective and the telescope.

    (12) Rotate nosepiece so that the 10X objective is again in place and turn off power switch.  Then, if finished for the day, inform supervisor who will put microscope away.  Otherwise, proceed to next exercise.


EXERCISE #7:  Aligning the phase rings without the "telescope"


     Nearly all instruction manuals on phase contrast give the impression that use of the phase ring telescope is a necessity.  Although it IS required to SEE the phase rings in practice, it is NOT needed to achieve phase ring alignment and good contrast.  You will demonstrate this to yourself in this exercise.



     (1) If necessary, complete Koehler illumination steps 1-9 in EXERCISE #5 (i.e., if you are starting here for the day).

     (2) With 10X objective in place, rotate condenser to position #1.

     (3) Unscrew the locking knob of condenser adjustment knob K-1 and purposely swing it far off center.  Similarly, rotate K-2 so that it is also far off center.

     (4) Observe image of test slide: contrast should now be relatively poor, and background fairly bright, with uneven illumination.

     (5) WHILE OBSERVING SPECIMEN, use the two condenser adjustment knobs, K-1 and K-2 to obtain maximum contrast.  Use them both at the same time, looking for positions at which they produce the darkest background, and then make smaller adjustments so that the specimen is symmetrically illuminated with an even halo around it.

     (6) Verify that this is approximately the correctly aligned phase ring position by replacing one eyepiece with the telescope, and checking alignment as in the exercise above.

     (7) Put eyepiece back in and put telescope away properly in the microscope tray.

     (8) Switch to 40X objective (Ph2); check Koehler illumination.

     (9) Repeat steps 3 - 7 (this exercise, above) for the 40X objective.


NOTE THAT YOU NOW KNOW HOW TO SET THE PHASE RINGS FOR MAXIMUM CONTRAST WITHOUT USING THE TELESCOPE.  If possible, continue right on to the next section and exercise #8.  If not, turn off power, rotate 10X objective into position, and ask supervisor to put the microscope away.


EXERCISE #8:  Phase contrast versus brightfield


     In this exercise you will prepare a slide of your own epithelial cells and compare the appearance of the cells in brightfield and phase contrast.



     (1) Obtain a fresh slide, holding it by its edges only.  Clean it briefly by rubbing with a Kimwipe (to conserve lens paper).

     (2) Obtain a small glass test tube (13mm culture tube) and a plastic transfer pipette.

     (3) Pour a very small volume of phosphate-buffered saline, or "PBS" into the tube.  The PBS will be found in a large stock bottle in the refrigerator.

            NOTE:  "saline = a sodium chloride solution; so PBS = phosphate buffer with a certain concentration of sodium chloride in it [0.9%] that is physiological for mammalian cells/tissues.

     (4) Place a small drop of PBS onto the middle of the slide.

     (5) Obtain a clean toothpick from box in microscope tray.

     (6) With the WIDE end of the toothpick, scrape the inside of one of your cheeks several times. SCRAPE; DO NOT POKE!

     (7) Mix the scraping into the drop of PBS on the slide; discard  toothpick properly (put it in the SHARPS hazardous waste!!)

     (8) Holding it by edges only, place a number 1 (thickness) 22mm square coverslip gently on top of the drop.  Avoid air bubbles (don't "plop" it on!).  Make sure that you do not have two coverslips stuck together (if you do, throw them away - in sharps waste).




     (9) "Mop up" any liquid that comes out around the edge of the coverslip (use a rolled-up Kimwipe edge).

    (10) Clamp slide in place on microscope stage.

    (11) Rotate 40X objective and condenser Ph2 ring into position.

    (12) Check Koehler illumination and adjust K-1 and K-2 to obtain maximum contrast.

    (13) The cells should have clearly visible boundaries and nuclei.  A slight bright phase "halo" will be visible at their edges, and possibly also around the nuclei.  this is normal.

    (14) Without changing specimen view or any other settings, turn down illumination (light intensity control) and rotate condenser to the "J" position.

    (15) Examine the cells in brightfield.  How does the contrast and visibility of structural detail compare?

    (16) Return to 40X phase contrast for another comparison.

    (17) If finished for the day, remove slide and discard it in the SHARPS disposal container.  Turn off power switch (rear), and rotate nosepiece so that the 10X objective is in place.  Ask supervisor to put microscope away.  If not finished, leave slide in place and continue.




     Oil immersion objectives are designed to produce high resolution images, but do so only when they are immersed in special oil called, fittingly, immersion oil.  The refractive index of this oil (a measure of extent of light path bending) closely matches that of glass.  This avoids alteration of the light path between specimen and objective lens.  In some microscopes there is also a very high resolution condenser that requires oil between it and the glass slide.  The Tech Facility phase contrast microscope does NOT have such a condenser (thus, DO NOT put oil on it!).


     The 100X objective (Ph3) of the Tech Facility phase contrast microscope is an oil immersion objective.  It is parfocal with the other objectives, so that the stage height should NOT be changed (by focusing knob) when switching to this lens.  Rather, focusing is first done with the 10X, then the 40X is rotated into place, and the object of interest is positioned precisely in the center of the field of view.  Without changing focus or moving the specimen, the 100X objective and phase ring #3 are then rotated into place.  Fine focusing (only) is done as necessary to re-focus the specimen, and then adjustments are made to the illumination and condenser. 


EXERCISE #9:  Using the 100X oil immersion objective



     (1) If you are continuing directly from exercise #8, go right to step 5.  If not, go to step 2.

     (2) Set microscope to 10X objective and condenser position #1.

     (3) Focus on a specimen, establish Koehler illumination, and adjust condenser knobs K-1 and K-2 for maximum contrast (if necessary).

     (4) Switch to 40X objective and Ph2 ring, and repeat step 3.

     (5) Position an object of interest precisely in the center of the field of view.

     (6) WITHOUT CHANGING ANY SETTINGS (i.e., do not change focus or move stage), rotate the nosepiece so that the 40X objective is out of the way, and you are clicked into the "no lens" position between the 40X and 100X objectives.

     (7) Obtain the little plastic dispenser bottle labeled "IMMERSION OIL" from the microscope tray, uncap it, and squeeze a drop of oil onto the test slide at the location where the objective lens will be. TIP:  You can find the spot by closing down the field stop diaphragm and noting where the light is centered on the slide.

     (8) AGAIN, WITHOUT CHANGING ANY SETTINGS, rotate the 100X (Ph3) objective into place.  It should contact the oil drop directly (observe this to make sure).  Be careful not to move slide or microscope stage; a slight movement at 100X can displace your specimen totally out of the field of view.

     (9) Rotate phase ring #3 into place. 

    (10) Use the Fine focus control (only) to re-focus the specimen.  The image should appear with only minor focusing adjustments.

    (11) Normally at this point you would establish Koehler illumination and center the condenser.  However, with this particular condenser (an old one) the Ph3 position is slightly flawed.  A good image of the field stop diaphragm is not obtained and the light sometimes appears reddish or other colors.  So - we will skip this and proceed to next step.

     (12) Turn up the illumination (light intensity control) if necessary.

     (13) Open the field stop diaphragm to so that it is not visible and field of view brightens.

     (14) Adjust condenser knobs K-1 and K-2 for maximum contrast (if necessary), as previously, and observe specimen.

     (15) When observations have been completed, turn light intensity all the way down (check this while looking at light source).

     (16) Turn off power switch (in rear).

     (17) You must proceed directly to the next section.




     You have now learned the basic steps for using an upright phase contrast microscope, but you are not yet finished!  The 10X and 40X objectives usually do not require daily cleaning, but the oil must be cleaned off the 100X objective immediately after use.  This is best done with a soft lens tissue (otherwise called lens paper), of which there are several types and brands.  The type preferred in the Tech Facility is Kodak lens tissue.  Solvents such as acetone SHOULD NOT BE USED on the lens or lens paper, as these can damage lens cementing materials.


     Nearly every time that a poor or blurry image is experienced when using any light microscope, it is due to something on the surface of the objective lens.  Sometimes, for example, oil used with the 100X objective manages to get onto the 40X lens.  This is "cured" by careful cleaning of the lens with lens paper.


EXERCISE #10 :  Cleaning the microscope lenses



     (1) Rotate the nosepiece so that no objective lens is in place.  The oil immersion lens should now be further toward the front.

     (2) Lower the condenser.

     (3) Take out a single sheet of the Kodak lens paper, which is found in the yellow packet in the microscope tray.

     (4) Fold it over on itself a few times, and gently rub the lens.

     (5) Move to a new location on the lens tissue several times, and continue rubbing gently until there is no oil coming off the lens as judged by no oil visible on the paper.




EXERCISE #11:  Putting the microscope away and cleaning up



     (1) Make sure that immersion oil has been cleaned off of the 100X objective lens (if used), and off the stage (which is frequently contaminated with oil accidentally).

     (2) Wipe oil carefully off of the test slide, and return test slide to the microscope tray.  Discard cheek cell slide in SHARPS container (if not done previously).

     (3) Discard used lens paper in hazardous lab waste receptacle.

     (4) Swing 10X objective and phase ring #1 into position.

     (5) Turn off power switch (if not already off).

     (6) Unplug power cord.

     (7) Locate microscope dust cover, but do not put it on the microscope (it is carried without the cover on).

     (8) Call your supervisor over and inform her/him that the microscope needs to be put away.

     (9) Clean up your work area (if necessary).  Return the test slide to the microscope tray, and discard your own epithelial cell slide in the SHARPS disposal container if not done previously.


EXERCISE #12:  Microscopy Videotape


     View the videotape labeled "Microscopy Video", which can be found in the file drawer marked "Tech. Facility Videotapes."  This illustrates the appearance of various cell types in cultures, using an inverted phase contrast microscope.  When finished, rewind the tape in the tape rewinder, and put it back in its proper file drawer.