Proper use of the Compound MicroscopeKohler Illumination & Staining
Although the light microscope is themost commonly used biological instrument, it is often used improperly. This may not matterso much with very thin commercial slides but proper alignment of the illumination systemis essential for viewing thick sections and whole mounts. It is also crucial forphotomicroscopy. You will be using microscopes throughout this class and for years tocome. If you learn the simple lessons we will teach you today you will do much better inyour work and see the exciting world of microscopy in a new light! (Sorry about the pun).The procedure we follow was developed by the German scientist, August Kohler (1866-1948),and it bears his name. Recently his ideas were used to make an advanced ElectronMicroscope by Zeiss. Thus, this procedure which was introduced in 1893 has been of lastingand value.
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Because the lens systems in amicroscope are composed of many lenses it is called compound. The typical illumination ofspecimens in which light passes through the specimen and travels to your eye is calledBright Field microscopy. Light has the following path.
Eye or Camera
Body or Tube ->
Condenser (Iris) ->
Condenser Lenses ->
Field (Iris) Diaphragm ->
Light Source ->
Locate these parts of your microscope by referring to Illustration above.
We will be using Leitz microscopes inthis class, however, the instructions for correct alignment of the condenser will beapplicable to other microscopes with adjustable condensers.
Light is provided by a built in bulbwhich is reflected through the field iris diaphragm, the condenser, the condenser irisdiaphragm, the specimen, the objective, the tube and the ocular.
There are various control knobs onthe microscope which affect the light path. In addition, there are knobs for coarse andfine focus, as well as knobs to move the stage.
Locate the coarse and fine focusingknobs on each side of your scope. Each knob does coarse and fine focusing. There is noseparate knob for fine focusing. You will see how this works later. The rotation of thisknob focuses the objective onto the specimen.
The knobs which control themechanical stage are on the right side of the microscope as it faces you.
The condenser aligns and focuses light on the specimen. It has a long verticalknob
Directly above this at aright angle to the condenser adjusting knob you will find a rod which controls a lenswhich can be swung in or out of place. This swinging lens is left out for low-powerillumination
In addition, there are two smallknobs on the front of the condenser, set at 45o which are used to center it.
Finally, there is a lever whichcontrols the aperture iris. This improves contrast (difference between light & dark)especially at intermediate and high magnifications.Do NOT use this to increase or decrease brightness!!!
Locateall of the controls for the condenser.
REMEMBER THIS !!!!!!!!!!!!!!!!!!!!!!!!!!
|The Objective focuses on the Specimales indigenous Above |
The Condenser focuses light onto the Specimales native Below.
The light source is housed in thebase of the microscope. It passes through the field iris diaphragm. The size of the fielddiaphragm is controlled by rotating a knurled ring which is concentric with it. The fielddiaphragm controls the area of illumination.
Locatethe field diaphragm and its knurled ring.
The magnification of an image isprimarily controlled by the objectives which are housed in a rotating nose piece. Tochange objectives you rotate the nosepiece, starting with the 4X objective. Do not startviewing by swinging in the 20 - 100 X objectives. These may be damaged if they hit thespecimen. The magnification is indicated by a number on each objective. Furthermore there is aprogression in size such that the longest objectives have greatest magnification. Thedistance between the objectives and the cover slip (working distance) decreasesdramatically as the magnification of the objective increases.
The 100 X objective is an oilimmersion lens. Note the black line near the tip of the objective. This is used toidentify an oil immersion lens. Place a small drop of oil on the objective lens.A small drop of oil must also be placed on the cover slip.
The lens should be carefully loweredinto the oil prior to focusing. Observe this with your naked eyes focusing on the objective and the specimen. Do NOT look through the oculars.Oil improves the optics because it unites the glass cover slip and the objective. Itreplaces air with oil. The oil has the same refractive index as glass. Thus less lightscattering & refraction occurs. Be sure that the specimen was in focus at 40X before switching to 100X. Avoid focusing down on the specimen with an oil immersion lens. Change the focus so thatthe objective is traveling away from the slide. If the image does not come into focus,reverse the direction until it does. When in doubt, STOP!!! & ask for HELP!!!The lens might be dirty or there may be some other problem.The oil also protects the objective lens from scratching.
Notice that we have 4, 10, 20, 40& 100 X objectives. Always start with the 4 X objective to prevent damage to the otherobjectives which may collide with the specimen.
Once one of the lenses is focused ana specimen, the others should also be in focus when they are swung into place. Thisproperty is referred to by the term parafocal. However, in actual practice some adjustmentis required when you switch from one objective to another. This usually presents littledifficulty. However, you must be especially careful when switching from 10X to 40X andfrom 40X to 100X. We will often be using fresh sections and whole mounts in the class. These can be thickand irregular. Consequently, greater care must be taken when changing objectives. When indoubt, play it safe and ask for help until you getacquainted with the material you are studying.
The oculars should be adjusted tosuit both of your eyes. Note that there is a scale on the tube holding both objectives. Wewill label microscopes so that each student can work with the same instrument throughoutthe course.Grasp the adjustable knurled ring below each ocular with your thumb and forefinger andgently rotate it so that each is set at 64 which is its midpoint.Before you make any adjustments, place a slide on the stage and focus on part of thespecimen.
Youare now ready to achieve Kohler illumination. Oh happy day!
The best resolution occurs when allelements of the microscope are in perfect alignment and the iris diaphragms are properlyadjusted to the best aperture. On simple microscopes you may not be able to alter thealignment of the different parts, but on these Leitz microscopes it is possible to alignand focus the condenser to achieve "Kohler Illumination".
Because we will be using a lot ofthick hand-sections in this class, it is vital that you learn how to achieve Kohlerillumination. Otherwise, you will not be able to analyze your specimens.
1> Place a commercially preparedslide on the stage.
2> Make sure the swinging lens is inthe light path (facing up) and focus on the specimen using the 10X objective.
3> Use only one eye
4> Use the knurled ring below theother ocular to focus it while looking through it with your other eye. You may not need tochange the focus. However, experiment by rotating the knurled focusing ring to see itseffect. My German friends have told me that the correct way to focus the second ocular isto make it more negative so it is out of focus, then rotate it in a positive directionuntil it is focused.
5> Having the oculars focused willimprove image quality and will decrease eye strain. Once this is done it need not be changed during a given session. However, it is a good habitto do this at the beginning of each lab. It is best done at 10X because there is lesschance for errors at this magnification compared to 4 X.
6> Make sure the aperture iris iscompletely open
7> Reduce the field of illuminationby rotating the knurled ring on the field diaphragm completelyclockwise. Be gentle with the field diaphragm. It should close without any effort.
8> You should see a small circle oflight. If you are lucky, it will be in the center of the field. However, it will mostlikely be off-center and out of focus. Let us know if you can"t find it!!
9> Use the vertical condenseradjusting knob to make the circle as small as possible by gently rotating it. This movesthe condenser up and down. Do this carefully so that the circle of light is not pushedlaterally. As you focus the field diaphragm you will notice that its halo turns from blueto red and red to blue. The best focus occurs when you adjust the condenser so that thehalo is just between red and blue. This is a little hard to do so don"t be too worried ifyou have some red or blue in the halo.
10> Expand the field diaphragm byrotating its knurled ring counter-clockwise, until the light touches one edge of thefield. If the light is perfectly centered it should touch the entire circumference of thefield. This is unlikely.
11> Center the circle of light byusing the two small adjustable knobs on the front of the condenser. When you aresatisfied, expand the field so that the light fills it completely. However, do not fullyopen the field diaphragm. Open it just enough to extend beyond the field of view.
12> Repeat this with the 20 or 40 Xobjective. For critical work this should be done for each objective. This is especiallyimportant for taking photographs and for examining minute, translucent specimens likefungi and algae. For our labs, it will be good to do this for the 10X objective at thestart of each session. You need not do this for 4X and 40X. However, if you are havingsome problems resolving details, check to be sure that you have the condenser aligned andfocused.
It may be difficult to do this withthe 100 X objective. However, if you achieve proper alignment with the 40 X objective, the100 X will be similar.
13> When working at 20 - 100 X it isimportant to adjust the condenser aperture iris. This is especially important fortranslucent structures. Closing this iris increases contrast. Thus something fuzzy becomessmooth and something faint becomes dark. It is usually possible to close the iris andjudge its effects subjectively. However,
14> Remove one of the oculars andlook directly down the tube at the light field. Close the iris so that it occludes 1/4 -1/3 of the area. This should give the best contrast. Examine a specimen before and afteradjusting the aperture iris. This should be done for each objective for critical viewing.In practice, you can experiment with this while viewing a specimen and adjust it withoutremoving the ocular. Closing the aperture iris also increases depth of focus up to apoint. Thus, more areas of a three dimensional specimen will be in focus If it is closedto much, a flat indistinct image results.The example shows part of a diatom frustule. There is little detail when the iris is wideopen (top). When it is fully closed (middle) the contrast is increased but there areaberrations which make the small holes appear larger than they are in actuality. Theoutline of the small holes is also indistinct. When the iris is closed 25 - 30 % there isimproved contrast and less aberration.
15> Experiment with the aperture iriswhile viewing a prepared slide. Once you have achieved what you think gives the best imagequality, remove one of the oculars and see how much of the field is occluded.
As part of the first lab, we will beusing different stains to study their effects an fresh specimens. Experiment with theaperture iris as you study these. Fresh sections are usually too thick for detailedexamination at high magnification, but the aperture iris can be used to great effect withthis type of material.
While these procedures may seemtedious, they will become routine as you progress in the course.
The ability to make free handsections will allow you to quickly analyze plant organs without resorting to laboriousprocedures. A tremendous amount of information can be derived from hand sections. These donot need to be extremely thin to be of use. In addition, hand sections of a structure donot need to be complete or uniformly thin to be useful. Your initial attempts at handsectioning will probably be frustrating, however, you will quickly become proficient. Handsections also provide 3-D information which is not available with most commercial slides.
1> Place a Band-Aid on the thumb ofyour left hand. Have the cotton portion on the bottom of your thumb. The thumb is abackstop for this operation.
2> Place another on the end of yourindex finger. The index finger will control the height go the specimen, and thus itsthickness.
3> Grasp the plant structure betweenyour thumb and forefinger so that the top of the specimen extends above the level of yourforefinger.
4> Take a single-edge razor blade inyour right hand. Be sure that it is wet.
5. Rest the blade on your forefingerand use a slicing motion to cut off the top of the specimen.
6. Try to avoid cutting your thumbwith the blade!!!
7> Raise the specimen slightly bymanipulating it with your fingers and repeat the slicing motion.
8> Thin sections can often beobtained by pressing the blade down on your forefinger and then slicing through thespecimen several times.
9> After several sections haveaccumulated on the blade, wash them off in a Petri dish of water.
10> Keep on slicing until you havesome thin sections. These will appear translucent when seen against the dark background ofyour lab bench. In most cases, the sections will have thin and thick regions. As long aspart of the section is thin, you may be able to use it, and thick sections are frequentlyOK.
11> Sections can be removed withforceps and placed in a drop of water or stain on a microscope slide.
12> It is a good idea to viewunstained sections prior to staining. Proper use of the aperture iris is important forthis.
It is essential that the sections becompletely immersed in water so that air is excluded.
Air bubbles or spaces will interferegreatly with your observations. To avoid these when adding a coverslip do the following.
a> Place your sections in 2-3 dropsof water or stain in the center of the slide.
b> Use a fine forceps to pick up alarge cover slip (20 x 40 or 20 x 50)
c> Place one end of the coverslip onthe slide (near boundary with frosting) without touching the solution containing thespecimens.
d> Steady this end with the fingersof your left hand.
e> Slowly lower the forceps until ittouches the slide. By this time the coverslip should have touched the solution on theslide.
f> Slowly remove the forceps so thatthe coverslip is gently lowered into its final resting position.
g> Remove excess solution by touchingthe side of a Kimwipe or paper towel to the narrow edges of the coverslip. Be careful notto drag out your sections with the excess solution.
h> if you have been using a stain,add water to one end of the coverslip while withdrawing the stain at the opposite end witha Kimwipe or towel. In most cases you do not need to get all of the stain out.
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i> Wipe excess fluid from the bottomof the slide or it will stick on the stage and make your life more miserable than italready is.