article - Microscopy/Microscopy Education
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1. rings around every little bit of dust and dirt Eyepieces revisited Each of us has a dominant eye It is the eye that guides us as we walk around the world and that we use when aiming in contests such as darts archery or shooting If both eyepieces are focusable leave the diopter setting for your dominant eye at zero Focus the eyepiece for the nondominant eye so that you see the specimen in the field of view clearly This small ad justment compensates for each eye s unique focus and avoids the terrible headaches and eyestrain often ex perienced by people who use microscopes for a living Four quick steps and 45 seconds to perfection Practice makes perfect By the time you have prac ticed these steps four or five times you should be able to establish Koehler illumination in well under 45 sec 1 EyepiecesSet IPD for one round field of view and diopter settings to 0 2 Objective Raise stage until nearly touching the sample view from outside then focus away view through microscope until the image of the sample is sharp To find your dominant eye make a triangular window by touching your two index fingers together and your two thumbs Hold the window at arm s length and use it to locate a distant object Look at the object with both eyes then close your right eye If the object stays in place or only moves slightly you are left eye dominant If it moves dramatically for example out of the window you2. PE Editor s Page Focus on microscopy KOEHLER ILLUMINATION AN AGE OLD TECHNIQUE IMPACTS MODERN DIGITAL IMAGING BY BARBARA FOSTER magine running a clinical sample without first learning and perfecting the protocol Ironically many pathologists and clinicians fall into that very trap with one of the oldest tools in the clinical laboratory the light microscope A simple process called Koehler illumination sets a baseline for imaging that minimizes artifacts reduces fatigue enhances productivity and significantly improves the quality of information fed into digital imaging and automated image analyses With a modest amount of practice the whole process takes under 45 sec a small time invest ment that reaps great rewards A quick anatomy lesson In its simplest form the light microscope can be thought of as three pieces of glass the objective con denser and eyepieces and two controls the field iris and condenser aperture iris The objective Figure 1c is the mastermind of the microscope A quick review of its engraving shows that it sets the first step of magnification the largest number and is a major contributor to resolution set by the numerical aperture the number that follows the magnification It may also have an internal compo nent that generates a special contrast technique anno tated with markings such as PH for phase contrast HMC for Hoffman modulation contrast or DIC for differential interfere
3. are right eye dominant 3 CondenserRaise condenser so that it barely touches the back of the sample view from outside Close the field iris view through microscope then fo cus away until edge of field iris is sharp Center con denser Open the field iris just outside of the field of view and set the condenser aperture iris for sharp edges and clean background 4 EyepiecesAdjust diopter setting for nondominant eye until image seen with that eye is sharp A few final tips Koehler illumination forms the baseline for all mi croscopy imaging Set it first thing in the morning then fine tune it whenever you change objective or sample These few swift adjustments are guaranteed to produce better photomicrographs and significantly improve information for automated imaging systems One final note Unlike any other analytical technology microscopists are part of the microscope system Es tablishing Koehler is also guaranteed to reduce neck and eye strain and improve your productivity at the microscope Ms Foster is President of Microscopy Microscopy Educa tion 125 Paridon St Ste 102 Springfield MA 01118 U S A tel 413 746 6931 fax 413 746 9311 e mail bfoster mmel com She encourages comments and in quiries about the new technologies presented in her articles AMERICAN CUNICALLABO RATO RY 13
4. etely Remove one of the ob jectives and rotate the nosepiece the assembly in The objective is the mastermind of the microscope which the objectives are mounted to create maximum access to the stage Fold the lower third of a business card back to make an L then rest the card on that short folded section just above where the light emerges through the opening in the stage When in po sition the card acts as a screen to capture the emerging light Opening and closing the condenser aperture continued L oF Cet ae Ps N 3 LA i s A gt aS X is y 3 bi oe A T Nee ase AY G r Ta x a a Ty x k 4 AAO gt 7 Aas Ras fil S08 4 Ea ARSS 1 AI AA NE ANE NY A SAL AONA K A a o a x ca vo Pr X gt Tor RES J SRA aS Figure 2 Cheek cells imaged with a normal brightfeld and b axial illumination 40x control Figure 1d opens and closes a cone of light When closed the beam forms a pencil of light axial illumination with little or no angle Axial illumination enhances edges compare Figure 2ato 2b and increases the depth of field making it the logical choice for imaging thicker samples Opening the condenser maximizes the angle and produces a narrow waist at the sample Providing that the specimen is well stained this setting is opti mum for imaging fine structures such as the endoplas mic reticulum or tiny particles such as those in granu locytes The condense
5. iris located in the light port in the base of the microscope i This control ad justs the size of the field of view and controls glare and haze If the sample scatters light creating a milky im age simply move the feature of interest to the center of the field of view and close the field iris around it This step assumes that the microscope has been set up for Koehler illumination Koehler illumination in detail Directions for setting up Koehler are as diverse as recipes for Mulligan stew The instructions shown here simplify the process making it easy to incorporate Koehler into the every day work flow They center on two key issues a The sample determines the setting and b the microscope has three pieces of glassware and two controls This discussion starts with the de tails then summarizes the process in four quick and easy steps Before we begin make sure that the rheo stat that controls the brightness of the light is set so that the field of view is comfortably bright and not yel low Your user s manual will tell you which voltage is optimum Eyepieces Start by setting the binoculars to the right distance so that you see one round field of view Most microscopes have a small scale a so that you can reset to this same distance every time you sit down at a new microscope Check to see if the eyepieces have focusing rings The interpupillary distance IPD is literally the distance in mil limeters from p
6. nce contrast frequently called No marksi Focus for the objective is controlled by the large knob Figure 1h The condenser Figure 1d and e is the second part ner in the optics scheme Located under the stage its A recent proprietary survey conducted by Microscopy Marketing amp Education indicates that over 75 of pathologists rarely or never set Koehler illumination In the mid 1960s Dr Georges Nomarski discovered a special method for cutting the prisms that are central to many DIC systems and that made the technique commercially viable His approach is widely used by Carl Zeiss Inc Thornwood NY However other companies may use other designs to achieve the same goal Leica Bannockburn IL for example uses a system designed by Francis Smith Despite these differences Nomarski has been adopted as a generic name just as Kleenex is often used to refer to facial tissues 8 JULY 2002 a interpupillary adjustment b eyepiece and diopter adjustment c objectives d condenser aperture control e condenser focus f condenser centration screw g fine focus h coarse focus i light port field iris adjustment Figure 1 The light microscope Image courtesy of Carl Zeiss Inc aperture opening or window establishes the angle at which light approaches the sample To demonstrate this effect use the coarse focus to lower the stage com pletely then use the condenser focus Figure 1e to raise the condenser compl
7. perture and field irises Using the condenser focus control the small knob under the stage e raise the condenser to the back of the slide Again note which direction you are turning the knob While the objective uses the sample as a ref erence for focus the condenser uses the field iris 7 as reference Close the field iris Figure 39 Using the con denser focus focus away until the edges of the field iris come into sharp focus Figure 3b Notice that the image of the field iris is off center at this point Locate the condenser centration screws Figure 1f Typically they are mounted at 5 and 7 o clock While peering through the microscope use the screws to walk the image of the field iris into the center of the field of view Figure 3c At first this process will seem awkward because these screws work against each other and along the diagonals rather than left and right up and down Microscopy involves strong eye hand brain coordination and this situation is one of those cases in which a little practice will improve that coordination considerably Unless the sample is highly scattering open the field iris i so that it is just outside the field of view Figure 3d Adjust the condenser aperture iris d so that the edges of the features are crisp and the back ground is clean If the condenser is set too far open the image will appear washed out If it is too far closed the edges will be too thick and there will be
8. r aperture control d is the most powerful control on the microscope and the most often overlooked Judicious use of this setting significantly improves the quality of images The condenser may also contribute to contrast en hancement Universal condensers for example have either sliders or rotating turrets with multiple loca tions for inserts for techniques such as phase contrast typically noted with regular numbers such as 1 2 or 3 which correspond to markings on the objective HMC DIC typically marked with Roman numerals 10 JULY 2002 or darkfield D Eyepieces Figure 1b complete the trio They are re sponsible for the second step of magnification again the larger engraved number and for setting the diam eter of the field of view the field number seen as the second engraved value given in millimeters To de termine the maximum diameter of the field seen with any particular objective simply multiply the field number by 1000 to convert to micrometers then divide by just the magnification of the objective For example if an eyepiece bears a field number of 25 mm and you are using a 10x objective 25 000 divided by 10 equals a field diameter of 2500 um This information is valuable for estimating the size of features in the field For instance if you estimate that 20 cells would fit across the diameter of your scene divide 2500 pm by 20 indicating that each cell is 125 pm wide The last control is the field
9. upil center to pupil center when you are looking straight ahead EDITO R S PAGE continued Figure 3 Field iris closed a Condenser uncentered and unfocused b condenser focused on field iris c condenser centered and fo cused d field iris opened outside the field of view 12 JULY 2002 EDITO R S PAGE continued diopter settings b If so set them both to zero or if there are no numbers so that the silver white or black ring just touches the eyepiece mount Ob jective Using the nosepiece rotate the 10x objective into place Put a sample on the stage Make sure that if there is a coverslip a small piece of glass on the specimen the sample is mounted with the coverslip toward the objec tive While observing the distance between the objective and sample from outside the microscope use the coarse focus h to carefully move the stage toward the objec tive Note which direction you are turning the knob Stop when the sample is as close as possible to the objec tive then while observing the sample through the eye pieces focus away That is turn the coarse focus the other direction until the specimen comes into sharp fo cus This procedure not only avoids damage to slide and objective it also guarantees that you will always find the specimen quickly an important productivity step Condenser This step encompasses four smaller steps focusing and centering the condenser then setting both the con denser a
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