Imagine running a clinical sample without first

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1 Editor s Pge Focus on microscopy KOEHLER ILLUMINATION: AN AGE-OLD TECHNIQUE IMPACTS MODERN DIGITAL IMAGING BY BARBARA FOSTER Imgine running clinicl smple without first lerning nd perfecting the protocol. Iro n i c l l y, mny pthologists nd clinicins fll into tht very trp with one of the oldest tools in the clinicl l o r t o r y : the light microscope. A simple pro c e s s clled Koehler illumintion sets seline for imging tht minimizes rtifcts, reduces ftigue, enhnces productivity, nd significntly improves the qulity of informtion fed into digitl imging nd utomted imge nlyses. With modest mount of prctice, the whole process tkes under 45 sec, smll time investment tht reps gret rewrds. A quick ntomy lesson In its simplest form, the light microscope cn e thought of s three pieces of glss (the ojective, cond e n s e r, nd eyepieces) nd two controls (the field iris nd condenser perture iris). The ojective (F i g u re 1c) is the mstermind of the m i c roscope. A quick review of its engrving shows tht it sets the first step of mgnifiction (the lrg e s t numer) nd is mjor contriutor to resolution (set y the numericl perture, the numer tht follows the mgnifiction). It my lso hve n internl component tht genertes specil contrst technique, nnotted with mrkings such s PH for phse contrst, HMC for Hoffmn modultion contrst, or DIC for differentil interference contrst (frequently clled Nom r k s i ). Focus for the ojective is controlled y the lrge kno (Figure 1h). The condenser (Figure 1d nd e) is the second prtner in the optics scheme. Locted under the stge, its A recent proprietry survey conducted y M i c r o s c o p y / M r k e t i n g & Eduction indictes tht over 75% of pthologists rrely or never set Koehler illumintion. In the mid 1960s, Dr. Georges Nomrski discovered specil method for cutting the prisms tht re centrl to mny DIC systems nd tht mde the technique commercilly vile. His pproch is widely used y Crl Zeiss, Inc. (Thornwood, NY). However, other compnies my use other designs to chieve the sme gol. L e i c (Bnnockurn, IL),for exmple, uses system designed y Frncis Smith. Despite these differences, Nomrski hs een dopted s generic nme, just s Kleenex is often used to refer to fcil tissues. 8 JULY 2002 g e h Figure 1 The light microscope. (Imge courtesy of Crl Zeiss, Inc.) perture (opening or window) estlishes the ngle t which light pproches the smple. To demonstrte this effect, use the corse focus to lower the stge comp l e t e l y, then use the condenser focus (Figure 1e) to rise the condenser completely. Remove one of the ojectives nd rotte the nosepiece (the ssemly in The ojective is the mstermind of the microscope. which the ojectives re mounted) to crete mximum ccess to the stge. Fold the lower third of usiness c rd ck to mke n L, then rest the crd on tht short, folded section, just ove where the light emerges through the opening in the stge. When in position, the crd cts s screen to cpture the emerging light. Opening nd closing the condenser perture c i interpupillry djustment eyepiece nd diopter djustment c ojectives d condenser perture control e condenser focus f`condenser centrtion screw g fine focus h corse focus i light port/field iris djustment d f continued

2 or drkfield (D). Eyepieces (Figure 1) complete the trio. They re responsile for the second step of mgnifiction (gin, the lrger engrved numer) nd for setting the dimeter of the field of view (the field numer, seen s the second engrved vlue, given in millimeters). To determine the mximum dimeter of the field seen with ny prticulr ojective, simply multiply the field numer y 1000 to convert to micrometers, then divide y just the mgnifiction of the ojective. For exmple, if n eyepiece ers field numer of 25 mm nd you re using 10 ojective: 25,000 divided y 10 equls field dimeter of 2500 µm. This informtion is vlule for estimting the size of fetures in the field. For instnce, if you estimte tht 20 cells would fit cross the dimeter of your scene, divide 2500 µm y 20, indicting tht ech cell is 125 µm wide. The lst control is the field iris, locted in the light port in the se of the microscope (i). This control djusts the size of the field of view nd controls glre nd hze. If the smple sctters light, creting milky imge, simply move the feture of interest to the center of the field of view nd close the field iris round it. (This step ssumes tht the microscope hs een set up for Koehler illumintion.) Figure 2 Cheek cells imged with ) norml rightfield, nd ) xil illumintion (40 ). control (Figure 1d) opens nd closes cone of light. When closed, the em forms pencil of light (xil illumintion) with little or no ngle. Axil illumintion enhnces edges (compre Figure 2to 2) nd increses the depth of field, mking it the logicl choice for imging thicker smples. Opening the condenser mximizes the ngle nd p roduces nrrow wist t the smple. Pro v i d i n g tht the specimen is well stined, this setting is optimum for imging fine structures such s the endoplsmic reticulum or tiny prticles such s those in grnulocytes. The condenser perture control (d) is the most powerful control on the microscope nd the most often overlooked. Judicious use of this setting significntly improves the qulity of imges. The condenser my lso contriute to contrst enhncement. Universl condensers, for exmple, hve either sliders or rotting turrets with multiple loctions for inserts for techniques such s phse contrst (typiclly noted with regulr numers such s 1, 2, or 3, which correspond to mrkings on the ojective), HMC, DIC (typiclly mrked with Romn numerls), Koehler illumintion in detil D i rections for setting up Koehler re s diverse s recipes for Mullign stew. The instructions shown here simplify the process, mking it esy to incorporte Koehler into the every-dy work flo w. They center on two key issues: ) The smple determines the setting, nd ) the microscope hs three pieces of glsswre nd two controls. This discussion strts with the detils then summrizes the process in four quick nd esy steps. Before we egin, mke sure tht the rh e o- stt tht controls the rightness of the light is set so tht the field of view is comfortly right nd not yell o w. Your user s mnul will tell you which voltge is optimum. Eyepieces Strt y setting the inoculrs to the right distnce c s o tht you see one, round field of view. Most micro s c o p e s hve smll scle [] so tht you cn reset to this sme distnce every time you sit down t new micro s c o p e. Check to see if the eyepieces hve focusing rings c The interpupillry distnce (IPD) is, literlly, the distnce in millimeters from pupil center to pupil center when you re looking stright hed. 10 JULY 2002

3 c d Figure 3 Field iris closed. ) Condenser uncentered nd unfocused, ) condenser focused on field iris, c) condenser centered nd fo - cused, d) field iris opened outside the field of view.

4 (diopter settings []). If so, set them oth to zero or, if there re no numers, so tht the silver, white, or lck ring just touches the eyepiece mount. Ojective Using the nosepiece, rotte the 10 ojective into plce. Put smple on the stge. Mke sure tht if there is coverslip ( smll piece of glss on the specimen) the smple is mounted with the coverslip towrd the ojective. While oserving the distnce etween the ojective nd smple from outside the microscope, use the corse focus (h) to crefully move the stge towrd the ojective. Note which direction you re turning the kno. Stop when the smple is s close s possile to the ojective, then, while oserving the smple through the eyepieces, focus wy. Tht is, turn the corse focus the other direction until the specimen comes into shrp focus. This pro c e d u re not only voids dmge to slide nd ojective, it lso gurntees tht you will lwys find the specimen quickly, n importnt productivity step. Condenser This step encompsses four smller steps: f o c u s i n g nd centering the condenser, then setting oth the condenser perture nd field irises. Using the condenser focus control (the smll kno under the stge, [e]), rise the condenser to the ck of the slide. Agin, note which direction you re turning the kno. While the ojective uses the smple s refe rence for focus, the condenser uses the field iris (i) s reference. Close the field iris (Figure 3). Using the condenser focus, focus wy until the edges of the field iris come into shrp focus (Figure 3). Notice tht the imge of the field iris is off-center t this point. Locte the condenser centrtion scre w s ( F i g u re 1f ). Ty p i c l l y, they re mounted t 5 nd 7 o clock. While peering through the microscope, use the s c rews to wlk the imge of the field iris into the center of the field of view (Figure 3c). At first, this p rocess will seem wkwrd ecuse these scre w s work ginst ech other nd long the digonls rther thn left nd right, up nd down. Micro s c o p y involves strong eye hnd rin coordintion nd this sitution is one of those cses in which little prctice will improve tht coordintion considerly. Unless the smple is highly scttering, open the field iris (i) so tht it is just outside the field of view ( F i g u re 3d). Adjust the condenser perture iris (d) so tht the edges of the fetures re crisp nd the ckground is clen. If the condenser is set too fr open, the imge will pper wshed out. If it is too fr closed, the edges will e too thick nd there will e rings 12 JULY 2002

5 round every little it of dust nd dirt. Eyepieces revisited Ech of us hs dominnt eye. d It is the eye tht guides us s we wlk round the world nd tht we use when iming in contests such s drts, rc h e r y, or shooting. If oth eyepieces re focusle, leve the diopter setting for your dominnt eye t zero. Focus the eyepiece for the nondominnt eye so tht you see the specimen in the field of view clerly. This smll djustment compenstes for ech eye s unique focus nd voids the terrile hedches nd eyestrin often experienced y people who use microscopes for living. Four quick steps nd 45 seconds to perfection Prctice mkes perfect. By the time you hve prcticed these steps four or five times, you should e le to estlish Koehler illumintion in well under 45 sec. 1. Eyepieces. Set IPD for one, round field of view nd diopter settings to Ojective. Rise stge until nerly touching the smple (view from outside), then focus wy (view t h rough microscope) until the imge of the smple is shrp. 3. C o n d e n s e. rrise condenser so tht it re l y touches the ck of the smple (view from outside). Close the field iris (view through microscope), then focus wy until edge of field iris is shrp. Center cond e n s e r. Open the field iris just outside of the field of view nd set the condenser perture iris for shrp edges nd clen ckground. 4. Eyepieces. Adjust diopter setting for nondominnt eye until imge seen with tht eye is shrp. A few finl tips Koehler illumintion forms the seline for ll mic roscopy imging. Set it first thing in the morning, then fine-tune it whenever you chnge ojective or smple. These few swift djustments re gurnteed to p roduce etter photomicrogrphs nd significntly i m p rove informtion for utomted imging systems. One finl note: Unlike ny other nlyticl technology, m i c roscopists re prt of the microscope system. Estlishing Koehler is lso gurnteed to reduce neck nd eye strin nd improve your p roductivity t the microscope. d To find your dominnt eye, mke tringulr window y touching your two index fingers together nd your two thums. Hold the window t rm s length nd use it to locte distnt oject. Look t the oject with oth eyes, then close your right eye. If the oject stys in plce or only moves slightly, you re left-eye dominnt. If it moves drmticlly (for exmple, out of the window), you re right-eye dominnt. Ms. Foster is President of Microscopy/ Microscopy Eduction, 125 Pridon St., Ste. 102, Springfield, MA 01118, U.S.A.; tel.: ; fx: ; e-mil: foster@mme1.com. She encourges comments nd inquiries out the new technologies presented in her rticles. AMERICAN CLINICAL LABORATORY 13