Microscopy F. K. Moellring fromthe verybeginning
CARLZEISS . Oberkochen/Wuern. i,;, ENCES AnJDTEC**oIOUO,.#O*^*, Microscopy from the very beginning
).
Dr. FriedrichK. Mijllring Dipl.-Biol. l member of scieniific staff, J Microscopy Laboratory l :l
I
'l Microscopy - from the very beginning
I st Siep: 1. Thisis a microscope 5 Microscopy In a nutshell 2. And now a bit of practice: we focus a specirnen 6 3. A glancebehind ihe scenes , t5 2A 5. Careand cleaning of ihe microscope 35
2nd Step: 6. Nurnericalapertqre, usefuT magnificatior Microscopy foriho serious worker and limit of resolution 37 7. Oilimmers'on 41 8. The question of cover glasses 43 9. Microscope illumifaiion for more exacting requirements(KohTer method) 10. Lengthmeasurements underthe microscope 51 11. Thecarnera on the microscope 53 12. N4icroprojection 51
More advancedliteraiure 59 60 60 Addiiional'nformation ZEISS- andthe art of microscopy 62 Preface
Whereasinstr!ction man!a s dealwiihihe operation of oneparticular microscope model, thisbrochure is lntendedto furnishgeneral information on rnicroscopy,ihat is, facts which apply equallyto a types and even rnakesof rnicroscope. Thebooklet serves ihree purposes:
1) lt ls intendedto be a guidefor the beginner, with the aid of whichmicroscope work shouldbe a pleas!ref.om the outset.
2) li will briefly recapliulatethe most essentiaifacts about m croscopy for the benefit of those who do noi usethe microscoperegularly.
3) lt is a so forthe Lrseofthe experencedm croscopist who wanis to !ntroduce oihers io ihe field of microscopy. He mayfind one or the otherof ihe hints conlainedin the booklethelpful for ieaclring- It shouldbe rememberedthat we iake manythings for granted,and all too quicklyforget the difficut!es we once hadwhen we madeour first acqualntance
We wish you a successand are sufeyou will enjoyworklng with your ZEISSmicroscope.
CABL ZEISS, Microscopy Depariment 6 7 8
-1o 1.This is a microscope
l) Eyepiece,s ippedinto upperend of tube. The engravedvalue, e. 9., 12.5x,lndicaies the magnification of the eyepjece;multiplied by the magnification of the objective, this gives the tota magnification
2) I ube (here an lnclinedtube for monocuar observation).
When the screwcollar is loosened andthe tube pressedagainst a sprlngin it, it can be lifted oui. j) Nosepieceto. q-icl e\cha-9eo-ob eci!es 4) Objecuve designation 40/0.65 160/0.17rneans: objecuvemagnlfication 40x, numericalapel1ure 0.65 (seeexplanation on page37), computedfor mechanicaltube lengthof 160mn and 0.17mm cover glassthickness. 5) Specimensiage wiih spring clips. 6) Condenser(correct position is a ways just below
Condenserfront lensswinging out for ill!minationof largefobjeci fields. 7l lris lever(condenser or aperiurediaphragm). 8) Swjng-out filier holder for filters of 32 rnrn diameter. 9) Fine adju€trnentknob. On STANDARD KF Mlcroscopes this aTlowsunlimlted rotationl on other rnicroscopesits rotation is limiled by iwo stops (lndex beside ihe knob, see Fig. 2). Coarse adjustrnentknob. With all Zeiss mlcroscopes, rotation in the direction of the arrow brings objectve and €pecirnencloser together (thesame applies to the fine adjustment). 10) Attachable simple substage illuminaior for direct connecuonto the mains (which can be exchangedfor mirror) with rotary switch and ground glass. 11)Base wiih ihree-poinisupport. On the !nderside, iapped holes for screwing down in cabinei as we l as two holes for mounting ihe connecilng brackeis STANDAFDKF M croscope of separateilluminators.
r 2. And now a bit of oractice: we focus a specimen
You have recelved your ZEISS m croscope and unpacked it according io the recomrnendauonsconiained in the pedjnent instrucijonmanual. lf you shoud be usinga mlcroscopefor the first ijme, we recommendihai you stari by making youF self familar with its most esseniialcomponents, such as objective, eyepiece,condenser, coarse and fine adjustments, usingFig. I as a guide.
Screwing in ihe objectiv€s Startby screwingthe objectivesinto ihe nosepiece,working from ow magnificationsupwards. Please avoid touch ng glass surfaces, parlicularly the froni lenses of the objectives. lf you touch them inadvertenily, remove the fingerprint imrne djately wiih ihe aid of a soft linen rag (see also page 35).
We need€pecimens Our first acquaintance with a rnicroscope rnay end on a rather cljsappoiniingnoie, because we may not have realized the fact that a normalmicroscope cannot simp y be usedfor magnifying any desired object - such as a fly - (for which thefe are ZEISS stereomicroscopes),but that the oblect we want to examineshould always be more or lesstransparent and as f at as possible.The wlng of a fly is a good example. These requirementsiherefore usually necessltaieihe so- calledpreparation of specimens,about wh ch deials can be gatheredfrom intioductorybooks on mlcroiechnq!e, seethe secuon on literaiure, page 59. For ihe purpose of the present descripiionwe must assumelhat suitabe specimens of which,incidentally, a wlde cholceis offered by companies specializingin teachingaids - are avalable. Our specimens are usuallymounted on a so-caled slde, a piece of gass about26x76 mm ln sizeand 1 mm lhick,which is coveredby an exiremely thln cover glass (various sizes; thickness as close as poscibleto 0.17 mm).- Be sure that the cover glassjs alwayson top,facing the objective
The microscopestands on a iable so thai you can took inro me eyeprecerrom a convenientposture. Microscopes with o-i a b-ilt-:n oest sei _o - f,onr oI a aTp w tf . ldrge J forn y en i! .9 su.{"ce.(Wo-< w-n specdl rnicroscopeiluminators is discussedon page45).
ileight of condenser, The condenseris in place and turnedor pLrshedup a the way.The condenserfronr lensis swungin. The eyepiecew be inseded later.At ihe beginningof an exarninaiion,we arways use a ow-power oblectve of abour6.3 o. 10x, becauseit perrnitscoverage of a wider fieldand is thus best sultedfor scanning.In addition,is depth of fjetd is greater ihan thai offered by objedives of higher power, and ii is ih!s easer to find the planeof besi definirion_
Whai is themagnificarion? The power of an objectiveis ndicaredby its nitialmagnifi- caiion. This s ihe f rst one of rhe values engraved on every objective (see page 28). The toral rnaqnificaiionof rhe micro scope s determinedby muliiplyng rhe magnificationof ihe objective with that of the ey--piece,which s engraved on the laiter. A 10x objectiveused in conjunctonwith a i2.5x eyepiecethus yie ds a totalmagnification of 125x.
Cauion - The specmen has been fxed on ihe stage,and we now in spiteof bu [-in sl de protecuonl cautiouslymove specirnenaid objectivec oser togerherby ihe coarse adjustment, uniil they are onty a few rii imoters apart (observe from the s de!). In ZEISSmicroscop€s, a stop and the springmolnt of rhe objectivesprevent ordlnary specimens (1 mm slide,0.17mrn cover gla6s)from being damagedby the objective.Onty a '"w specal p rposeoble(t.-. a e e(ep-o^s
'l The objectiveis uminated Before nseiringthe eyepieceof lowest powe. jnto the iube, we open the jrs diaphragmof the condenserand turn the illurninatingmirror until the backlens of the objeciive,which is visibleai the lower end of the tube, is evenlyi uminared. When ihe atiachablesirnp e substagej lurninatorfor direct connectiofto the ma ns shownin Ftg.1is used,thisoperaiion is not required.After the eyepiecehas been inserted,we cose the condenserlrs about halfway,look into rhe eye- piece and increasethe d stancebetween the objectiveand the specjmenby the coarseadjustm€nt unt I detats of the specmen can be recognzed, even if they are st ll bllred.
Exact foclsing is now achievedwth the aid of the fine adjustment. n ihe case of spec mens of very low conrrasi, finding the foca pane rnay occasionay be somewhar dlfflcLrlt;it is fac ltated if during operationof rhe coarse adjusimeniihe specinrenis slowly displacedon the stage, becausemovjng sir!ctures can be recognizedmore readily. Closingthe condenseriris furihermay a so be helpf! . When the areaio be examlnedis in ihe cerier of the fie d of view, ihe objectiveof rext higherrrragnificaiion may be movedtnto position,for whichon y thefine controlis neededforfoc!sing (parfocal objectives see page 29).
Do .ot siopdown too far The adjustmentof the condenseris a particulartyfrequent sourceof eror. The condenseris correctlyadjusred when ii s in lts highesiposltion of di.ectlybelow this and when its lrs lras bee. cosed oily far enoughro obiainjust suffclent conirast The beg nnerus!a y slopsthe condenser downfar ioo much.Never use the condenser rstodmthe imagelOihermeans,e.g. grayflters, mustbe Lrsedfor ihis "io purpose. Lowerng ihe condenser jncreasecontrast' whlch s popuar wjth manym croscopistsonly has the same effectas furiherclosing the condenserirjs and s therefore
lf wth objeciivesof very low power ihe urninatedfeld shouldbe too sma , sw ng olt the f.ont ens ofihe condenser and open is djaphragmiully.
ALwayskeep a hand Thedepth of fieldln the specimencorrespond ng to a certan on ihe fineadjustment settingofthe fineadjustment js extraordnar y shallow(more pronouncedwjth high-powerobjectves than with lower powers,more wjth high-qualiiyoptcs than when these are less good).This is ihe reasonwhy n mcroscopicwork ihe fine adjusimentis continuouslyrnoved up and down n order to coverihe entjredepth of the specirnen. Whenthe f ne adjustmentfails lvlost microscopes{excepi theSTANDARD KF)have a I rnited f ne adjusidrentrange, and it may happentha! ure upperor ower siop is reachedduring observation.A gtancear ihe side ot the rackand p nionw confirmthis (Fig.2a). tn ihis case,rotate ihe fine adjustmenrknob uirit ihe i.dex I ne ies rougrrlyin beiweenthe iwo orhers(Fig. 2b). Then refocus wlthihe coa.seadjustrneni, and yo! wittagain have s!fficienr L.rpperand lowermargin for fine adlusrmenr. Two rnportantpoinis: When !s ng optcal instrurnents,such as a microscopeor a lelescopetwo thingsare ol greatimportance and sho! d be
' ) rhe e/e 'L ould be e "'ed when lookinginlo t5'e/eo e
2) the eye should be kept ai a certaindisiance from the
re axedaccommodation Beglnnersoften nake ihe mistakeoffocusing the microscope as if the magnified mage ihey want to view were ocated very closeto the eye,as c ose as a poslagestamp whlch we want 10 studyin deia for instance.Th s nearsetung ot the eye (= accorhmodaion) is howeverextreme y tiringll rnain tainedfor a longeriime. We thereforerecommend working wiih perfectLyre axed eyes. At the beginningthls is done more easilylf we imagne that we are viewingthe mageas "into if it were at inf nity.Thus do not look ihe m croscope but thro!gh the microscope". lf you are not quile ceriajn as io whetheryou are really workingwith re axedeyes, make ihe fo owingtesl: Firstfocus the specmen. Ihen hold a rnirror!n front of your secondeye, which is not lookinginto ihe rnicroscope,so thai yo! can look olt of the windowor ai an objectal the other ero ol Lheroon W-e_ you L" - Io rqodtis e.e "- _ e fal object,the other eye mlst at the sameume see ihe micro- scopc lmagewlih rnaximumdef niiion.lf this is the case,you are slre that you can work n an absolutely relaxed'siate. Alwaysremember lhat the microscopecan be refoc!sedwith the a d of the fine adjusimentand can tlrusreLeve your eye of accommodationlt is characteristicof the experlenced rncroscopjst that he keepsplayLng w th the fine adjustment. ln ihi6 way hls eyeslvl neverget t red,or acila y h!t1
and good coniact"of the pup s The secondimportant po nt is the correctdlslance of the eye frornthe eyeplece.Shou d ihe eye be broughias closeto the eyepieceas posslbe as w th a rnagnifier?Genera y not, becausethe correct eye point as this dsiance is ca ed, variesaccording to the eyepece used.The best procedure s to move in from a few inches distanceuntiL the field of view is widest and sharply defined. In thi€ ca€e, all the lighi rays emerging from the microscope find their way into the eye. As is evident from Fig. 3, this is possible orly if the eye pupil is brought very exactly to the point of constringenceof the m'croscopes light rays, whic$ has therefore been named the exii pupil of the microscope . When you have acquired a cei.ain amount of pract ce in the use of a miiroscope, you will automatically find and keep the right distance from the
coirecr dr3tonceof 6yerrom eyepece
Eyepuprr redu.es rhe fierd. Let !s iow pa!se to considerwhere you may slill enco!nter dis.ull. in.o.rri-5-dq.ai,rcn(e rr. er '.o op-
is Laterallyreversed You have,of colrrse,noiiced that the microscopicmage s upsde down. f yo! displacethe specrnen 1c' one side,the lmaqemoves in the opposited recuon.You w ge1accus- tonredto this so quckly ihat yo! w soon forget the fact
You c€nnotobtain a sha-pimage Lt may happenihat you cannot get a sharp inrageof the specimenin spile of everyihng yo! do. n th s case,the t ne "d.J"_ne_Lc erLe red ed " stoor'ered. -e prge 9l o tr - ob e r.e ro cFe, rhe ,oeuin"n be'o_e" s'" o -rage s obtained.Then the cover g ass is eilher far loo thick,or yo! have lnadvertenllyplaced the specrnen wrong sjde up on ihe slage.Whatever the causemay be, lhe spr ng mount of your ZE SS objectve preventsdamage to lhe specmen.
Ovalfleldof v ew? It s noi only the begliner who suddeny finds that the f eld of v ew is not rolnd. In thiscase, e therthe obtecuvehas not fu y engagedits cl ck siop post on or the fi ter ho de. below the condenseror n fronl of the I lnrinatoris not swungin
'Mouches!o a.tes' Another phenomenonmay at first be nexpicabe to the beginner Specmens with a wide clear backgroundmay show strange,irregular patches, shaped rather lke worrns, whichremain stationary when the eye ls f xed,but which start rnovingwhen the v ewlngd fection s changed.These are ihe shadowsof inhomogeneites in the aqueoushurnor ofihe eye, wh ch becomevisible whenever the ex t pupil of lhe m cfo scope becomesve'./ smell: jf the condenseriris is closed down too far or f a hgh-powereyepece s !sed. (lncden ta y the sameshadows can be seen I you look at a un torm sky. However,ihe sky musi be bright becauseony then do the eye p!pils closefar enough.)
Ob lque! urninaton A strangeeffecl may occur when the imagein the mlcro- scope adopts a ceriainreief appearingaLmost to have a 'shlft thlrd dirnenson, and focusng seems to ihe image. Th s a ways happensif you have ob que I um naton". A glen.e into the tube wth the eyepece removedwii! show
12 you that the back lens of the objectve is not uniformy illumnated. Th s can be coffectedby readjusringrhe minor
Keepboth eyes open I you are not uckyenolgh to havea b noculartube from the very start (so that you can look into the m croscopewiih both eyesl, observationwiih ony one eye wiil at first be somewhatdffc!lt. We therefore advse €verybodywho ihrows rnorethan lust an oc.aslonalg ance nto the m cro scooeto keep his s€condeye open fronrthe very start.The imageseen by the secondeye, whjch is naturallysuperim- posed on the mic.oscopc mage will be found dlsiurbig on y in tlre beg nn ng and w I later be subconscous y s!p- pressed.ln order not to hafm the unlsed eye by lhis fornr of discornection, the eyesshou d be Lsedalternately.
A. accessor: theere s(reen Cosing one eye for a longerperiod of lme s very lirjng. Ll you canrot get acclsiomed io leav.g ihe second eye openand if the acq!is i on of a binoclrlartube s notpossibe a cardboardscreen may be slpped onio the tube n slch a manier that the secondeye is covered.Some peoplemake h",ro.\ bi.o .p-o1 (F g. 4, not commercally avalable).
Any ZE SS mjcroscopecan easily be converiedfof b nocular observations nceih s only meanschanging the lube
Wjlh or witho!t eyeglasses? Here a so the qlest on ariseshow eyeglasswearers sho! d /ork TtsFb". Lofe-p e gtor.- o and to lse specla eyepeces for eyeglasswearers. Blbber guardsavold scratchlng of the eyeglasses.
Lfthis is nct possb e, you sho! d firsrof a f nd olt wherher your eyegasses have spherica or toric enses.This can be detem ned very easly. Ho d you. glassesin front of yo! at arm's engih:the ensesw e rhef reduceor magntfyan objecton ihe iable.Then rotaie the glassesabour the center of the ensthrolgh wh ch you afe ook ns. lf the shapeof ihe ob 6. dob oi cha od d r'- g as 13 hason y splrericalpowert however, if lengthand width of the objectchange dur ng rotaiion,the g asseshave roric enses (Fig.5).ln the lattercase yo! will haveio keepthe g asses on or have auxliary lensesmade from your eyeglasspre- scription,which are then slippedonro the eyepieces(ensure correct orentaton!) lf your glasses have only sphertca enses/o. on p o.e Lher .o ^orl n rl Lhe . o. ope and no oss of sharpnesswill be observed.In rhs caseihe vjsual defect is compensatedby the adjustmeniof rhe 3. A brief glance behind the scenes After you have foclsed the specirnensrn accoroancewrth the explanationsln ihe precedingsection, you wi know that your m croscopedoes work. However,you w tl orty derive full sausfactonfrom it f yoLrknow at leastsomeihing about "gtance how it works. lt is naturalrhai ihts behind the only be a brief one, and lsers studythe iheory olthe m croscopejn morederatare referred to flrelterature indicaied on page59. As an opUca instrumenrrhe microscopeforms a magnjfied lmageof a sm€liobject. Howls af irnageformed? We mustfirst of all !ndersrandth€ formauonof an image,for whichwe shal choosea very primtive example.f you are standingin a dark corrdor and the keyholeof a door ts of approprate size you can clearlysee a pjciureof the window in that room proleciedon the opposiiewall of rhe coridor tpin hole canrera).You immediatelyrecognize what s happenlng:every point of ihe wlndow emils lighrrays n ali d rections.While most of them end on ihe wa s or ai the door, a few of them pass through the keyhole (Fig.6). Consequenily,a br ght part of the wtndowproduces a bright patchon the wail, ard a dark part producesa dark parch And becauseall ight paths can just as well be followedin ihe oppositedirect on, we may also say thai every po ft on thewaliofthecotridor(=unitof areal sees amoreorress brightspot of the windowthrough ihe keyhole(Fig. 7). The drawbackof such an mage is abovealits low brighi brightnessand €harpness nes€ whichwould inrmediatelybe increasedif the holewere larger.So lets makeit arger Throughihe biggerho e, a unil of wall area now sees" noi only ihe dark flower pot, blt aso ihe bright window pane beside t (Fig.81. As a reslli, ihis !nt is now neitherdark nor brlght,bLrt gray - the dividingline betweenthe pot and the pane is now blured in ihe imageon the wal . In other words,thetwopointsatadsianced are no onger resolved' l. e., ihey can no longerbe recognjzedseparately. Nalurally, ihe b ggerthe holeihe moreblurred s the lmage.This is due to the fact that in a p n-hole camera ihe rectilinear propa- gationof light does not makea pointcorrespond to another t5 l l a v/t I I 16 point, but io an area whjch increases in size, the bigger the opening is (Fig. 9). A lensis the solution A lens has ihe extrernelyvaluable properiy of refraciing ihe light, thus counteracting ihe principle of the rectilinear propagation of lighi (Fis. 10). In an ideal case it therefore becomes po€sible to combine both fequirements,viz., bright image due to large aperiure and imaging of a point as a true point (the latier with cedain restrictions; see page 37). L There s onlyone plane As an example,let us considera camerataking a pictureof a plant.The lghi travelsfrorn any poini on the plant jn al directons (Fig.1l). A part of the llght rays is lnterceptedby theobjective lens, refracted and recombined in the filmplane lf behindthe plantthere is, for exampe, a window,then the ray6 emanatingfrom there do not recombine exactly in the film plane but in front ol it, aid on the filrna circle of dia- meterI is inaged insteadot the point:the windowis out ot "Focuslng focus(Fig.12). can be achievedonly by a relaiive displacemenibetween ens and film plane.This shows that !n an imageformlng system a given objectp ane is a wavs .o're dteoro " d. r' le planeo'"Fa p Inagel Howbig will the imagebe? Apart from the locaiion of the irnage pLane,however, ihe objeciimage rato is likewisefixed. This raiio is normally exactlyidenticalwlih ihe re at onshipbetween objectdistance (objeci-lens d stance) and lmase distance (lens'image distance)(Fis. 13). The rorrnula *""a ,r'r" -"d, 4-i Slnceihe imagedistance b in the camerais smaller,usually muchsmaller than the objectdistance a, this opticaljnetru- meni of coursef!rnishes reduced pictures. We builda modelmicroscoPe We now know enoughio try and build a rnode m croscope frorn the cornponenis we have Learntto understand - a concretemodel if possible,or otheMlsean irnaginary,'one The microscopeis io magnify;therefore the cameraglvjng and f rst imageJorming system reduced mages is not suitabe as a basis for our model microscope.We are ooki.g fo. an opiica inslrurnentwhich providesenlarged images, i. e., n which the imageis mlch further away from lhe lens than the object SLrchan instru- ment is folnd in the famiiar slide projector(Flg 14r A= object,ihe slidei B = screen imagei L = proiection ensl Slnce in this case the object the slide,does not ernitLight itself,it mlst be i luminatedfrom behind Let us iherefore placea Lampbehind lt (FlS.15). However, the side js much largerthan the lampf arneniand ihe lers diameter,so ihat only the ighi rays passingthrolgh ihe cenier of the slide 18 I reachthe lens.Besult: the f e d s noi fltly umjnaied,ihe marginalzones remaindark - Thts can be remediedby a ight source which is at leasi as arge as ihe stde. An iliuminatedground-glass screen is suirabe for this purpose. The lght strikingihs screen is deflecredin a direcitons (Flg.16)- Such a screenalso a ows ihe jght raystransmiited by the peripheralparts of rhe slide to enler rhe tens.How ever,thi6 is ratheruneconom cal becausethe majorparr of ihe ight rays s defecied in direcrionsin whch we do noi need any light. A better solution is a tens (here called condenser)wh ch directsall inctdentrays exctusivey to the pointwhere they are requtred:to ihe projectionens(Fig.j7). We thls havethe faci ihat in the firsi placethe lightsource s irnagedby the condenserin the projecrionlens, white in the secondpace an rnageof ihe stide is formed by rhe projeciionlens on the screen:a so catled nrertockng lghr path such as we find in the microscopewth the so,ca ed Kdhlermethod of ill!m nation(see page 451. Thesecond maqeJo m ng system Let Ls now turn to the enlarged mage on the projection screen.How big we see this magedepends qlire decisivey on lhe dstance from whichwe vew ir. f we move hatfway up to the screen,we seethe imageexactly rwice as largeas before,i. e., wiih 2x rnagnficaton". t we draw a line,as shownin Frg.18, frorn one edse of the picture1o the eye of ihe obseruerand from thereback io ihe other edse, an angle(c) is subtended,which s the angleof view.The rnagnificationis dererrninedsolety by the magnl tude of th s angle.For normalslide projecron we shall nor move too c ose to the screen,because the unsharpnessof ihe imase(e 9., due to srain)wo! d be disturbing.For our experiments we therefore Lrse a microscopc specimen nsieadof a slide,for exampe a f ee, which,contrary to the side reveas ever new deta s even Lrnderconsiderable magnflcauon. n the ifterest of high magnificarionwe may, of course,move !p as cose as possibleto rhe rnage,but eventua y ou. own shadow w ll nierfere We therefore look ai tlrepicture from the backof the lransparentpaper screen. The fact thai ihe picture is thus aierally reversed need not worry us.The next siep: we exchangethe paperscreen for a ground-glassscreen and, n addition,use a magnifierfor vlewingtlre picture.A magnifiercan increasethe ange of view and ihus the magnlflcatonconslderably. An object (Fig. 19) viewed once with the naked eye from a reading distance" of 10 ins. and then through a magnifier is seen under considerablyhigher magnificationdue to the refraction of rays. But ihe magnifier also magnifies rhe texture of the groundglass so that itbecomesdisturb'ng (Figs.20 and 20a), and we therefore investigate whai happens if we simpty remove rheground glass. The imagewitt stitt exist in rhe same plane in the form of a so-called aertal image. Through our magnifier this aerial irnage will, however, be visibte onty in the direction of the illuminated projection lens and around ir there will be darkness, because ihe rays miss the magntfier. This is illustrated in Fig.21. ln rhis case, rhe remedy consists once more in the use of a lens in exactly the pjane in which the image 's formed. Just as a magnifier placed directty on writing does noi magnify the letiers, a lens located exacity or almost exactly in the image plane has no effeci on image 22 sharpness, bui con.r'oLresto bnghteningthe ,rd,g,ra,zones or rnenatd bac€userhe rays passingtfese zu,re6are rrans -nill-edInio th€ (T,9.22). eye fhese trerdtenses, , as r!ey are ca eo. are rhe.ero.e becer tha- Srourd ana ,n @orflon,irey Srass"s do not.inlroduce vo-btesome g.ain irlo rtr€g€ tte trn other words we have here a case which:s very srnlar lo rhe one nenr,onedabove, wne-e we had praced a tens as a condenserbetween ,anp and s,ioe_ tikewise as a ground-gJasssubsiitute). Tle- effrciency ol our noder p.oveo by rhe ." fte ro ow:rs p€se. rhese are 1ol mr.roscop;cli-*:l-: iTa9es senurne b.t inages ac.L€JJyproduced by a 2r2 proj-ctor two and soectacteterses. One spFctac,erens was usao as a tietd iers, lhe orher as ar eyetens.rle eye was (see the corespondinsrLtudraudns represented by a reftex camera wjth its jens focused at inf'nity. ihs swhairheitermedrate maqe ookrk. a.c ob3eiy:dsr.!qE a m.sn fer tF n 20) remore.r(Fiq,r)rrra srah i6s d epp*E opina res!ts,re prodrcedoi ywhen!3 ns 24 :rom the nrodeltothe Veryfew changesare reqLrtredfor the stepfrorn thjs projecior wth magniie.vewng to the rncroscope. Thus rhe image d stanceol severa Jeet s ioo awkward.We sholld atso ike to have both optcal sysiems.v:.. ihe one caLsng rhe firsr magntcaton(the objectve)and ihe one maqnfyngthis rmagea secondt me combned n one ns!r!neni. The ent re seiup can be kept mlch sma er by appropraresee.iion of b,l.!.w6- e. e-do,o. .-o - ot.- o.op" aroLrndth s ight pathand keep in m nd thai the path oi rays s us!aly fcded' for greaterconvenieice aidwehave a rea micfoscope{Fs.23) o @ Theprinciple in a nutshe The principleof ihe microscopenray ihus be sumrnarzed as The microscopeobject ve first forrns n the tlbe a magnfled aeral image( nlerrnediateimage) of the specimeni uminated from below by the lamp and the condenser.However, this image is not intercepted,bur viewed under magnification througha magiifier(c€lled eyepiece) Thedesign of ihe lighipath n the foregong paragraphswe have trted to expa n ihe basicfacts of the mlcroscopeas c eary as possible;in doing so, however,we would give no exaci descripton.tn ihe fot- lowing,ihe readerwho s morefamilar with opticalsubjects will find instructionsfor the design of a rnicroscopetight path.In prjnciple,th s is rea y qutteeasy. First magejormingsystem Fig. 24 shows the objective Ob with rhe two foc F I and F 2. Thespec men of sizeA B is iocaredsltghtty ouiside the foca lengih. From A two rays are traced: one paraltelto rhe opi cal axisthrolgh ihe focus F 2, and ihe oiherihrough the lens center, which is therefore noi refracred.The iniersection of both rays g ves the irnageof A, A'j in the same manner, B' resultsas the imageof B. The imageA'B'rhus obtained ls enlarged reai (i.e., it can be intercepted)ano reverseo. Thiscorresponds io ihe paih of raysin the m croscopeup to theflrst aeria inrage. ... andsecond magn fcaton n anothercase [Fjs. 25) we aranse a lens Ok n such a mannerin relaiiorto an objectB'A' thai the lalreris shifted slighilyoff the focaLpoint n the directionof the lens. The designofthe pathof raysis analogous:After beingrefracted, the paralel ray passesihrough the focal poinl,wh le the ray ihroughthe ens cenierts not deflected.Sill, the two rays do nor intersectr only their extensions ro ihe rear have a po nt of intersecuonai B" or A".The mageB"A" is likewise enlarged,but erectand virtual,i. e., t cannotbe intercepted becausethe lighirays do noi reallyintersect at the pointsB" and A". - Thisis the lightpath of a magnifierand thus a so ofamcroscopeeyepiece Lfwe connecithese two lightpaths in sucha mannerthai the mage formed by Ob s at the same iime the objeci for Oc, then we havethe two-stagelmage formai on of a compound microscope(Fig 26). 26 Geometricaloptics, wave optics We must confess thai with the geometric optical light parh discusEedup to now we have investigated only one aspect of image formation in ihe microscope.Another is the wave- optical aspect which is, however, beyond the scope of ihis booklet. A concise description, in which this second aspect is also laken into account, will be found, for example, in "How to use a Microscope by W- G. Hariley, American edition edited by John L Lee and Bernard Friedmann,The Natural History Press, Garden City, New York lg n. 2T 4. Practical hints After a brief exc!rsion nio the theory of the rnicroscope therestl remaina few facts the understandjngofwhich s ot lmportancefor practicalwork. Objective The objeciive is engraved wth a value (Fig.27) which Engravingand other characterisi cs ndicatesfar mofe than initia magnlfica1!onatone. Ler us repeatihe exampe on page 5 40/0.65 I60/0.17 means, nilial magnfcation 40x (rnulip ed by eyepiece magnlficaiion= total nragnfcationof microscope);numer ca aperture0.6s (for explanaton, see page 37Jr for use wth m croscopeswith a mechancal iube ength of 160mrn rl".qe (d *d-.e oe.^ae' ob,.'r - cnd e.eo.ce s". 9 face)and w th a coverg ass of 0.171 0 01 mm thckness. nsteadof 10.17the engravng may also read / = insensl- tive to arger deviallonsof the cove.g ass ihickness /0 = correctedfor"zerocover-q assthickness .e. foruncovered Objeciiveswitho!t any furthe. engravng are ihe wldely !sed achrornats.Other engravedabbreviaiions frequently Plan = P anachrornati.e flat-fied achromat Neofuar = high'aperiure objective (see pase 37) wiih mprovedcolor rendition,hlgh contrasi, b!t w thouif at field. Planapo= P anapochrornatrobjectjve of optm!m coor correcton, maximlm€pertlre and f at fie d. LJlt.afuar= specia objective for Lrtrav olet rl1croscopy cotrectedfor waveengths between 230 and 700nrn. n Eppan = fat-feld objectivefo. vertica brght'fed iu- Epplan HD - flat'fied objectivefor vertca brght fLed and dark-feld iliuminaton (in conj!ncto. with concentrc ref ectingor .ef.actingcondenser). POL = strainfree objectivefor polarzed ght m croscopy. Ph 2 = phasecontrasi objeciivefor diaphragm2 of the pnaseconrrasr conoensers. m. l. = objectivew th iris diaphfagma so su ted for trans rntted darkJied I urnination. Oel 515)black ring .9 .t 333)whiie r ng Glyz glycerin 455) orange rins E 7401yel ow rlnq E Kon. - hish aperture oblectve with correctionco ar io allowfor differenicoveFg ass thicknesses(see pase 43). n addition,colored r.gs near the knurled rng faciliiate recogniiionof ihe different magnificatjonsiblack - 1x, brown 2.5, Formerly,changlng objectives was rather inconvenieni, becausethe tube had to be lfted beforeiurning ihe nose pece, and every objectivehad io be focused separately. Today, tl.e objectives are parfocal . Th s means thai they are optjcaly and mechancaly desgned so thatihe dlsiance between thd specrnen and the aeria lmage is always constant.Slight refocusingwith the aid of ihe fine adjust nrent is therefore s!fficient to resiore critical sharpness of the image aftei changingfrom one objectiveto another, while the coarse adjustmentneed no longer be operated. This featureis particularyvaluable when working with oii immersion,in whichcase the depthof focus is so smallthat focusing wlthout padoca ized objeciives usually presents considerabledifflcuity. (Only the especiailyong Planachro mat 1 objecuve ls not parfoca ). andihelr prerequielies It should,however, be notedthat a few conduons musi be satsfied to maintainthe parfoca settingof the objectives: "CARL jn the first place,on y eyepiecesengraved ZEISS' must be used (oiher eyep eces are des gned for a different locaiionof the aerlal jmage);secondly, the observerm!st have normalvsion (or the visua defect musi be cotrected by eyegasses or a focuslngeyelens)i and finally,coreci tube engrhis essentia(with brnoculariubes,identica vatues mlst be set at the eyepiecetubes and the whee between the eyepieces.See page 32.). Normalmedium to h gh-powerobjectives have a retatively shortworking d stanceand may knockagainst the specirnen unlessspec al care is taken. n order to prevenidamage ro the specinren or the front lens of the objective, these objectives therefore have a built'in spring so thar the objective as a whole wi y eld io pressure.Sorne objectives, paric!lary those for oil irnmersion,can be lockedin the retractedpositon by c ockwise rolaion. Ths is io avoid soilingof the specrnen wth o lwhenihe nosepece isturned. A very important h ni regarding h gh dry objecrives wt I be fo!nd on page39 (correctioncollar). Wh le a few year6 ago il was necessary to use differefl A singletype for all objectves types of eyep ece for olv power and high-power objectives (Huygensand compensatinseyepieces), ZEISS m croscope objectives are now generaliy used with compensaung ocuars. Consequenty eyepeces need no longer be ex changedwhen chafs ns over from ofe objectiveto anoiher. ln spte of thls, two classes of eyepieceare sit being manufactured.While ihe s mple C+ype eyepieces are primarly usedln conj!ncion with achromatc objeciivesihe Kpl (compensatingf at f eld) eyepeces afe intendedfor the other objectivesof higher correction blt rnay,of course, also be conrbinedwiih achromats. Eyeplecesfor spectaclewearers The mosl widey used eyepiecemagnfcatons are 8x and 12.5r. Fg. 28 shoyTsa lsi of all resuliantmagnlficauone which can be oblainedwlth our mjcroscopeobjectves and Particuar meniionshould be madeofthe eyepiecesspecially designedfor spectaclewearers. As was mentioieda ready on page 10,ihe exit pup of the microscopeand the eye pupilmust cojncide if ihe entirefie d of v ew isio be covefed. Wiih normaleyepieces, lhe distancebetween ihe edge of ihe eyepieceand the exit pupil (eye point) is so short that eyeglassescannot bekept on ifthetwopupilsareto coincide. The hgh eye polnt of the specieleyepieces for eyegass wearersrnakes this possibleeven when the eyeglassesare r0t obledve,3x ayepiece= not removed.The rubber guardssupplied with these eye- piecesprevent scratching of the spectaclelenses. - fthese 30 (1600) 1250 1000 600 630 500 100 320 250 200 160 125 t00 60 OJ 50 10 32 25 20 125 t0 0 63 5 specal typesot eyepieceare usedby obseruefsnor wear ig g asses,care mLr6t be taken1o keep the eyesat an adequaie distancefrom the eyepieces.lf necessary,flnd f9 sre correcr dlstancecan be facilitatedby stippingeyecups onto rhe Field-of-vew nurnber Apart from ts in t al magnfrcat on, an eyepece s chafac- terized by rs fied ofvew nlmber. Wiih the ad of ihs number li is easy io calcuate rhe diameterof ihe field coveredin the specimenplane,becauserhisis(in m imerers) eq!a to the fleldof-vtew number djvjded by the ini!al rnagnificationof the objedive.The fletd-ofview numDersor ihe mostfrequently used eyep ece types are givenbelowj 5x 20 6.3r 18 8X 16 8)< 18 10 )< 16 l0 r t6 12.5.1 12.5' ( Kpl w de-argleeyep ece 12.5).. 18 16 I 10 25X 6.3 Exanrpleia 10). objectivein combnation wth a l2.Sx eye- p ece perrnitsa fjeld of 1.25mm diameterto llre specmen plane. The m croscopecan easiiybe equippedfor binocularobser vaiion by exchangng ihe ordinaryiube for a binoculartube. Theeyeplece sleeves of ihlsi!be canbe dispaced in relarion io each other on a slde to allow for adaptarionto rhe indv dlal interpup arydistanceofthe observer.lfyouknow your interpupilary d siance,yoLr may sel ihis value on the whee jn the cenierby drawingthe tubesapart. f you do not kfow this value,look into the eyepiecesand vary ihe distancebeiween the tubes uniil the fields of view seen by botheyes co ncideexact y. Thevaluetndicated on theceniral 32 wheel rnust then also be set on each of the eyepiece sleeves.This restoresthe co rrect .nechan ical tu be lengthwhich has been changed by the inierpupillary disiance setting, at the same time ensuring that the objectjves are padocal (see page29). lf you shouldhave the impressionihat your visualacu ty is different in the two eyepieces, adjusi the eyepiece s eeve onihepoor side untilbotheyes seethesame, sharply defined Because ol its great imporiance we want to repeat once more that the correct pos uon of the condenser is always at iis upper stop; it is lowered slightly only for the Kohler methodof illuminailon{see page 45). I urninatinglarge fields The requirernentsmade of a condenserare partly contra dictory, because it is Jrot only ihe large object fields of the low-power objectives which have to be illumjnaied, but also the pupils of high-power sy€iems of high aperiure. Some condensers are therefore equipped with a swing out froni lens: as soon as a owpower objeciive reproduces more thanthe condenserilluminates, the condenserlris is opened fu y and the front ens swung out. Condensers without a swing-oullens usua ly allowthe front ens to be unscrewedl if eventhis is irnpossibe, then lt is necessaryto changethe Obliqueillum nation For phase-contrastjll!mination a specialcondenser is used, the design and operation of which are described in the per tinent instrucuonmanual. These phase condensers are also su ted 'o so-ca ed obliquei u-rinalon . and for dark-feld illuminaton.After the microscopehas been adjustedunder brlght-feldillumination, turn the turrei plate "l" frorn its click-stopposition (iris,i.e., brightfield) to the eft or right, and you wlll immediaielynotice the three dimensionaleffeci which is typical of obliquejlumination. Shortly before ihe field of view is completely obscured as you keep turningthe p ate, one-sideddarkjield illumination I Nofrna i.e., darkjie d ilumiraton from all sdes can be obtaned with ow and medum powerobjectves by choosns ann!lar d aphragmsof ihe phase'contfastcondenser wh ch, d!e io the r size,remaln outside the oblectiveapedlret this can eas y be checkedby lookng into the tlbe after having removeo rne eyep ece. Forfudherdetals on speciadarkJ e d condensers,reference s madeto the broch!reG 4C-165. 5. Care and cleaning of the microscope An instrurneniwhlch has to sat sfy the most exacring requ re, ments regardlngrnechantca and opticalprecsion naturay demandsa certainamount of care. EnemyNo. 1: dust Dust is practcally everywhereand may becornea solrce of trolbe wherever- as n the nricroscope- .fechanical parts are mov ng in precisionguides. When the tracksare clrty they need washing and regreasing whlch shoutd pfefe.ablybe done by the manufacrurer. Dust on op!cal elernentswill degradethe imagequal ty to a greater or esser exteni. lt is true rhai a surfaces exposed to dlst are also easiy accessb e for c eanng, but no g ass 'cleaning', surJaceis irnprovedby so that ihe best advice is alwaysio avoid exposLreof the m croscopeto dust by coveringit up with a hoodwhef not n lse or by keepingit in a cablner.Specral care should be rakenro ensurethat the tlbes of the m croscopeare always cosed ether by an eyepece or a dustp ug. D rty objectve fro.i e.s While dlst partlces on the eyeplecew only g ve r se to paichesin ihe image - which noi every observerfind6 d stu.bng - a dirty objectjvefroni lens may hopelessly reducethe sharpnessof the rnage,or at leastits contrast. DLreto the faci that jt ls closeto the specmen and possibly to ihe lmmersjonoil of ihe coverglass,but partculary to the handoperat ng the nosepiece,the front lensof the objective - -l is .p.ri"l d.9e- o" gerr.9 soled. t.- e Snres- fingerprintmay have grave consequences(Figs.29,30J. Beforesiariing important work t s advsab e particuarly if the microscopels not used by one observera one - to lnscrew every oblectve and check t carefuy wiih the ad of a magnifier.Drt is eas y re.ognzed f the objeciiveis held so that the imageof a lght solrce is reflectedfrom its planesurface. lnthe caseof objectveswhichhavea concave fronl ens,a dlfferentapproach s indicated,viz., io examjne a.re6n objecrivefroni lens siheftur ihe surfaceof the front lensfrom the screwthread side. The prerequi3ie forb. tiad masas(ropl remaininglens e ernentscan a so be exarninedeas ly and "start Evena;nsefpl rt oi rhef.ont Lens any fauls (cracks, ng of the cenrenOdetected with- w rru n rhe contrud (bordh). 35 Hintson rroube-shoot ng Shouldstr!ctures be foufd inthe imagewhichare suspected of belngextraneous to the speclmen,the fa!lt nay be traced lf the troube can be ellminatedby s ight adjustmentof the condenser,the causemust be soughiin ihe bulbof the amp, ihe lamp condenseror the flter in front of t. Flowever,it adjustrnentof the condenserdoes not producea.y result, the nexistep s to turnthefocusing adjlrstment, wh ich should eiirninatea I taultsdue io soilingof the condenserfront ens or the specimen.lf thls does not leadanywhere, slight v turn f rst the objeciiveand then ihe eyepece, and yoLrwill lmrne- diately notice in which case ihe foreign body lollows ihe Dust partc es are mos! cleary seenwhen the apertlre stop has been fuLLyclosed, because in ihis case lhe depth of focus is al its greatest, Cleaning,but how? ln alrnostall cases t will be sufficientto cleanthe ouler lens faces wlth the a d of a srease'free brush (jf necessarv,wash n ethe. first) or wlth a freqlentv washed'absolutelv dust- ffee inenrag and dist ed water,produc€d easily bv breath- ing uponthe surfaceto be c eaned lf an organc solvenicannot be dlspensedwith,t is advlsabe ro usevery littleether or benzinejnstead of water,but never alcoho which mght desiroy the cernent between ens Etheris usua ypreferredbecause it evaporatesrnostqulckLv and any harrnfuleffect js thus less ikely Finaly, residLres are aLwaysremoved with water as descrlbed above. Shouldcornpressed air be ava ablefor cleaning,be sure to use a f lter of cotton wool. lf the alr in the work roorn conlin!ousy has a relative hurnidityof more than 600/0certa n precautionsshould be takento avoldfungus growih on the opticale ernents.Do not keep micfoscopesunder plast c covers,do not siore them!n cabineis,but ensuregood ventilalion,i necessarywith the aid of a fan. In a partjculaiyhumid clrnate it s advisableto keep opncalparis n pefectly a rproof containersprovided with a desscant or ln whjch for ifstance,a amp and a fan c rcllate a r of 40 +0 o C (100-1 20 o F) - Our agents have specal instru.ions and w ll be p €asedto assistyou. 6. Numericalaperture, useful magnification and limit of resolution ihegalrinthesde To underslandthis questionbetter, lei us now think agan oi the prolector and the projecied sl!de described n sectjon3. At first we obserueihe landscapeon ihe screen from far behnd the projector.ln this case,magnification js still low, becausethe angle of vew is qute small. As we move closerto the screen,rnagfification increases and we beglnto recognjzedeiajs whlch had originallyescaped our ailention.But fina y a point s reachedwhere higher magnl- fication (= movns closer) does noi gve better resuts. lnsteadol singe eaveson the trees we then only see the skuctureof ihe color film,an acc!m!laljonof dye particles, the so-calledgrain. By furtherreduc ng ihe viewingdistance we could,of course,furthef ncreasemagnificarion but thts wou d be so ca ed emptymagnificat on", i.e., rnagffication whichdoes noi revea any newdeial . In the m cfoscope,the stuaiion s ihe same.As we have to a diffracuongrain seen,the pfolectedirnage coresponds to ihe aerialimage formedby the m croscopeobjectve in the tube.iust as wlth ihe oro_ecrd(lda haF r- "l o " iTi !o-.Le pro_e.leo "usef!l aerial niage where rnagnificationends and "empty magnificaton" b€g ns. t is trle that the microscopicimage wl not exhibiiany actualgrain struciure, blt here aLsowe may speakof grain , becausethere s no opiica imagein which ai objectpoint ls reallyreproduced as a poini (i.e with no rneaslrabled ameter). Due to ihe phenomenaof diffracton, an objecipoini ls a ways iransformedjnto a sma disk n the mage. i is these so ca ed Airy disks which 'nrage forrnthe gra n". Thjs type of gra n can be found in any optca image f the magnfcationis high enough.lt ls obviols that imagedeta s will no onger be ceary recog n zed as soon as they reachthe size of lhe Airy dlsk - lust as we wefe unableto recognlzethe eavesin the s ide when theywere as smallas the dye gra n. Another example:Let us examinea mosaic frorn a long distance(i. e., with low rnagnification).As we move closer we recognizemore details.As soon as we beginio recog- nize individualelemenis of ihe mosaic,however, we pass "u€eful the limjtof magnificaiion.ln this case,the elements of ihe mosaiccorrespond to the Airy dlsks maklngup the aerial!mage. Bewareof pseudostructuresl lf we vlew a side or a mosaic with higher than lsefu magnficauon,no harm is actuallydone, becalse we know very well that the above rneniionedgra n structure does not be ong to the object, for insiance the landscape. Flowever, in microscopythe siluationis dlfferent: so ca ed empty magnificationrnay show struclures in the imagewhlch do not exjstjn the specrnen. But sincewe do no1know ihe speci- men,there is a dangerof etroneo!sconclusions. And n fact there are exarnplesof th ngs being dlscussedin sclenufic Iteraturewhjch did not exjsl ln realitybut eveniually revealed thenrsevesio be a klndof nragegrain, ie ther€sutot d lfraclionphenomena. Whatis the hjghesipower li followsfrom the aboveremarks that we shouldknow the pe fo,na e in.of .henco""ooeob.e .e.^euse.i.' ihe point at whch thelr usefulmagnlfcaion ends. This ls jndicatedby a valueengraved on everyobjective and cal!ed 'numercal apedure'. The tremendols importanceof thls characteristicwl rnmediatelybe recognized lt we say that ihe numerlcalapertufe is lhe decidingfactor \ryhen deier.nin- ing the slze of ihe A ry disksln ihe m croscopicimage, i e , "screen the size of ihe . There is a defjnte relationshp beiween the nume.ical aperture and ihe s ze of the A ry disk fo'the no'rent.yo 'e"o only-ro^ he'olo$-S: The tola magnificaiionof the microscope(i. e., lhe magnl- fication of the objective multip ied by ihat of the eyepiece m!ltiplied by any tr.rbe or OPTOVAF factor) should noi exceedone thousandi rnesthe numericalaperture. Nlicroscopsts who have partc!lafly good vsual acuty shoud even use fjve hundredlimes ihe numericalaperture as a limt. Exceptons to this r!]lemay be allowe.lin orderio facilitatemeasuring and countingwork 38 We st owe yo! an expanatoi of th€ term iumercal aperiure. F g 31 g ves a dlagranrmaticv ew of an objeciive front ens and specmen. The ange u' subtendedby rhe optical axs and the outefrnostrays st covered by the objectives a measureof the apedureof the objectve;it is the apertureangle. However, the magntude of ih s angteis nol ndicaled n clegreesbut n the form of a s ne valle, ihat is a n!mericalval!e. This expa ns the crig n ''n!meri.a of the ie.m apedure . t s the s ie of rhe haf apertureangte rnutip led by ihe fefractivende)i n of rhe mediurnft ing the spacebetvr'een the cover giassand the froni lens. NLrmeicaaperi!re = n sin!' s. -"- " a rendr - o-. o- I - Ta. o€ "q- reo when dry oblectlvesafe used. mmersionoil, which in the case of an o imnrersor obtectve f s ihe space between the cover qlassand the froit ens has a refractivendex of 1.515,a.d ii s obvous that s!ch an objectivenrakes it possb e to achleve a consderab e gain in nurnerical *..mpned ! th a dry obF.r L: i ?:lj ta 39 Whatjs the resolvinglirnit? Wiih ihe aid of ihe numericalaperiure we can also compute ihe limjt of resolution or maximlm resolving power. By ihis we u_de's.a-dlha smdlle5t"epa-dtion - desg-a.ad o which two skuct!ral elernents,e.9., two adjacentflagella, may have in o.der to be irnaged as two separate elemenis And howls it determined? lf n. a."bj and n. a. condare the nlrmericaapertures of objec- tve and condenserand l- is ihe ,ravelengrhof ihe tightused for observation(in 1/1000mm or microns),then ihe srna est resovabLe separaiion between two object poinis (likewjse n. a. obt + n. a. oond Example: Using a green filter, the waveength is 0-55 p; assumingthe objectiveused io havean apertureof 1.25,and the condenserone of 0-Srthen d = 0.25rr, i.e., the fourth part of a thousandth of a millimeter. - However, slructural elements which are just reso ved are not reproduced wjth f!llfidelity. 4A 7. Oil immersion Whatis oil immersion? Oil immerslonobjecuves are objectiveswhich are lsed w th immersion oil (instead of alr) between objeciive froni ens and cover glass.As was explainedin the previoussection, this method eads to a gain in numericaaperture. For the same image scale, an oil immersionobjective is aLways superiortoa comparabe dry objectveas regardscorrection. Thereare, o; course,cases which do not permilihe applica- ion of the ojl immersiontech. q!e. 100x objectivesafe a ways oil jmrnerslonobjectives. The usershould, if possb e, ernployon y the type of imrner correctapplication of the rightoil sion oil s!pplied by the objectivemanlfact!rer, because not only the refractiveindex must havea certainvalue, bui also the dispersionof the oil.The imrnerslon ojL slrpplied byZElSS s synthetic,non-fluorescing and non-resjningoil with a relractiveindex of 1.515and a dispersionof 43.96.The oiler sLrppliedby lrs with every jmrnersronobjective is designed io facilitateapplication of the oil to the cover glasswithoui air bubbes: turn oiler upsde down, wait until the air has risenand applya itte oil.The objectivernay be rnovedinto positionas usLra- In thls case,excessive oil and, posslby, bubbes are pushedaside due to the shortworking d stance. ln addition,objectives w th a planefront ens can be pushed upward in their spring rnount, locked by clockwise rotation and loweredveltcally into the oi only after the nosepiece hasbeenturned and engagedthenotch. Personal experience wll Facl-you $Lr^h o lFase L\ro nerhods i .ore _on lmmersionobjectives mostLy !f focusingthe speclmenwlth oj irnmersionshoud prove havea shorlworkingdistance impossibieremember the prevlouslyrnentioned possib lliy ihat ihe cover gla6€ may be too thick for the shori working distanceavailable or that the spacebetween ihe specimen and the cover glass,which is filled with rnountingmedium, maybe loo wide,whlch comes io the samething. lf your condenser has a numerical aperture higher than 0.9, alsorequire immersion ii is advisable also to immerse the condenser (if not wiih oll, at leasi with water) for use in combinaiionwith an oil immer- sion objective. For this pu|pose, a drop of oil is applied to the underside of the specimen slide and another one io the condenser front lens. This will effectively reduce ihe danger of bubbles forning when the condenser is immersed. - Wheiher both oil films are aciually free lrom bubbles can easily be determined by looking into the objective from the top afier having removed ihe eyepiece. The ceniering tele- scope of the phase contrasi equipment simplifies this quite Although ZEISS immersion oil is of the non resining iype, it should be removed at the end of ihe day by means of a clean rag of the kind used for cleaning opiical elements- Paidchromat63i/0.9o yih coreciron carrar Top: carefullyadlusied for coversrassthickness eolibaderiaand.raphylococc {cram s sia n) 42 8. The question of cover glasses Knowingthe high precs on requred for the producion oi " the f rst ens" ofthe objectve mlcroscopeobjectives which are to gjve first-rate mages we shoud not be surprlsedihat the cover glass,which s locatedbetween the specimenand the objeciivefront lens nay not be jlst any cover glass.We shouldnoi forget that s rnpleg ass p ates,that js planep ates,aLso have an optical power and ihat the op!cal power of ihe cover glass was taken lnto accountln the computaton and design of ihe objectve. Wih normal rnicroscopeobjectves, tlre cover glass is pari of the lmageforming system lt s a lens element (tho!gh wth infnite rad of c!Nature), which is locatedoutside the objectlvemount but st fornis a part of the opticalsystern - The opumlm thlcknessof a covef Thehigherlhe aperture Strictly speaking,ihs applies only to objectiveswhose the .noreaccurale !he cover g assL numerlcalaperture exceeds a certainvalue. Wth objectives having an apedure not exceedlng0.3 specimensmay be examned boih uncoveredor w th coverglasses as thickas a millrneter, and the sphericalaberration wl not be notice able. Wth aperilres between03 and 07, ihe cover-gass th cknessshould not deviaiefrom the nomjnalva ue bv more ihan 0.03 mm. With higherapertures, even a devation of 0.01mrn will consderab y impairthe quaity ot the rnage Objectivesw th a cotreciioncol ar ln order not 1o be resirlctedto specf ed coveFglassthlck_ arefor carefulusers on y nesseswhen uslng dry objectivesof padicuary hlgh aper- ture,obj€ctives were designedin whlchlhe opt mum cover- glass th cknessls not a fixed 0.17 mrn, but can be varied wlthina rangeof 0.12to 0.22mrn (objectives with correction collar).lf the cover-gass ihicknessof the specjmento be examinedis unknown,first set the correcuon co ar at 0.17mm and f nd a hjgh-contrastarea ln the specmen. Then try whelherth€ conlrastis mprovedlf the coreciion co ar s set for greateror esser cover_glassthlckness This a so requiresrefocus ng. Ltis obvous that ln ihe handsof inex pefenced users who usua y leavethe corre.ton .ollar at one end of lts range of adjustme ni, ihese objeci ves rnay give very disappointing.esults (Fg 32J. 43 Oi immersionis less criiical The situaiion is d ffereni with oj mmerslon objecilves, becausethen a rnedum of high refractve ndex,namey oil, is in front of the first lens.ln ihis case,ihe thicknessof the coverglass is not very crilical.Stil , it shouldbe remembered "homogeneoLrs" thatimmersions are today no longer enough (i. e-,glass and o I of dentcal refracliveindex) to allowthe cover glass io be dispensed with as a matter of course. When the cover glass is omitted,fo. very crilicalwork a correctionls requlredeither by increasingihe mechanlcal tube lengthor by lsing a specialimmersion oil (for more details,see for instance,lVichel, Venna 1964,page 176). Howto obtaingood cover glasses? For pfact cal work, ii ls thus necessary always io use cover 9lasseswhich are exacily0.17 mm thick,for specimenswhich are to give opumumlmages even when examinedwlth the aid ofhighdry objectives. M crometers,so-called cover glass gages,are avalable for measuring.Appropriate covers may thus e ther be selected by ihe microscop isi himself, or others usedwhich have been specjally selecied bythe manufacturer. It shou d be mentioned ihat in those cases where the cover glass ls not in direct contactwith the specmen,ihe inter medate ayer of mountingmediurn has, of course,the same effeci as additional thlckness of cover. lt is therefore advis- able io weigh down the coversof specmens duringdrylng. 9. Microscope illumination for more exacting requirements Two importantrequiremenis: For less criticalwork, the microscopemiror may be j umi- objectfieldand pupilmusi be brighi nated either by daylight preferably d ffused llght, such as given by a white cloud - or an ordinarydesk lamp with inside frosted bulb. More convenieni are the attachable simpe subsiageilluminaiors for direci connectionto the mains fo. teachinsmlcroscopes (see Fig. 1). In all these cases optimum cond tions will have io be deiermined by trial and eror. Two things are impodant: the field of view shouldbe evenlyillum nated and - when the condenseriris is open and the condenserin its upperrnostposition - the back lers of ihe objective should be filled with lighi as compleiely as possible. This latter condition is checked after the eyepiece has been taken out of its tube. Kdhlerillumination For microscopic research work, phoiomlcrography, mjcro- projection, eic., preclse control of ihe lighi paih should a ready start before the light reaches ihe specirnen,i. e., at the llght source. Prof. August Kdhler fjrst used this exaci controlofthe lightpath in the illumnaiing beam ofihe mlcro- scope,and the methodis ioday callecl Kdhler lllmination" (lournalof the RoyalMlcr. Soc.). Use of the Kdhler method depends on ihe availability of a microscopeillum nator with a Iamp condenserand an iris dlaphragmin front of it (Fig.33). The lampsused in such illuminators usually have a sErall,concentraied filameni and operate on 6 or 12 volts; they are therefore called low- voliage lamp€. Contrary to normal bulbs for electric lighting it is not their wattage which is declsive for their efficiency, but their luminance. The low-voltage illuminatorinco|porated ln the base of !arger microscopesis, of course a so suitedfor applyingKijhleis rulesof illumnation. The procedurefor applyingthis methodof ill!mlnationis as folows (Fig.34): I 46 l. Aign rnicroscopeillLrm nator with pane mtror of the rnicroscopeand adjust t so that a small,bright spor of ight is projectedonto the mrror or an image of the f laorent formed(this adjlstrnentis faclitaied f a sheet of paper s he d overthe slrface of the m rro, ' o"ch -o rL" - _opo-o r.lF out of the tube.Keep adjustng ihe mjrrorunt the backlens of lhe objectve sf edwiih ght.Dmrning the lghi by a .eutraldensity f ter wou d be advlsabe. nsertthe eyepiece andfocLrs the specmen wlth a l0;l or l6: objecuve. 3. Close ihe iris daphragm of the iilumnator(fied stop) amost compelely end move its (!nshafp) mage oi the specmen nto the centerof the fie ci of vlew by rotatng and 4. Focusthe edge of this diaphragmimage by s ghtly owe. ing ihe condenser.Both the specimenand the f e d stop are now sharpy def ned.Then open the f eld siop lnt I ihe enlire riedofvew s justc ear 5. Slghtly .eadjlsi ihe lamp socketor the lamp condenser Lrnti the f e d of view ls evenly umlnaied. 6. At first openthe condenseri.is (aperturestop) compLete y (imagevery brightland closeit on y far enoughto el minate glare jn the rnost lmportantimage e ernentsand to rnake them appearw th satsfactory conirast. With sta ned speci- mens lhis is the case elen al a reatvely wde aperture, wh e fresh specmens requires ighty more sioppng down Slopp ng down f!rther than s absolutelynecessary (for exanpe, to redlce ihe brghiness of the mage)is one of thebiggest mistakes a microscopst can make,sjnce it entails a oss of resolvng power.- lf tre intensityof the ghi s too hlgh red!ce the lamp voltageor insert gr.y or green flte16 ito the filter holder. 7. lf wjth low power objectlvesonly part of the feld is ll!m nated swingout the condenserfront lensand openthe condenserr s ful y Contrast s then controlledw ih the aid of ihe ampd aphragm. ln the case ol m croscopeswiih a bu lt-inilllm nator step l and party aso step 2 ar€ omiited.Under3, centerlngs noi acheved w th th€ aid of a mltror,but by the c€nteringscrews of the condenser.Piease consu t the operaiinginsiruci ois suppied w th ihe mlcroscope Let us now examne the pathof rayswth Kdhleri urninaton. t conslstsof two sup-.rmposedbeans- For greaterclarity we shallshow both beamsseparate y and ony on one sde (in the case of ihe pup beam,for insiance,ony the rays originatng at one end of the fi ament). Fg.35 sholvsthe pair of the rays originatngand meeung n the panes whch are in sharp focls iogetherwith ihe specmen, i. e the so-calledw ndows.The rays emanaung from the field diaphragmof the arnp are cornbinedln the specimenby the condenser(in otherwords, an imageof the field diaphragmis fo.m€don the specirnen).Ffom there, the two imagesare projectedonto the feld of view diaphragm of ihe eyepiece(aerja image).When the adjustrnenis coF rect, the magesof the field and field of vew djaphragms appearsharp both are centered,i. e they are concentrc andihey are of identicas ze forthe apedufestops Fig. 35a shows the rays which combne at the ap€dure diaphragmplanes. Cosing down these diaphragmswoud affectthe d ameterof the prevolsly menijonedex t pupil of the microscopeabove the eyepjece.Due to refractionby the condenser,the rays emanatingfrom the sourcemeet n the aperiurediaphra-qm of the condenserand are then imaged by condenserand objectve jn the exitpupil of the objectve, whence they are iransmiied by the eyepieceto the exil pupL of ihe mlcroscope.At a theseponts,anmageofthe lghtsource L is formed. What advantagesdoes the KohLermeihod offer? of Kdhleril uminaton 1. In spite of srnalfilaments the entire area of the arnp diaphragmhas the sar.e lumlnanceas the fiament, so ihat onlya ow power nputis requiredfor the amp. 2. Dle to ihe adtustmentof the anrp diaphragm,the afea LLlmlnaiedin the speclmencorresponcls io the one whch ' r oq-d @..o d.ncp ^., Lhe'edsooof 66160a^e. The speclmenis thus not subjectedto ufnecessaryirradia- ton (examinauonofliving organisnrsl) and conirastreducing stray ighi from specinrenareas outs de the field of view is 3. The apedureand thus contrastas well as depthof focus arecontro ed by the condenseriris. 4. Viewed from the specimer, the light source is at infinitv (parallel rays in the aperture ray path, Fig 35al Anv irre- gularity in the source is therefore least effective in ihe 5. Sincethe fieLdstop is jlst fullyfi led with ight due io the adjustmentof the lampd!aphragm, no lightwhich couLd give rise to disturbing flare reaches the inner walls of ihe tube As a res!lt, pariicularly brilliant images are obtained When adjustingihe equipmentfor KijhLerlllumination it mav happen that a cetain granularity !s noied which is super lmposed on the specimen over the entire field of view This is thetexture of aground glass screen or an etched collecior lens whici are lntended io cover up the iregularitv of ihe source.lf this planeshould accidentally be in sharpfocus as well, it is only necessaryio alter the heighi of ihe condenser slighily. With high-aperturecondensers sharp imagingof the lamp diaphra;mis frequentlypossible onlv lf the specirnerslide is not thicker than 1 mrn Why aLrxlljarylenses? ln microscopeswith built-inllluminaior the field djaphragm s sometimes located very close to the condenser' When imagjngthis diaphragmon the specimenin accordancewjlh Kcihlerls nrles, ihe condenser has to be lowered to such an js extenl that a considerable Lossof aperiure ensues This avoided by a so-called auxlliarv lens which ls suppLiedto- qether with the microscope and is ocated dlrectlv below the ;ondenser. lts function is illustraied ln Fig 36 lf a separate hiqhperformance illuminator is used the auxi!iary lens sn-ouidbe swunq out or, letter siill, replaced with ihe longeF "auxiliary '. focus lensl! WithKdhle. illuniiatron lowennsthe condenserwould lead io on exessive ros ofapsbre(refD. 50 10.Length measurements under the microscope Meas!rernentof rncroscoprc objecrs js re ai ve y 5 mpre,ou1 d6o4o oa 6...t"bt. ot r6 oor q measuringeyeplece, €yep ece m crorneteranu sraljem cro meter(Fig.37). A rolnd gass dsk wih a scae -ihe mcro,,ererosK can be inseriedtn the measurng eyepece. and ihe foc!€ing eyeens ensuresthat evenobservers wiih defec,ve eyesgnr can seea sharp mageof rhesca e _ Thestage nricromerer is a scae graduaredin uiirs of 11100nrm on a coiveniional o' \ 3. .q I ,ll lllll llti il lil l ,lllrl{/,1r11ll,tIll'rlrl jlnI lrlllirl l ,1, (\ \t tat (o N 51 The procedureis as fo ows: 4 1 Post on the fo!nd eyepece m crometer n the measuring eyepece. For this plrpose unscrewthe Lowerpari of the eyepece rernovethe ring above the eyepiecedaphragm and -eplaceit after havng insertedthe m cronreterdjsk {scale lpwards).Then screw the eyePece togetherasain 2. Focusthe eyeens in sucha mannerthat the scae appeafs 5 w th nraxim!msharpnessi then lnsertthe eyeplece nto tfre 3. Placethe stage n-rcrometeron the stage and focus ihe microscope on the sca1e.Both scaes will then appear shafpy d€fined. By t!rning the eyeplece tlrev are iaid pafale io eachother. 4. Flnd olt how rnanydlvis ons of the eyeplecem cron-reter correspondto a certaindistanc€ on the siage micrometer and cal.u aie the lengihwh ch cotrespondsto one d v slon of the eyepece m crometer. Example(Fig.38) 80 divsionsconespond to I3s nrm onoc .i. o - 0.069 _.' o, so 5 The ir crometerva ue thls detefrnlnedapp ies on y ror the oblectve wilh wh ch the ca ibraton was rnacre S.. e.oitias: n. om*"f Subsequenily,it s only necessarvto m! up v the nlmber of d v sjons coveredby an interestng deta ln ihe specf'ef 0 bv the mlcromelerva te in order to determne ihe lengthoi tnl" a"tnl 'n tt-,"ofj""t plane (Forsenal m€asuremenisthe rnicromeiervaLue need oily be set once on the slide r! e \lhereuponthe dlfferentlengths can sirnpv be readoff ) Slncethe ir crometerva ue also dependson the r.echanlca 1 iube eiqth. ii s advsableto keep an eve on the settng of ihe eyepece sle€vesof the binoculartube wh ch 'nust not Lfthe nreaslre.nentsextend over a onger perod of trne or f the specmens themse ves exhblt a fine lneariexture we -ecommerdlhat ? conirast, lne_contrastor net micromeier dsk be !sed nstead of the nof.na evepiecemlcrometer {Fs.39al. A m€as!rig eyepiececan. of course be used LntTre same mainer as any ordinaryeyep ece f the m crometerdisk is laken olt Lnthis case an obseruerwho has normalvison sholld set the zero f.ark on the evelensscale at the black .stead of the red lne. 52 'll. The camera on the microscope I necessary,it canbe s mple The s mplesiway of takingphotom crographs is to mounta camerawith ts lens sel at tnfiniiy as closey aoove rne m croscopeeyepiece as possble and to open the camera stop fully. f the nricroscopewas fi.si foclsed w rh re axed accornrnodaton,then the pholonrtcrographw be sharp, bui lslally v gnetted.Th s s due to the fact rhai in conrbi natoi wlth a cameralens of normalfocal tength{50 mm for 24x36 mm, 75 to 80 mrnfor 6).6 cnr t05 rim for 6:i9 cm) only w de-ange microscopeeyepieces cover the enrirefie d of view. - Any doubts regardng critcat iocLrscan be e m natedif a slngle-lensreflex carnera semp oyed(Fjg.40). Focls ng beafl sp illefsare deal Snce a poriion of ihe lghl s lost bythetime t reaches .o mal ,efe^ carneras{ground-g ass screen, spl t-lmagera.sefinder, m cro- prsm focusingring etc.l are not very convenienl.Consder- ably rnoreaccurate and brghler are speca focusirg beam ' i..es ^ o" "i,. 'o fo -.irg oa I Wilh cameraswth focal pane shutter such as Contarex Conlax etc. t is advisablero removeih€ lensand aiiachthe camerabody with a specal adepiefto a so-ca ed aitachment camerawhich is avalab e e ther w iholt (Fig.41 a) or with photoelecirc expos!redev ce (Fig.41 b). What imagescale ls obtaned The imagescale on ihe film differsby a cedainfacror from with a photomicrographiccarnera? the rnag.itjcationgiven by the microscopeduring visual work.Thjs factor is found by dividingihe foca engthof the cameralens (in lnillrneters)by 2s0 mm. (Example:A Conra- flex camefaas shownin Fig.40 has a focal engrhof 50 mrn. Consequenly, the factor is 1/5. Objecrive magnification )< eyepece magnjucationx 1/5thus givesthe imagesca e on the 35 mrn negative.)The specia ZE SS attachmentcamera shownin Fig.41 has camerafactors dependent on the indi Deiermnation of ihe correci€xposure for photomicrography is very convenientif specia photoeeckic exposuredevces are used(Fig.41 b) Anothersimple and accuraternerhod is directmeasurernent of the finder irnage of a reflex camera, preferab y by means of a photoelectrc exposuremeter which has a smattaccept- ance angle and measuringsurface (e. ''IKOPHOT 9., ihe ZETSSIKON CD ). Not even calibrauonphoios are reqlired, if, for instancethe fo owingprocedure is useo: Point camerawith lens set at f/2.8,for exarnpteai a brght wall.Take a meterreading of the wall,w th, say,f12.8,1150 sec.Then iake a readtngwirhthe nreterpointing atrheground g ass or ihe fifder inrageand readthe f siop correspondjng to l/50 sec, e. g., f/1.4.When rakingthe photomicrographs, a feadingsshould then be takenwith the meterpoinring at the finderimagei the exposuretirne is thenthe valuecores, lf working condiiionsare aways identical(oblecrve, eye- piece, position of condenseriris, lamp, filrn speed),then exposure measlrements are not required. A iew rest exposures nray be made, after which the optimum exposure time determined can always be used. High-contrastfilm With normal development,h gh-conirastflm sho!ld be or energeticdeveloperl employed,because contrast in photomicrographsis !sually "particu lowerthan in outdoorphotography. - Filrnsof arly highresoiving powel' are not necessary,because espectally 56 in high-power$,ork the resout on of eventhe eastsutable flrns is still higherthan ihat which s physlcay possiblein the rrricroscope.The s mplestand one of the best methods s to use ord narya l-roundfl m and to developt i a papef Whenis 35 nrmtoo small? t has beenfolnd in praciicalwork that negatives zes afger than 24x36 mm are of advantageonly f smaLlscale photo- micrographsand pariicularlyphotornacrographs, predomi nate. n addition, arger sjres should be used if coor photos have io be taken fof printng purposes.ZELSS supples specal phoiomlcrographicattachment cameras for 2r"/r") 12.Micro.proiection llicro-projectionis a valuabletechnique if severalpeople want to observea m croscopicinrage s mutaneolsly. For smaller groups a viewing screen is ernpioyed,which s s mply exchangedfor the observaion tlbe and whichoffers an imagesca e of 10: ihe objectivemagnification (Ftg. 42). A lessexpensive so utionistoprojectthe microscoptc image onio awh te sldacewththe ajd of adevlatngprisrn attached -o .hp h6 e.eo,o.a wL F, ob.pn6. ..ano< oqsioe ha aya plece,the magnificationis ihe sameas in the microscope.f the viewingdistance s halved,magn f caton sdolbled etc. Due to the frequently arge mage diameters and ihe relatively o.r efficiencyof pfolecuon in refected ight, light so!rces of higher lumnance are then req!ired, with whichabove all the specialequipment for m cro projectionis p.ovided.For furtherdeiails, reference s madeio the cotr respondingZE SS iterature. 5T 53 Annex Bibtiography For more comprehensivenformaiion on the fie d of m cros copy we give betowthe tjtes ot a nurnberof works. ''Using the tVicroscope,,by A. L. E. Barron Champmana Ha l, Londoi, 1965 'Microscopy by W. c. Harfley EnglishUniverstes press Lrd.,London, 1962 Anrericanedition edited byjohn J. Lee and Berna.d Friedman The NaturatHisrory press, Carden Ciiy, New york, 1964 Lecr!renotes on the use of the rnicroscopeby R. Barer Blackwe Scientificpubtcarons, Oxford, 1953 and F. A. DaviesCo., ph tadelpha, pa. ''An lntroductjonio the princtpes of the M croscope a.d irs App tcaiionto the practiceof phoiomcrography.. by F. Schenkand c. K siler Chapman& Hatt,London, 1962 ''phoiomicrography by R. M. A en D. Van NosrrandCo., Inc.prtnceron, New lersey,j95B "Ana ytica Cytotogy Editedby F. C. Me ors 2nd Ediiion 1959,Mccraw Nilt,London "Progress in Microscopy'by M Francon pergamonpress, London, 1961 ''Handbook of Chemica Microscopy, by E M. Chamotard C W. wason rohnW ley ard Sons,Inc. Newyork, N.y. 19S8 Comprehensiveworks in cerman and Ffench: ''Die Crundagen der Theoriedes Mikroskops,by K. Mtchel 2nd editioniWissensch. Ver agsgese sch.,Siuttgart, 1964 Die Mjkrophotographe,, by K. M che 2nd edtionr Springer,Venna, 1964 'Tratte de Microscopie" s nomo urrvensAvt-*.p" by A. Policard,M. Bessus and lvl.Locqlin Massonet Cie Pars, 1957 59 ZEISS literature The MicroscopyDepartment has distributeda whole 6erie6 of caialogswhich gives a good ideaof almostthe entirellne of equipmentsupplied by thern;the rnostimpodant of these Routineand BesearchlVicroscopes 4A-120 STANDARDKF Mlcroscopes 40 130 STANDARD UNIVERSALM lVicroscopefor Veriicalll umination 40 143 PhaseContrast and InierferenceContrasi 40 160 40-215 nve.ted lvlicroscope 40-254 lvlicroscopeI uminaiorsand Lighi Fiiers 4A-344 Insirumenisfor the Photographic,Graphic and Projectjve Reproduciion 40-400 40-430 ULTFAPHOTII 4A-452 ResearchMeta ograph ULTFAPHOTll lV 40-453 Instrumentsfor PolarlzlngMicroscopy 40-550 ntederence lvlicroscopyw th TransmitiedL ghi 40-560 Epi-Microscope 40-655 STANDARDMeta lurgical Microscope 40-660 40-700 ZEISSreprints Reprinlsof partic!larlyinteresting anlc es are Likewiseavai- able. However,ihe stock of such maierial is llm ied, and very oftenthe articleswerereprlnted only inthe anguagein whiclr they orlginallyappeared. oi request,schoo s, instltutes,etc., may receivean lnstruc- tionaldiagramln the form of ro er posters,showingthe cross sectionof a microscopeand the correspondinglght path (Cat. No. 40-020). 60 Additional information The staff of our guesi laboratory at the Oberkochen rnain factory as well as our subsldiarjesor representativesall over the wond are entirelyat your servce for consultaiion on your particuLarproblems. May we also call your attenuonto ihe fact that anyone who understandsGerman and is interestedin photomlcro gfaphy rnay participate in a five-day introductory course whichls held everyyear ln Marchor April at Cdiiingen.For applications,please write to oLrrZEISS agent or djrectlyio the MicroscopyDepartment, CARL ZEISS, 7082 Oberkochen- Wuertt. - West Gerrnany. Delivery programmefor ZEISS l,/lcroscopes and SupplementaryAttachments Routineand FesearchMjcroscopes Microscopesw th bu ft-inautomalic camera Attachrnentcarneras wth mechanicaland automatcshutler for photornic.ography Micro c ne cameras Polarizingrnicroscopes and accessories Equipmentfor lnterferencernicroscopy w th lransrnittedllght Phase-contrastrnicroscopes Fluorescencemlcroscopes and flLrorescenceI lurninators Special.nicroscopes for rnetallographicexarninations EpiMlcroscopes,Epl Technoscopes Microscopesfor depth measurements (rnicroscopesfor measuringthe depth of eich ns we s) Dlfferent types of Stereomicroscopes Microscopesfor planktonand t ssue examinations Cytopherometerfor electrophoreiicexaminations of cells Un versallVicro-Spectrophotorneter UMSP I Mic.oscopePhotomeier IVPM Micro projection devices Microscopellunrinaiors and jght f ters Tubes for conneciing the micfoscope !o the TV camera Drawinginstru.nents for riicroscopes,heaiing stages and olheraccessor es Diffracton apparatusfor m croscopicdemonstrat ons 6l ZEISSand the art of microscopy The origln of your ZEISS miffoscope les n the workshopsof the firrn of CARL ZEISS. n 1846 the universitynrechanic of lhat nameestablished a workshop for opica mechanjcsat Jena,whlch gainedword renown afterDr. ErnstAbbe hadjoined him €s scientf c collaboraior in 1866.ErnstAbbe,who was also professorof rnathematical physicsat lena LJnversity, a d the scientificbas s for the entirefeld of m croscopy.After the death of Cad Zejss he set up the CARLZEISS Foundation (18891, transferred his entirefortune to ii and plt nto practicesocial ideaswhch at thatume were sensatona (e. g., penslonr ghts and proft sharing).Ever slnce ZE SS has beenworld famousfor top qualty opiicsfor use inihef eldsof m cfoscopy,photography astronorny,etc AfterWord War ll the fjrm of CABL ZEISS was dismanted and expropriated.The Managementand about 100leading personne of the companybegan to blild a new ZEISS factory at Oberkochen (Wueirtemberg/ West 1". Cer ".)l $f. " ^..- q dd . y Jo edb. nanr.pe.i.- lsts from the old works. ln ihe new productionprogram, wh ch rangesfrom spectace ensesto planetarlaand from magnfjersto e eciron rnicroscopes,ight mtcroscopesare cornnrerciallythe most imporrantteor. They are being bu t n an effod to carryon the tradiiionof a greatname. ZEISS makes microscope h;story Ihe ZEISS works have made decslve contributionsto ihe deveioprnentof the modernmicroscope. Frorn the long isi of innovaiionsjntroduced by this company,wehavese ected thosepo nis whjchmay be of particLrar jnterest1o you. 1812 Forthe first t rnein the hisioryof the m croscopeobjectives "trylng were offeredwhich had not been nradesimpy by lt ot't bli on the baslsof rnathematcaland oplicalfesearclr. The CARLZEISS ist No. 19 (1872)opens wth the sentence: "The m croscopesystems below are excusive y based on theoretica complrtaiions carried out by Professor Abbe at Ai the same time, the bis Abbe illuminator appeared for rhe "combjned first time in all its parts exclusivelyon rhe basis of theorerical considerations _ 1886 In the ZEISS works, Ernsi Abbe succeeded in making inicroscope objectives of unusuallyhigh correction,wh ich he called Apochromais- At the same iime, rhe corresponding compensatingeyepieces were introduced. 1893 ProfessorA. Kdhler,who joined ZEISS in 1900,published lLe m.-hod o" illuTinato- ale callaodhe hirn. 1896 ZEISS built the first Greenoughstereomicroscope. 1904 Professor K0hler published his work on ihe uliraviolei I911 Followinga suggestionby Kohler,ZEISS introducedpaF focallzed microscope objectives. 1933 zL S:i e.orulo i/ed rne corst.uctjonol Ticroscopesby deeigning ihe L stand (not iiltable, stage always horizontaT, r935 For mllitaryreasons, the anti-reflectioncoating of lens sur faces ('bloomlng) inventedby ZEISS had to be classified r936 ZEISS built a firsi prototype of the phase contrast micro- scope based on sussestions by the later NobeTPrize winner, 1938 After prolonged developmentwork, ZEISS were able to offer the first flat-field objectives (Planachrornats). 1943 The mic.oscopy iaboratory of the ZEISS works produced the first motion picture on cell divisiof r.rsingthe phase- contrasi microscope (Dr. K. Michel). Wjth this examination method, cell research entered a new phase. 63 1944 ZEISS bulli the first stereomicroscopewith magnificauon changer{eliminat ng exchangeof objectives,offerlns con stant workjng di6tance). 1950 The ZELSSSTANDARD Microscope ntroduced coaxrar controls and spring- oaded objectives (specirnenprotection) into microscopeconstruction. 1950 ZEISSobiained a paientfor a magnificationchanger desig- nated as OPTOVAF. 1955 The first photomicrographicdevjce wiih f! y autornaticex posureconirol was sold (ZEISSPhotornicroscope). 1959 Wilh their Ultrafuars,ZEISS succeededin makingrefract- ing objecuvessuilab e for boih u travolet and visible llght (imporlantfor m crospectrophotomeiryand cancer researchl. Thls book is copyrghi. li may not be reproducedor irans' lated by any rneansln whole or in part withoutpermiss on. Applicalionswlthregardto copyright shor.rld be addressedto; CARL ZEISS, Oberkochen/West Germany