!I*"[''QK5519 16, 8, 6or3 mm.focus. Forthe260 PROJECTIONMICROSCOPE[CH.IXthreekepton the nose-piece most constantly a64,42 and 6mmis asgooda combination asonecan get. Otherpowersshouldbeavailable,however, and for specialspecimensandoccasions onemayemploythetwomm. oil immersion. Forpowersabove20 mm.,ordinaryobjectivesaremoresatisfactory than mostprojection objectives.S41S.Projection Oculars(Fig. 185). If one usestheapoch-romaticobjectivesof 16 and S mm. focus the projection oculars areusedwiththem. Theycan also beusedwith wide angle achromaticobjectives. Withthemonecangetan enormous magnification evenwith the 16mm.objective. (Seethe table below,421.)These oculars restrict the field greatly and in thewriter'sexper-ience it seemedonthe wholemore desirable formostobjectsnottousethe projoction oculars. They cannot be used advantageously withobjectives higher than S or 6 mm.and are most satisfactory withoneof 16mm.In using the projection ocular with a 16mm. objectiveonecanplace the special achromaticcondensernextthestage, butiftheocularis notusedonemusteither do without a specimen condenser, or takeoneof lontrer focus, if hehastwoor three asshownin Fis;. 207.lililllllllllllL,,,Fig. 211.Fig. an. Zeiss' Micro-Planarforprojection. (Cutloanedby Bausch y aconvergingconeoflight, and itshouldbelighted bytheentireconeoflighttraversingthe lampcondenser. If oneuses a white card it iseasy todeterminetheposition andsize of the cone of light. If it istoolarge for thespecimen, eitherthelampcondenser is too near theradiantorthespecimen is too close to thelamp condenser.If oneusesanachromaticcondenserthe lamp condenserand theachromaticcondensershould be so arranged that the entire cone oflight traversingthelamp condenser canentertheachromaticcondenser.If the cone is too large they are too close together, orthelampcon-denser is toonearthe radiant. If thecone is too smallthenthelampcondenseris too far from the radiant or the achromatic condenser,orperhapsbothfaults are present. Onemustrememberin all hisex-perimentsthat aconvergingconeof light should be used and not adivergingone. Thespecimenmustthen not be beyond the focus ofthelampcondenser.If onewishesto make micro-projection a success it will be nec-essary to givetheapparatusthe requisite time and thought. Tr}' tounderstandtheconditionsofsuccessandcontinueexperimentinguntilyouhavelearnedtomakeit possible for themachinetodo its best for3-ou. Thesatisfaction of showinga class real things is sufficient re-ward for all the trouble.420.ScreenandScreenDistance.Fora screen nothing issogoodas a dead-white, smoothwall. Alusterless,whitecloth screenanswerswellalso. It is anadvantagetohavethisentiretyopaque, sothatnoneof thelight canpassthrough it. Onemust remember thatthelight passingthroughtheminutelenses of the objective must bespread out over a great space even with low powers, and over amuchgreaterwithhighpowers,sothat onecannotaffordtohaveanyofthelight lostbytransmissionthroughthescreen. Allareagreedthatfor micro-projection a translucentscreenwiththeprojectionapparatusbehind it is notdesirable,althoughforordinarylanternslide projectionit answers fairly well.Thedistanceof thescreenfromthemicroscope dependslargelyonthesize ofone'saudience. Thewriter has found a distanceof eightmeters (26 feet) goodfor bothlow andhigh powerprojection. Thisdistanceanswerswell for a classof 200persons.262 PROJECTIONMICROSCOPE\_CH. IXFortheminutedetailsof aprojectedspecimen it is recommendedthattheaudienceuseoperaglasses. Theseare also usefulfortheoper-atorin focusingtheimageonthescreen.TABLESHOWINGTHESIZEOFOBJECT,THEMAGNIFICATIONANDTHESIZEOFTHESCREEN IMAGEWITHVARIOUSOBJECTIVES ANDPROJECTIONOCULARSDistanceojthescreen Jromthe stageojthe microscope, 8meters (26feet+).Arclight, 12amperecurrent, forillumination.ObjectiveProjectionOcularAchromaticCondenserActual Size ofFieldMagnificationDiameterof ScreenImage64 mm. none none 20 mm. 125 250 cm.42mm. none none 10 mm. 185 185 cm.35mm. none none9 mm. 230 207 cm.24mm. none none 7mm.335235cm.18mm."mm."mm.none noneX2 condenserX 4 1condenser6mm.1.5mm.1.5mm.4408601640264 cm.130 cm.245cm.16 mm."mm."mm.noneX2X 4nonecondensercondenser4 mm.1.25mm.1 mm.5310902040200 cm.136 cm.204 cm.8 mm."mm."mm.noneX 2X4condensercondensercondenser3 imm.0.60mm.0.60mm.100022504500300 cm.140 cm.270 cm.6mm."mm."mm.noneX2X 4condensercondensercondenser2mm.0.50mm.0.50mm.120025505000240 cm.130 cm.250 cm.5 mm-"mm.none2condensercondenser1.80mm.0.40 mm.17503500300 cm.140 cm.4mm. none | condenser j 1.50 mm. | 2150 320 cm.3mm. none | condenser | 1 mm. | 2350 235 cm.2mm. none | condenser | o.6oxmm. 4000 300 cm.421.Thistableshowsapproximatelythe size of objectwhicheachobjectivewill projectuponthe screen. Thiswasdeterminedex-actly asdescribed in50. Forthemagnificationonesimplymeasuresthe distancebetweentwoormorelines of the image of the microm-eteronthe screen, anddivides the size of the image by the knownsize of theobject(155).Anygood stage micrometer will answer.It is necessary, however, to useonewithcoarse lines for thelowpow-ers( 159, 170).Bycomparingthemagnificationwith and without the projectionoculars, andalsocomparingthe size of objectwhich can be projectedCM.IX] PROJECTIONMICROSCOPE 263withandwithouttheocularsonecan decidequite accurately the bestcombinationto select. On thewholethewriterhasfound it better toemploya sufficient variety of objectives and not use the projectionoculars. It issomewhateasier to obtain a brilliant image without theoculars.422.Darkeningthe Room.It is impossible to succeed inmicro- projection unlessthe room can be made dark, the darker thebetter. It is especially importantthat thescreenshouldbe free fromall lightexceptthatprojectedupon it in formingtheimage.423.Enclosing the Projection Apparatus.It is desirabletohavethe projection apparatus closed as completely as possibletoavoiddiffusing light through the room and thus vitiatingthe mostcarefuldarkeningof all windowsandskylights. It is also desirableto shutin thelight fromtheapparatus, as it dazzles the eyes of theoperatorandof thosenear it in theaudiencesothat theimage ou thescreencannotbesatisfactorily seen. Someformsofapparatus areen-closed in a metal box, othershaveaframe over them upon which isspreadblackcloth like silesia. If this is madefireproofbysoaking itthoroughlyin a solutionofalum, boraxand sodium tungstate it willnotreadily catch fire. Theclothshouldnotbetoothick, otherwise itwill retaintoomuchheataround theapparatus.One should remember the fundamental law of vision, viz, thatotherthingsbeingequal, theclearestimagesareobtained whennolightreachestheeyeexceptfromtheobject.424.Preparations Suitable for Micro-Projection. As ageneralization it maybesaid thatanyspecimen which shows clearlyandsharplyunder the microscope with a 16 mm. objective will alsogive an excellent projection image. Details which are not visiblewith the 16mm. objective arerarely well brought outwith sufficientclearnessonthe screen foroneortwohundredpeopleto see.(A) Thestains showingbestarethosewhichareverytranspar-ent, or pure differential stainslike hematoxylin. Admirable resultshavebeenobtained with hematoxylinandeosin, andthevariouscar-mines when differentiated. Every method of staining which giveseither sharply differentiated results or transparent colors producespreparationsadaptedto projection. Aweak,orwashedoutappearanceunderthemicroscope is suretobeevenless satisfactory on thescreen.(B) Thethicknessofthe sectionsmayvaryfrom l/< to40^.Butonemust remember that thick sections are adapted for low powers264PROJECTIONMICROSCOPE\_CH.IXonly, whilethin sections, if well stained, maybe used with bothhighandlowobjectives.Thesize of theobjectwhichonewishesto project determines theobjectives tobeused. Byconsultingthe table onecan get a fair ideaof the size of object which each objective will satisfactorily project.Anexcellent plan to follow is that for ordinary microscopic study (seep. 102),thatis, use first a lowpowertoshowtheobjectasawhole,thena higherone fordetails.(C)For minute objects like white blood corpuscles, etc., it isnecessary to use a high powerandto havea small audiencewhichcanbeclose to the screen, or a somewhatlargeraudiencecan see well byusingopera glasses.(D) Forthe circulation of theblood it is necessary to eliminatetheheat rays almost entirely. Nothing has proven so good as thesecond or specimen cooler(413).The conditions are still morefavorable if a circulation of cold water is established in the large waterbath also. This is easily doneby the use of two large bottles. Thecold water can be siphoned or aspirated from an upperoneandthewarmwater allowed to flowoffinto a lowerone. Forthis it is ofmuchadvantage to havea tube in the bottom of thewater bath in which tointroducethe cold water. Thewarmwaterwill then flowoff througha tube in thetop. Onemustrememberthat perfectly clean watermustbeused for the waterbath especially whena circulation is established,for opaqueparticles in the waterbath give undesirable shadowsin theimage.( B) Apractical suggestion is madebyLewisWrightin his bookonoptical projection, andthat is towarmtheobjective before using itforshowingthecirculation of theblood or in any casewhena moistobject is under it. If the objective iscold the vapor fromthe objectwill be condensedon the objective and make satisfactory projectionimpossible.425.Masksfor ProjectionPreparations.Thelightusedforprojection is so brilliant that it is practically impossible to arrange theobject underthe objective with rapidity andcertainty unless there issome kindof guide. Thebest onefoundso far is a maskonthebackof theslide with anopeningfor the preparationLto be shown. Thismaskshouldbemadeof black paper. Onecan cuttheholesin it withscissors orwithticket punches. Withthespecimensproperlymasked,andthe partsof theapparatus lusterless black, as suggested by Dr.CH.IX]PROJECTIONMICROSCOPE265Coplin, the operatorcan work with rapidity, certainty and also withcomfort. (Fig.213.)79oF51086T0193o|Homo 5Slide 10See's7920m 1001901Fig. 213. Slideofserial sectionswith ablackmask,perforated over the sec-tionsto bedemonstratedwith the projection microscope. Thismask is put on thebackoftheslide, noton thecover-glass.Unlessonehasa masksomethinglike this the light is so dazzling that it isalmostimpossibletofindthe propersections. It is easily removed byplacingtheslideon wetblottingpaper.S426.Howto demonstratewiththe Micro-Projection Ap-paratus. Microscopical preparationsare not so easily usedasarelan-tern slides. Thewriter hasfound that themostsuccessful method isforthe teacherhimself to stand bytheapparatus,insertthespecimens,and find exactswhat he wishes his pupils to see. Thento pointthemout abamboofish pole with sharpend is used. This should be2-3 meterslongandif heldout in thedivergingcone of light leavingthemicroscope, a sharpshadowwill be cast upon the image. Withthispointeronecan indicate the part to be demonstrated even moresatisfactorily than as if he pointedthem out directly on the screen.While it is not possible to delegatethe finding of the specimen to anassistanthe is of great help in keepingthecarbonsof thelampin ex-actlytherightposition. If the light is kept perfect the teacher hasvery little troublewiththerest of themanipulation.427.Cleaningthe Glass Surfacesof the Micro-projectionApparatus.Inasmuch asit issodifficulttomakethelight sufficientlybrilliant for micro-projection, it is of the greatest importancethat allglasssurfacesbekeptas clean as possible. The lenses of the lampcondensershouldbecarefullywipedoccasionally;andthe water bathshouldbe opened and the plane glass faces thoroughlycleaned. Itis desirable tosoakthem in thecleaningmixturefor glass. There isalwaysacertainamountof depositon theglasseven though distilledwateris used. Everygradeof opacityrenderstheimageonthescreenless excellent. Cleanliness is oneof the mostimportantrequirementsfor successful micro-projection.266 PROJECTIONMICROSCOPE [CH.IXEachpreparation shouldbewipedoff before it is put in positiononthestage. Anyparticlesof dustarepainfully evident in the pro-jectedimage.VFig. 214. ZeissEpidiascopeforOpaque Objects, andforTransparent Objectsin a HorizontalPosition (Zeiss' Special Catalog.)Asshownin this jiguretheapparatus is set upforopaqueobjects. Fortrans-parentobjects M2(mirror2) is removed, when the light striking Mjis reflectedtoIII'andthenceupthroughtheobject toM'andto thescreen.Commencingattheright : R.Parabolicreflector, whichprojectsthelightfromthecraterthrough( W ) thewaterbath,to M2the mirrorwhich is atthe properan-gleforreflectingthelightdownupontheopaqueobject. Fromtheopaqueobjectthelight is irregularlyreflectedup through the objectiveto 31'.M'serves to reflecttheraysfromtheobjectivetothescreen.V. Ventilator. M;andHI'arcmirrorsforusein reflectingthelight throughhorizontaltransparentobjects.CH. IX]PROJECTIONMICROSCOPE 267Thisapparatusis designedtoprojectopaqueobjectsas large as 22centimetersin diameter, ata magnificationof5to 10witha30amperecurrent. Forasmallerobjectonemaymagnifyashighas25diameters. Witha50amperecurrentandalargerreflectorthemagnificationmaybe from14tip to37diameters.PROJECTION OF OPAQUE OBJECTS\42S. Episcope.Fortheprojectionof opaqueobjectslikeanatomicalprep-arations, figuresinbooks, coinsorindeedanyopaqueobjectanapparatus on theprincipleoftheonefigured(Fig. 214) is used. Thatis, apowerfullightisthrownupontheopaqueobjectandtherays reflected from theobjectarethenprojecteduponthescreenby anobjectiveasfor a lantern slide. Astheobjectsaremostlyina horizontalpositiontheobjectivepoints directlyupward,andtheraysfromitmustbemadehorizontalbymeansofa45degreemirrororprism.Thisapparatusis veryold. Its first namewas"aphengescope" oropaquelan-tern. Nowit is called an episcope, or megascope,and if for both opaque andtransparentobjects (Fig. 214) it is designatedasanepidiascope.Forits satisfactoryuseexceedinglypowerful lightmustbeused. Someopti-ciansemploytworadiants, othersbutone. In anycasecurrentsof30 to 50 am-peresareused. Oneshouldweardeeplystainedglassesinworkingwith it.Theapparatusworkswellwith flat objects, and ratherbrilliantobjects, likethemovementsof awatch, etc. It is also more satisfactory forobjectsof slightthickness. Forobjectslikebones, etc., onemustfocusupand down for thedif-ferentlevels.REFERENCES TO CHAPTER IXLewisWright, Optical Projection;Carpenter-Dallinger;Leiss, Die optischenInstrumentederFirtna R. Fuess;The works on Photo-Micrography;Thelatestcatalogsorspecialcatalogsonprojectionapparatusissuedbytheopticians,especial-ally Zeiss,ReichertandLeitz. Thevolumesof the microscopical periodicals forthe lastfewyears, especiallythe JournaloftheRoyalMicroscopicalSociety, andtheZeitschriftfurwissentschaftlicheMikroskopie.CHAPTER XTHEABBETEST PLATE ANDAPERTOMETER;EQUIVA-LENTFOCUSOFOBJECTIVESANDOCULARS;DRAW-INGS FORPHOTO-ENGRAVING ;WANMODELS2 429.On the Method of Using Abbe's Test-Plate. This test-plate is in-tended for theexamination of objectives with reference to their corrections forsphericalandchromaticaberration and forestimatingthethicknessof the cover-glass forwhichthesphericalaberration is best corrected."Thetest-plate consistsof a series of cover-glassesrangingin thicknessfrom0.09mm.to0.24mm.,silvered ontheundersurfaceandcementedsidebysideona slide. Thethickness of each is writtenonthesilver film. Groupsof parallellinesare cut through the film andtheseare so coarselyruled that theyare easilyresolved by the lowest powers, yetfromtheextreme thinnessof thesilver the}'forma very delicate test for objectives of even the highestpower and widestaperture. To examineanobjectiveof largeaperturetheplatesaretobefocusedin succession observingeachtimethequality of theimage in the center of thefield andthevariationproduced by using alternately centra) anil very obliqueillumination. Whenthe objective is perfectly corrected forspheircalaberrationfortheparticularthicknessof cover-glass under examination, thecontourof thelines in the centerof the field will lie perfectly sharpbyobliqueilluminationwithoutanynebulous doubling or indistinctness of the minute irregularitiesofthe edges. If after exactly adjusting the objective for oblique light, centralillumination is usednoalteration of theadjustmentshould be necessarytoshowthecontourswithequalsharpness.""If anobjectivefulfills theseconditions withanyoneof the plates it is freefromsphericalaberrationwhenused with cover-glassesof thatthickness ; ontheotherhandif everyplateshowsnebulousdoublingoran indistinct appearanceoftheedgesofthesilver lines, withobliqueillumination,orif theobjectiverequiresadifferent adjustmentto get equal sharpness with central aswithoblique light,thenthesphericalcorrection ismoreorlessimperfect.""Nebulousdoublingwithobliqueillumination indicatesovercorrectionofthemarginal zone, want of the edges without marked nebulosity indicates under-correction of thiszone;analterationof theadjustment for oblique and centralillumination, that is, adifferenceof planebetween the image in the peripheralandcentralportionsof theobjectivepointstoanabsenceof concurrentactionoftheseparatezones,whichmaybeduetoeitheranaverageunderorovercorrectionor to irregularityin the convergenceof theravs.""Thetest ofchromaticcorrectionis basedonthecharacterofthecolorbands,whichare visiblebyobliqueillumination. Withgoodcorrection theedgesoftheCH.A']TESTPLATEANDAPER TOMETER 269silverlinesinthecenterof the field should show but narrowcolorbandsin thecomplementarycolorsof the secondary spectrum, namely,on one side yellow-greento apple-greenontheotherviolettorose. Themoreperfectthecorrectionof thespherical aberrationtheclearerthiscolorbandappears.""To obtain obliquity of illumination extendingtothe marginalzoneof theobjectiveandarapidinterchangefromobliqueto central light Abbe'silluminat-ingapparatusis veryefficient, as it is only necessarytomovethediaphragm inusenearertoorfurther from the axis by the rack and pinion providedforthepurpose. For the examination of immersion objectives, whose aperture as arule is greater than 1S0 in air and those homogeneous-immersion objectives,whichconsiderablyexceedthis, it will benecessarytobringtheundersurface oftheTest-plateintocontactwiththe upperlens of theillluminatorbymeans ofadropof water, glycerin oroil.""Inthiscasethechangefromcentral to obliquelightmaybeeasily effectedbytheordinaryconcavemirrorbutwith immersionlensesof large aperture it isim-possibletoreachthe marginal zonebythismethod,andthebest effecthastobesearchedforaftereachalteration of thedirectionof themirror.""Forthetheexaminationofobjectivesofsmalleraperture(lessthan4o-50)wemayobtain all thenecessarydatafor the the estimationof thespherical andchromaticcorrectionsbyplacingtheconcavemirrorso far laterally, that itsedgeis nearlyin thelineof theoptic axis the incident coneof raysthenonlyfillingone-halfof theapertureoftheobjective. Thesharpnessof thecontoursandthecharacterof thecolorbandscanbeeasily estimated. Differencesinthethicknessof thecover-glasswithin the ordinary limits are scarcely noticeable withsuchobjectives.""Itis of fundamentalimportancein employingthe lest asabovedescribed tohavebrilliant illuminationandto usean eye-pieceof highpower.""Whenfrompracticethe ej^ehaslearnt to recognize the finer differences inthequalityof thecontourimagesthis method of investigation gives very trust-worthyresults. Differences in thethicknessof coverglassesof 0.01 or 0.02mm.canberecognizedwithobjectivesof 2 or3mm.focus.""Withobliqueilluminationthelightmustalwaysbe thrown perpendicularlytothedirection of the lines.Fig. 215. The Abbe Test Plate."Thequalityof theimageoutsidetheaxisis notdependenton spherical andchromaticcorrectioninthestrict sense of the term. Indistinctness of the con-tourstowardsthebordersof thefield of viewarisesasarule, from unequal mag-nification of the different zones of the objective;colorbandsintheperipheral270 PESTPLATEANOAPER TOMETER\_CH. Xportion (withgoodcolorcorrectioninthemiddle)arecausedbyunequalmagnifi-cation of thedifferentcoloredimages.""Imperfectionsof thiskind, improperly called "curvature of the field," areshowntoagreaterorlessextentinthebestobjectives, wheretheapertureis con-siderable."ii&*...".1615 *'-*%,>--CarlZeLS^Apert^ejep Ienj^__Fig. 216. Abbe Apertometer.\430.Determinationof the Apertureof Objectives with anApertometer.Excellentdirections forusingtheAbbeapertometer may be found in the Jour.Roy. Micr. Soc, 1S78, p. 19,and 1S80,p.20 ; in Dippel,ZimmermannandCzapski.Thefollowingdirectionsarebutslightly modified from Carpenter-Dallinger,pp.394-396.TheAbbeapertometerinvolvesthesameprincipleas thatof Tolles, butit iscarriedoutinasimplermanner;itis shownin Fig. 216. Asseenbythisfigureit consists of a semi-circularplateof glass. Alongthestraightedgeorchord theglassisbeveledat45, andnearthisstraightedgeis a small, perforatedcircle,theperforationbeingin thecenterof thecircle. Touse the apertometer the micro-scope is placedin averticalposition,andtheperforatedcircleisputunderthe mi-croscopeandaccuratelyfocused. Thecircularedgeof theapertometeris turnedtoward a window or plenty of artificial lightsothatthewholeedgeis lighted.Whenthe objectiveis carefullyfocusedontheperforated circle the draw-tube isremovedandin its lowerendisinserted thespecialobjectivewhich accompaniestheapertometer. Thisobjectiveandtheocularformalow power compound mi-croscope, andwith itthebacklensof theobjective, whoseaperatureistobe meas-ured,is observed. Thedraw-tube is insertedandlowered until the back lens oftheobjectiveis in focus. "In theimageof theback lens will be seen stretchedacross, as it were, theimageof the circular part of the apertometer. It will ap-pearasabrightband, becausethelightwhichentersnormallyat thesurfaceis re-flectedbythebeveledpart ofthechordin averticaldirectionso that in reality afanof 180 in air is formed. Therearetwoslidingscreensseenon either side oftheapertometer; theyslide on the vertical circular portion of the instrument.Theimagesofthesescreenscanbeseen in theimage of the bright band. Thesescreensshould nowbemovedso thattheiredges justtouch the peripheryofthebacklens. Theyact, asit were, as adiaphragmto cutthefanandreduceit, sothatitsangle justequalsthe aperature'of the objective and no more." "Thisangle isnowdeterminedbythearcof glassbetweenthescreens; thuswegetan angle inglasstheexactequivalentof theaperatureof theobjective. Asthenumericalap-erturesofthesearcsareengravedontheapertometertheycanbereadoffbyinspec-tion. Neverthelessadifficulty is experienced, fromthe fact that it is not easy toCH.X] TESTPLATEANDAPERTOMETER 271determinetheexactpointatwhichtheedgeof thescreen touches the peripheryof thebacklens, orasweprefertodesignate it, thelimitofaperture, for curiousastheexpressionmayappearwehavefoundattimesthatthebacklensof an ob-jective is largerthan the aperture of the objective requires. In thatcase theedgesof thescreen refusetotouchthe periphery."Indeterminingtheapertureof homogeneousimmersionobjectivestheproperimmersionfluid shouldbeusedasinordinaryobservation. So, also, withglycerinorwaterimmersionobjectives.I431.TestingHomogeneous Immersion Liquid.In order that one shallrealizethefullbenefit of the homogeneous immersion principle it is necessarythatthe homogeneous immersion liquid shall be trulyhomogeneous. In orderthatthe ordinary worker may beable to test theliquidusedbyhim, ProfessorHamiltonL. Smithdevised a tester composed of a slip ofglass in which wasgroundaccurately asmall concavity and another perfectly plain slip to act ascover. (See Proc. Amer. Micr. Soc; 1S85,p. 83). It will be readily seen thatthis concavity, if filled withairor anyliquid of less refractiveindexthan glass,willactasaconcaveordispersinglens. If filledwith aliquidofgreaterrefractiveindexthanglass, theconcavity would act likeaconvexlens, butif filled with aliquidof thesamerefractiveindexasglass, that is, liquid optically homogeneouswithglass, thentherewouldbenoeffectwhatever.Inusingthistester theliquid is placedin theconcavityandthecoverputon.Thisis best applied by sliding it over the glass with the concavity. Asmallamountof theliquidwillrunbetweenthe two slips, making optical contact onbothsurfaces. One shouldbecareful notto includeairbubblesintheconcavity.Thesurfacesof the glass are carefully wiped so that the image will notbeob-scured. Anadapterwithsocietyscrewis putonthemicroscopeandtheobjectiveisattachedto its lowerend. Inthisadapateraslot is cutoutof the right widthanddepth to receive the tester whichis justabovetheobjective. As object itis welltoemployastagemicrometerandtomeasurecarefullythediameterofthefield withoutthetester, then withthetesterfarenough inserted topermit of thepassage of rays throughtheglass butnotthroughthe concavity, andfinally theconcavity is brought directly over the back lens ofthe objective. Thiscan beeasilydeterminedbyremovingtheocularandlookingdownthetube.FollowingProfessorSmith'sdirections it is agoodplantomarkinsomewaytheexactpositionof thetubeof themicroscopewhenthemicrometeris in focuswithoutthetester, thenwiththetesterpushedinjustfarenoughtoallowthelighttopassthroughtheplaneglassandfinallywhenthelight traversestheconcavity.Thesizeof thefield shouldbe notedalsointhethreeconditions (ji50-52.)Itwill beseenby glancingatthefollowingtablethat whenever theliquidin thetester is of lowerindexthan glass, that the concavitywith theliquid acts as aconcavelens, orinother wordslikeanamplifier(p. 109),andthefield issmallerthanwhennotesteris used. It will alsobeseenthatastheliquidintheconcav-ityapproaches the glassinrefractive index that the field approaches the sizewhennotesteris present. It is alsoplainlyshownbythetable that the greaterthedifferencein refractiveindexof thesubstance in theconcavityandtheglass,themoremustthetubeof themicroscopeberaised to restorethefocus.If asubstance of greater refraction than glassis usedinthetester the fieldwouldbelarger, i. e., themagnificationless, andonewouldhavetoturn thetubedowninsteadof up to restore the focus.272TESTPLATEANDAPERTOMETER CH.A']Thetablegivenbelowindicatesthepointswithatesterpreparedbythe Gund-lach Optical Co., and used with a 16 mm.apochromatic objective of Zeiss, ,/\compensationocular, achromaticcondenser, i.ooN. A. (Fig.41 ):Sizeof theIElevation oftheTubeTester and Liquidin theConcavity'...necessarytoFieldRestoretheFocusNo tester used 1.825 mm._.Standardposition.Wholethicknessof the tester at one end,notoverthecavity1.S5mm.___Nochangeof focus.Testerwith water 1.075""--.Tuberaised3J2mm.Testerwith95%alcohol 1.15mm...J . . . . 3mm.Tester withkerosene 1.4mm. .... 2mm.TesterwithGundlachOpt. Co's'hom.liquid t.825mm._. .... ,'" mm.Bausch&LombOpt. Co.'shum.liquid .1.825mm.__ . $5mm-Leitz'hom.liquid [.825mm._..... ,-" mm.Zeiss' hom. liquid 1.825mm.__ .... ,-" mm.\4^2.EquivalentFocusof ObjectivesandOculars.Toworkout inpropermathematicalform or to ascertain experimentally the equivalentfoci of thesecomplexparts with real accuracywouldrequirean amountof knowledge and ofapparatus possessedonly byanoptician ora physicist. The work may bedone,however, with sufficient accuracy to supply most of the needsof the workingmicroscopist. Theoptical lawon whichthe following is basedis:"The sizeofobjectandimagevarusdirectlyastheir distance fromthecenteroj thelens."By referring to Figs. 14, 16, 21, it will be seen that this law holdsgood.Whenoneconsiders compoundlens-systemstheproblembecomesinvolved,asthecentreof thelenssystems is not easily ascertainable henceit is notattempted,andonlyanapproximatelyaccurateresult is sought.\433.Determination of Equivalent Focus of Objectives. Look into theupperend of theobjectiveandlocatethepositionof thebacklens. Indicatethelevel in somewayoutsideof theobjective. This is notthecenterof the object-ive butserves as an arbitrary approximation. Screwtheobjectiveintothetubeof themicroscope. If aHuygenianocular is used with the ocularmicrometer,screwoff the field lensandusethe eye-lensonly. If a positive ocular is usednochangeneedbemade. Pull outthedraw-tubeuntil the distance betweentheocularmicrometerandthebacklensis250millimeters. Useastagemicrometerasobject and focus carefullv. Make the lines of the two micrometers parallel(Fig. 10S). Note the numberof spaces on the ocular micrometer requiredtomeasure one ormorespacesonthestagemicrometer. Supposethetwomicrom-etersareruled in/,mm.andthat it required 10spacesontheocular micrometertoenclose 2 spacesonthe stage micrometer, evidentlythen5spaces would coverone. Theimage, A'B'Fig21 in thiscase is five timesaslongastheobject, A,B.Nowif the size of objectandimagearedirectly as theirdistancefromthe lens itfollows that as the size of object is known(,- mm.),thatof theimagedirectlymeasured( j ]Jmm.),thedistancefrom the lensto the image also determined inthebeginning, thereremains to be foundthedistancebetweentheobjective andtheobject, which will representapproximatelytheequivalentfocus. Thegeneralformula is, Object, O: Image, I : : equivalentfocus,F 1250. SupplyingtheknownCH.X] TESTPLATEANDAPERTOMETER273values, O-,-, I -];| then |^m.:i 111111. ::F: 250whence F50mm. Thatis, theequivalentfocusis approximately50millimeters.'{434.DeterminationofInitial or Independent Magnificationof the Objec-tive. Theinitial magnificationmeanssimplythemagnificationof the realimage(A'B', Fig. 21) unaffectedbytheocular. It may be determined experimentallyexactlyasdescribed in\ 433.For example, the image of the object ( -j-,, mm.)measuredbytheocularmicrometer, at adistanceof 250mm.is\%mm., i. e., it isfive times magnified, hence the initial magnification of the50mm.objectiveisapproximately live.Knowingtheequivalentfocusof an objective, one can determine its initialmagnificationbydividing 250mm.bytheequivalentfocusin millimeters. Thustheinitial magnificationof a5 mm.objectiveis- T:"-=50;ofa3mm.,-5"=83.3;of a 2 mm.,-}'i =125, etc.\ 435.Determiningthe Equivalent Focusof an Ocular.If one knows theinitial magnificationof theobjective (''/,434)theapproximate equivalent focus oftheocularcanbedeterminedas follows :The field lens must not beremovedin thiscase. Thedistancebetweentheposition of the realimage, apositionindicatedin theocularbyadiaphragm,andthebacklensof theobjectiveshouldbemade250mm.,asdescribedin\ 433, 434,thenbvtheaid of Wollaston'scameralucidathemagnification of the whole mi-croscope is obtained, as described in { 160. Asthe initial power of the objectiveis known,thepowerof thewholemicroscopemustbe due to that initial powermultipliedbvthepowerof theocular, theocularactinglike asimple microscopetomagnifytherealimage (Fig. 21 ).Supposeonehasa 50mm.objective, its initialpowerwillbeapproximately5.If withthisobjectiveandanocularofunknownequivalentfocusthemagnificationof thewholemicroscopeis 50,thentherealimageor initialpoweroftheobjectivemusthavebeenmultiplied 10 fold. Nowif theocular multiplies the real image10fold it hasthesamemultiplyingpoweras a simple lens of 25 mm.focus, for,using the same formula asbefore : =5:1 =50 : : F:250whenceF =25. Thematterasstatedaboveis really very muchmorecomplexthanthis, but this givesanapproximation.For a discussion of the equivalent focus of compound lens-systems, seemodernworksonphysics ;see also C. R. Cross, on the Focal Lengthof Micro-scopic Objectives, Franklin Institute Jour., 1S70,pp.401-402; Monthly Micr.Jour., 1S70, pp. 149-159J. J.Woodward on the Nomenclature of AchromaticObjectives, Amer.Jour.Science, 1S72,pp.406-414 ;MonthlyMicr.Jour., 1872,pp.66-74. W. S.Franklin, method for determining focal lengths of microscopelenses. Physical Review, Vol. I, 1S93,p. 142. Seepp.1119-1131 of Carpenter-Dallingerformathematicalformulas ;also Daniell, Physicsformedical students;Czapski, Theorie der optischenInstrumente;Dippell, NageliundSchwendener,Zimmermann. E. M. Nelson,J.R. M. S. 1S9S,p. 362, 1900, pp.162-169.Jour-QuekettMicr. Club, vol. V.pp. 456,462.\436.Drawings for Photo-Engraving.Theinexpensiveprocessesofrepro-ducingdrawingsbringwithin thereach of every writer upon scientific subjectsthe possibility of presenting to theeyebydiagramsanddrawingsthefacts dis-cussed inthetext. Though artistic ability is necessary forperfectrepresentationof anobject,neatnessandcarewillenableanyoneto makea simpleillustrativedraw-ing, fromwhichan exactcopycanbeobtained andaplatepreparedforprinting.274APPARATUSFORSECTIONING{CH. XAcarefulstudy of thecuts orplates used to illustratethesameclassof factsas one wishes to show will enable one to produce similar effects. Out-lineswhicharetransferredto the drawing paper maybeobtainedbythecameralucida or from a photograph. The drawing shouldbe madesothatit can bereducedanywherefrom one-eighth to one-half. For ordinary photo-engravingfor such line drawings as are usedto illustrate this book, use perfectly blackcarbon ink. A shaded or wash drawing can be reproduced by the half-toneprocess, alsophotographsas is illustratedbyfigures1 90-191. A crayon drawingonstipple paper withshadowsre-enforcedbyinklinesandhighlightsscratchedoutwith asharpknifegiveadmirableresults for anatomical figuresbythehalf-toneprocess. (SeeforexampletheworkofMaxBroedelinContributionstotheScienceof Medicine, (Welch Book) Baltimore, 1900).Forphoto-engravingsof line worktheletters, figuresorwordsusedto desig-natethe different parts canbeputonthe drawingbypastingletters, etc., of thepropersize in the right position. In preparing the block the photo-engravereliminates all shadowsandtheletterslook as if printedonthe drawings.'4437.Wax Models.Largewaxmodels of the objects which one studiesunderthemicroscopeare helpful both to the teacher and to the investigator.These models are becoming more and more appreciated for embr3'ologicandmorphologicinvestigations, for, asonecanreadilyappreciate,theeffortto producea representationof the embryo or organ in threedimensionshelpstoovercomedifficulties whicharealmostinsurmountableif studiedinthesectionsalone.Theyaremadefromwax plates, theprincipleinvolvedbeingthatthediame-terof thedrawingonthewaxplate is asmuchgreaterthantheobjectasthewaxplate is thickerthanthesection.Thewaxplateis cut with a sharpinstrument, following the outlinesof theobjectwhichhasbeentracedupon it bytheaid of acameralucida or theprojec-tionmicroscope. The sections are piled together,some line or lines obtainedfrom a drawingorphotographof the spceimen beforeit was imbeddedandsec-tionedbeingusedasaguidebywhichthecorrect formof thepile of sectionscanbetested. Finally thewholeiswelded into one by theuseof hotwaxorahotinstrument. Modelswhichillustratecomplex internal structures are difficult toprepare, butnumerous devices will occurtotheworkerasthe representationofbloodvesselsandnervesbystringsorwires. Alargemodelwillneed muchsup-portwhichcan be given by wire gauze, wires, pins or paper accordingto thespecial needs.Apracticalmethodforwaxmodeling wasfirst publishedbyG. Born, Arch. f.Mikr. Anat., Bd. xxii, 1S83,p. 5S4. The most detailed statements of improve-mentsof themethodhavebeenpublishedbyBorn(Bohru u. Oppel) 1900, andbyDr. F. P. Mallandhis assistants. See contributionstothe Science of Medicine,pp.926-1045. Proceedings of the Amer. Assoc. Anatomists,1901, 14th session(1900) p. 193.\438.SomeApparatus for Imbedding and Sectioning.AsasupplementtoChapterVIII, the following figures of imbedding and sectioning apparatus areappended. It willbenoticedthatthe microtomes are complexandconsequentlyexpensive. Oneis figured in whichtheknife is movedbythehandsoftheoper-ator (Fig.217). Thisform of instrument is excellent, and with it one can doall kindsofwork, bothwith collodion andparaffin. OnecannotworksorapidlyCH.X] APPARATUSFORSECTIONING275norwiththesame precision. For much of the workonema3'sectionfree-hand,withoutamicrotome. Indeed the great basis of histologicalandembryologicalknowledgewas gained bystudying free-hand sections and dissections. At thepresenttime there is a strong reaction against the exclusivestudyof sections,andatendencyto combine with theserial sectionsdissections suchas the olderanatomists and embryologists made and gainedsomuchfrom.Fig. 217. A Microtomeforall kinds ofsectioning; theknife is guidedbythetopofthemicrotome, but moved by thehands oftheoperator(Bausch &LombOptical Co.)Fig. 218. TheMinotmicrotomeforribbon sections asmadebyBauschandLombOpticalCo. It isarrangedforsectionsfromin to 2511andanyintermedi-ate thickness.276APPARATUSFORSECTIONINGI[CH. XFig 219. TheMinotmicrotomeforribbonsections as madeby theFranklinLaboratorySupply Co.,Boston.This is to bemadeforzn,6u, ion, 14U, 20H,andjoit sections.AFig. 220.Fig. 221. A.CH.A"] APPARATUSFORSECTIONING277Fig. 221. B.Fig. 221.A BFig. 222.Fig. 220-222. A paraffinholder clampanda razorsupportfortheMinotMi-crotome. ( Trans. Amer.Micr. Soc,igoi).Fig. 220. Clampforthe paraffinblock holder. In Ait isshowninsection, ina sideview. Withthisclamponecan use stove boltsaswellastheexpensive par-affin holders furnishedwith theinstrument. Alaboratorycanhaveas many par-affinblockholdersasnecessarywithoutundueexpense.Fig. 22i. RazorSupportandRazor.{A) Supportwithheavybaseand -vertical piece. Thebaseshouldbecapableof movingendwiseoneortwocentimetersto bring theopening in the verticalpartoppositethe paraffin block.(B) Front pieceto therazor(seeFig. 222 A).(C) Razorwithstraight back and edge. Bymovingthisbackandforthonthesupportnearlytheentirecuttingedgecan be utilized.Fig. 222. The knife supportofthe microtome with the razor support andrazorinposition.(A) Frontview;(B) Backview. Inthe inclinationof theknifetoward theparaffin block isshown.278 APPARATUSFORSECTIONING[CH. XFig. 223. Sliding microtomeadaptedespeciallyforcollodionsectioning. {TheBausch &LomhOptical Co.).CH. X] APPARATUSFORSECTIONING279AFig.224ParaffindishforinfiltratingintheLillieoven. Itis made0/cop-per andasshown has a handleforease in transference. Athe wholedish,Bthedish in section.| four. Appl. Micr.iSgg,p. 266).Fig. 225. TheLilliecompartment,paraffin ovenforinfiltratingtissueswithparaffin. Varioussizesofthisaremade I 8, r6and_=y compartments). Exceptforthelargestlaporaloriestheonewith 16compartmentsand trayswill be foundofsuf-ficientcapacity. ( BauschcrLombOpticalCo.).28oAPPARATUSFORSECTIONING \_CH. XFig. 226. Circulation board, especiallyforNeclurus. This is preparedfromaboardaboutS x20centimeters. Nearone edge it hasaholefora perforatedcork.Onthetopofthecork iscementeda thickcover-glasswithshellacorrubbercement.The corkcan be raised or lowered in the board. The gillsofNecturus orthewebof afrog's footcan be spread outon glassover thecork. (four. Appl Micr1898,p. 131.Fig.22;embryologicmaterialandsmallaquaiespecially designedfor collecting with a bicycle (four.Appl. Micr., iSgS,p./j/).227. Copper can with screw topforcollectingJ^icmaterialandsmall aquaticanimals. Itwas