Optical Systems for the Microscope

General remarks

Planapochromats Phase-contrast obiectives

A few wordson the workingprinciple of the microscope

P O L Zo b i e c t i v e s

4 Parfocalizationof objectivesandeyepieces10 Appropriateselection o f i n i t i a lm a g n i f i c a t i o n s of objectivesand eyepieces 12 F i e l do f v i e wa n d v i e w i n ga n g l e 14 Correctionof aberrations 16 The coverglassand its importancefor i m a g eq u a l i t yi n t h e m i c r o s c o p e The immersionmethod 20

51 52

54 56 UD Achromats 57 Special-purposeobjectives 58 ULTRAFLUARS Jamin-Lebedeff transmitted-light interferenceequipment 59 microscopy 60 Objectivesfor reflected-light E P I P L A NH D o b i e c t i v e s 61 62 E P I P L A No

63 64

EPIPLANPOL objectives

65 ZEISSobiectives Classificationof objectives Achromats Planachromats NEOFLUARS Planapochromats ULTRAFLUARS Obiectivemounts ZEISSobjectivesfor photomicrography LUMINARS

23

25 26 27 27

68

Photomicrographic objectives LUMINARS w i t h i r i sd i a p h r a g m Epi-LUMINARS

69 71

28 29 32

Eyepieces C-typeand Kpl eyepieces Eyepiecesfor spectaclewearers Eyepiecesfor micrometerdisks Micrometerdisks Object markerwith diamondtip

72 73

36

Pointereyepieces

74 75 76 77 78

Eyepiecefor stereomicroscopes

80

42

Microprojectioneyepieces

B1

46

Special-purpose eyepieces Grain-sizedisks accordingto ASTM E and VDE h standards

82

Intermediate systemschanging

Stagemicrometers

M ZEISScondensers D a r k -ife l d i l l u m i n a t i o n

EPIP Pho Antifleximmersionobjectives

Integrating micrometer disks Doubleeyepiecewith pointer Centeringtelescope,Klein magnifier, ctrosco

84 86 86

Ing

systems NEOFLUARS

Condensers

88 89

remarks General A few words on the working principle lf objectsor detailsare to be seen clearly, of the microscope a sufficientlylarge image must be formed on the retina of the eye. A measureof the size o f t h i s i m a g e - w h i c hc a n n o tb e m e a s u r e dd i r e c t l y - i s t h e s o - c a l l e da n g l eo f v i e w .I n m a n y c a s e st h i s i s s o s m a l lt h a t t h e d e s i r e dc l a r i t y c a n n o l o n g e rb e a c h i e v e dT. o o b t a i ng r e a t e r clearness,means must thereforebe used to i n c r e a s et h e v i e w i n g a n g l e a n d t h u s t h e i m a o ef o r m e do n t h e r e t i n a . fhe simplest solution consists in approachingthe eye as closelyto the object as p o s s i b l e .T h i s . h o w e v e r ,i s f e a s i b l eo n l y t o a c e r t a i ne x t e n tb e c a u s eo f t h e l i m i t e da b i l i t y of the eye to accommodate.To overcome this difficultywe have to avail ourselvesof lensesor lens systems.The effectof such an auxiliarymeansis, in each case,that the object located a short distance in front of the eye-or even an image of such an object-is imagedat a greaterviewingangle and a sufficient distancefrom the eye to allow observation without any particularstress on our m e c h a n i s mo f a c c o m m o d a t i o n . T h e a i d u s u a l l ye m p l o y e df o r t h i s p u r p o s e is an ordinaryconverginglens of sufficiently , h i c hw i l lf o r m a m a g n iife d s h o r tf o c a ll e n g t hw image of an object located in its focal plane at a distancefar enough for the eye looking t h r o u g ht h e l e n s t o v i e w i t w i t h o u td i f f i c u l t y . T h e e y e w i l l t h e ns e et h e o b j e c tu n d e ra w i d e r viewing angle as if it were viewed from a normal distance.Such a lens is called a magnifier.lts magnificationis definedas the relationshipbetweenthe tangentfunctionsof the two viewing angles,a certainvalue having been adoptedas the normalviewingdist a n c e ,v i z . 2 5 0m m . T h i s i s t h e s o - c a l l e dd i s tance of distinctvision. Using this value,we o b t a i nf o r t h e n u m e r i c avl a l u eV Lo f t h e m a g n i ficationof a magnifierof focal lengthfL: ,r, - -250 v L

-

f ,

lf higher magnificationsare required, simplelensesare no longersufficient.In this case,the requirementsto be made of image quality can only be satisfiedby lens combinations. However,the magnificationwhich can be achieved in a single magnification stage, as in a magnifier,is limited because technicalproblemsonly allowthefocal length to be reducedto a certaindegree:the lenses haveto be curvedever moresteeplyand their diametersthus become smallerand smaller. This results in difficultiesnot only in manuf a c t u r i n g ,b u t p r i m a r i l yi n u s i n g t h e m : t h e viewing distance is greatly reduced, the image brightnessis low and the visual field small. These disadvantagescan be overcomeif two successiveimage-formingsystems are used as is the case in the compound microscope: The first stage of image formation consists of a lens system,the microscopeobjective, located close to the object, which forms a real and magnifiedaerial image of the object, usually at a certain distance from the latter. The relationshipbetween image size and object size,the scale of the image M,ni, is governed by optical laws and is represented by the relationshipof the separation between the image and the primary focal point of the objective,which is called the "optical tube length t", and the objective focal lengthfoui,viz. M o b i=

fobi

The contentof this aerialimageformedby the objective,i. e. the detail of fine object structuresit contains,however,has nothing to do with the scale at which the objective reproducesthe object. On the contrary-as Abbel was the first to prove-it dependsnot

only on the wavelengthof the light used for observation,but primarilyon the light-admitting propertiesof the objective. These are determinedby the apertureangleof the cone of rays from the pencil originatingat the object, which is able to enter the instrument through the aperture of the objective. The measure of this angle is-likewise since Abbe-the numericalvalue of the sine function of half the apertureangle. lf the pencil originating at the object does not pass through air before it reachesthe objective, but through another medium of differentrefractive index, the angle must be multiplied by this index. Becauseof its importancefor image formation in the microscope,Abbe coined the term numerical aperture for the product of the sine of half the aperture angle and refractiveindex. Thus if this rerm is abbreviatedN.A.,as is customaryin microscopy, and o insertedfor half the aperture angle,then we have N.A,:n.sino With regardto the magnifiedimageformed by a microscopeobjectiveit must be noted that two adjacentobject detailswill only be separated,or resolvedas it is called in optical language,if the expression

)'

)'

>a > N . A .: - : 2 N . A .

applieswith regardto their separationd. Consequently, the magnitudeof the separation d to be resolvedis always betweenthe limits ), I ano *-A2NA I II u s t r a t i o n : l l l u m i n a t i n gc o n e m a d e v i s i b l e . The cone of light varies as a function of the numerical aperture.

I Ernst Abbe (1840-1905), physicist and professorat Jena. From 1867worked for the ZEISS Optical Works. Became a Dartnerin t h e c o m p a n yi n 1 8 7 5P . i o n e e ro f m i c r o s c o p ec o n s t r u c t i o nE. s l a b l i s h e dt h e Z E I S SF o u n d a t i o n .

Resolvingpoweral J"= 550nm Table 1 Objective aperture

0.10 0.30 0.65 0.95 1.25 1.40

Separationd (p) N.A. 5.5 1.83 0.84 0.58 0.44 0.39

2 N.A. 2.75

0.92 0.42

0.29 0.22

0.20

An importantfact which is frequentlyoverlooked is that nearthe lirnit of resolutiongiven by the aboveformulait is exclusivelythe distance betweenthe object details which is reproduced correctly. Nothing can be said about their shape. To reproduceobject configurationswith a reasonabledegreeof similarity, we must remain considerably-by a factor of about 5 to 1O-above the limit of resolutionfor the detail in question. The second stage of image formation in the compound microscope is exclusively designedto spread the image produced by the first stage so that all detailscan be conveniently recognized by the eye. For this purposethe aerial image formed by the objective is viewed through a lens systemacting like a magnifierand called the eyepiece. It is natural that even the highest eyepiece magnificationcannot show the eye more than the aerial image in accordance with the resolving power of the objective. There is thus no point using a higher eyepiece magnificationthan is requiredto make the resolveddetailof the aerialimagevisible. Abbe realizedthat it was sufficientto use a total magnificationof the objective-eyepiece system of 500 to 1000x ihe objectiveaperture. He coined the term usefulmagnification

for thisrange. The total magnificatiohVmicris just the p r o d u c to f t h e s c a l eo f t h e a e r i a li m a g eM o o i produced by the objectiveand the eyepiece magnificationVoct, namely 250

Vmicr:+

focI

O n t h e b a s i so f t h i s f o r m u l aw e o b t a i nf o r the total focal length of a compound microscope 250 250 \/ v m r c r-

f"b . f""

-

f micr

t , lmicr:

E y e p ie c e

Intermediate lmage

Objective

Specimen Schematic drawing of the light path in the microscope

fobi ' focl 1

A c l o s e rl o o ka t t h i sf o r m u l ac l e a r l ys h o w s the advantageswhich the compoundsystem has over the simplemagnifyingsystem: D u e t o t h e p o s s i b i l i t yo f c o m b i n a t i o na, multitude of total focal lengths can be achievedwith only a few elementsof different focallength. Since the focal lengths of the different components remain large as compared to the focal length of the overall system, it is possible by appropriate selection of the formerto obtain practicallyany desiredshort overallfocal length. This meansno lessthan that the magnifyingpower of a microscopeis a c t u a l l yu n l i m i t e d .T h a t t h i s u n l i m i t e dm a g n i f y i n gp o w e r c a n n o t b e f u l l y u t i l i z e di n t h e c o m p o u n dm i c r o s c o p ei s d u e t o t h e l i m i t e d resolving power of the objective,which is determinedby the wavelengthof the light u s e d a n d t h e n u m e r i c a al p e r t u r e . However,sufficientlyhigh resolvingpower a n d a m a g n i f y i n gp o w e r h i g he n o u g h t om a k e the resolvedimage detail clearly visible still do not enablea compoundsystemto present the eye with an undistortedimage of this

10 detail. In addition,the errors inherentto a greateror lesserdegreein any imageformed by the lenses have to be eliminatedto such an extent that the overall system guarantees a largely unaberrated reproduction of resolved image detail. While-as we have seen-the resolving powerat a givenwavelengthof light depends exclusivelyon the objective,the image-forming properties are determined jointty by the objective and the eyepiece, although the primaryinfluenceis the type of objective. The magnifying power is determined on the one hand by the objectiveand the eyepiece,but on the other alsobythe mechanical system of the microscope connecting the two,becauseits Iengthdeterminesthe optical tube length. Since the demands made with regardto resolvingpower,magnifyingpower and image-forming properties cannot be satisfiedwith a singleobjectiveand eyepiece system, the connecting element, the body tube, must be designedso that the two componentscan be easilydetachedfrom it. This is why the lenselementsof both the objective and the eyepieceare accommodatedin special "mounts". The former have a standard threadby whichthey can be screwedinto the lower end of the body tube. The simpler, tubular mounts of the eyepiece lenses are slipped from above into the suitablyshaped eyepiecetube. Parfocalizationof objectivesand eyepieces

To facilitate the exchange of objectives, so-called "objective changers" are usually . inserted between the objective and the body tube. The location of the plane separaiing the body tube from the objectiveon the one hand and the eyepieceon the other is determined by practicalconsiderations: The tube should always be in the same positionin relationto the specimen. The image must remain in focus when

11

m : mechanicaltube length a : object-to-image distance b : object distanceof objective : intermediateimagedistance of eyepiece - working distanceof the objective

objectivesor eyepiecesare changed. ln microscope language,this is called "parfocalizationof objectivesand eyepieces". To satisfythe latter requirement,which is essentialfor undisturbedwork with the microscope,the optical tube length cannot be the same for all types of objectives.lt is the distancebetweenthe specimenand the aerial image that must be kept constant. With a given mechanicaltube length this can only be achievedby appropriatedesignof the objective mount. The length of this must be chosen so that the separationbetween the object plane and the undersideof the body tube againstwhich the objectiverests when it is screwed in is the same, and the lens systemis positionedso that the aerial image will always be formed in the same plane in the tube, regardlessof the objective focal length.The measureof the distancebetween objectiveplaneand objectivescrew flangeis the so-calledobject distanceof the objective. ln order that the image will remain in focus when the eyepiecesare exchanged, the eyepiecefocal plane must always coincide with the real aerialimage. In otherwords,the eyepiece flange must also be located at a fixed distance (intermediateimage distance of the eyepiece)from the plane of the aerial image in the tube, and the eyepiecemounts must be so designedthat their focal plane is alwaysat a fixed distancefrom their seating. Severalpoints have to be taken into account when fixing the three mechanical dimensions:ihe o b j e c td i s t a n c eo f t h e o b j e c tive,the mechanicaltube lengthand the intermediateimagedistanceof the eyepiece. The object distance of the objective is preferablychosen so that the objective of lowest power that is used frequentlyas well as objectivesof particulariylong construction can still be parfocalized.On the other hand, however,the objectivesmust not be made

12 excessivelylong or centeringwill become+oo difficult. A reasonablevalue has been found to be 45 mm, which we have been using for all our transmitted-light objectivessince1950. The intermediate image distance ol the eyepieceshould be chosenas short as possible to allow eyepiecesof short focal length also to be parfocalizedwithout difficulty. With our eyepiecesit is 10 mm. The mechanicaltube length should above all be chosenso that the microscopecan be dimensionedto suit its purposewithout bec o m i n gu n w i e l d yT. h e m e c h a n i c a l t u blee n g t h o f o u r m i c r o s c o p e iss 1 6 0m m . For the normal transmitted-lightmicroscope,the combinationof thesethree magnitudes results in a distance of 195 mm between the specimenand the aerial image. Object distance of objective

45 mm

mechanical tube length 1 6 0m m

intermediateimage planeof eyepiece

1 0m m

objectto-image distance 1 9 5m m

Once the tube length has been fixed, the total magnificationof the microscope only depends,in addition,on the focal lengthsof objectivesand eyepieces,i. e. the scale of the aerial image and the eyepiece magnification. Appropriateselection The user of the microscopewill find it of of inilial magnifications advantageif these two factors are chosen so of objectives and eyepieces that a Iarge number of total magnifications can be achievedwith a minimumequipment outlay. Such a series can be consideredas well coordinatedonly if it satisfiesthe followingconditions: The relationshipof every component in the serieswith the one precedingit and the o n e f o l l o w i n gi t s h o u l d b e o f e q u a l m a g n i tude. It should be possibleto obtainserieswith larger increments by leaving components with smallerincrementsout of a basicseries.

13 The values of the different components s h o u l d b e r o u n d f i g u r e so r s h o u l d a t l e a s t b e c l o s e e n o u g ht o r o u n d f i g u r e st o b e e x pressedby them with sufficientaccuracy. The seriesshould be so establishedthat once fixed for a power of ten it can be e x t e n d e da s d e s i r e d b y m u l t i p l y i n gb y 1 0 , 1 0 0 ,e t c . Theseconditionsare well satisfiedby the d e c i m a lg e o m e t r i cs e r i e so n w h i c h t h e G e r m a n l n d u s t r i a lS t a n d a r dD I N 3 2 3 i s b a s e d . T h e w o r k i n g g r o u p o n m i c r o s c o p yi n t h e standards committee of the German Prec i s i o nE n g i n e e r i n g a n d O p t i c a ll n d u s t r yh a s therefore proposed that the R 10 standard seriesfrom this standardbe adooted as the b a s i sf o r a m i c r o s c o p em a g n i f i c a t i o sne r i e s . It is containedin the draft standardon microscope magnifications,DIN 58,886,and is composedas follows: StandardSeries

10 100 1000

l.tc

t.o

12.5 125

to

160

1250

1600

20 200 2000

2.5 25 250

2500

3.2

32 320 3200

6.3

40 400 4000

50 500

o,J

630

80 800

etc.

When deciding on the characteristic values of objectivesand eyepieceson the b a s i s o f s u c h a s e r i e s ,t h e f o l l o w i n gp o i n t s mustbe takeninto consideration: There should be 4 to 5 main objectives with the aid of whichthe rangeof total magnifications requiredfor practicalwork can be c o v e r e ds, i n c eo n l yt h i s n u m b e ro f o b j e c t i v e s can be mountedon the conventionaltype of objectivechanger(revolvingnosepieces). T h e v a l u e sf o r t h e i n i t i a lm a g n i f i c a t i oonf the objectivesand eyepiecesmust also be taken from the aboveseries. Only then is the a d v a n t a go e f t h es t a n d a r ds e r i e sf u l l yu t i l i z e d , viz. that the product of any two figures is a g a i n a s t a n d a r df i g u r e .

14 In view of these considerationswe have chosen the following magnification incrementsfor our objectives. Only in rare cases and in the case of special-purpose objectives have important reasons prompted certain deviations. Series of initial magnifications of objectives Basic series Main series S i m p l i f i e ds e r i e s

2.5 4 2.5 2.5 (2.5\

Seriesof initial magnificationsof eyepieces

4 x 5 x 6 . 3 X8 X 1 0 x 1 2 . 5 x1 6 x 2 0 x 2 5 )