Microscopy fromthe verybeginning
CARLZEISS . Oberkochen/Wuern.
F. K. Moellring
i,;,ENCES AnJD TEC**oIOUO,.#O*^*, Microscopy from the very beginning
). Dr. FriedrichK. Mijllring Dipl.-Biol. l
J l :l
I
'l
member of scieniific staff, Microscopy Laboratory
Microscopy - from the very beginning I st Siep: Microscopy In a nutshell
1 . T h i si s a m i c r o s c o p e 2. And now a bit of practice: we focus a specirnen 3 . A g l a n c eb e h i n di h e s c e n e s , 5 . C a r e a n d c l e a n i n go f i h e m i c r o s c o p e
2nd Step: Microscopy foriho serious worker
6. Nurnericalapertqre, usefuTmagnificatior and limit of resolution 7. Oilimmers'on 8. The question of cover glasses 9. Microscope illumifaiion for more exacting requirements(KohTermethod) 1 0 . L e n g t hm e a s u r e m e n tusn d e r t h em i c r o s c o p e 1 1 . T h e c a r n e r ao n t h e m i c r o s c o p e 12. N4icroprojection
More advanced literaiure
Addiiional'nformation Z E I S S - a n dt h e a r t o f m i c r o s c o p y
5 6 t5 2A 35
37 41 43 51 53 51
59 60 60 62
Preface W h e r e a si n s t r ! c t i o nm a n ! a s d e a l w i i hi h e o p e r a t i o n r i c r o s c o p em o d e l , o f o n e p a r t i c u l am t h i s b r o c h u r ei s l n t e n d e dt o f u r n i s hg e n e r a li n f o r m a t i o n on rnicroscopy,ihat is, facts which apply equallyto a types and even rnakes of rnicroscope. T h e b o o k l e ts e r v e si h r e e p u r p o s e s : 1 ) l t l s i n t e n d e dt o b e a g u i d ef o r t h e b e g i n n e r , w i t h t h e a i d o f w h i c h m i c r o s c o p ew o r k s h o u l db e a p l e a s ! r ef . o m t h e o u t s e t . 2) li will briefly recapliulatethe most essentiaifacts about m croscopy for the benefit of those w h o d o n o i u s e t h e m i c r o s c o p er e g u l a r l y . 3 ) l t i s a s o f o r t h e L r s eo f t h e e x p e r e n c e dm c r o s c o p i s t who wanis to !ntroduce oihers io ihe field of microscopy. H e m a yf i n d o n e o r t h e o t h e ro f i h e h i n t s c o n l a i n e di n t h e b o o k l e th e l p f u lf o r i e a c l r i n g I t s h o u l db e r e m e m b e r e dt h a t w e i a k e m a n y t h i n g s , n d a l l t o o q u i c k l yf o r g e tt h e d i f f i c ut ! e s f o r g r a n t e da w e o n c e h a d w h e n w e m a d eo u r f i r s t a c q u a l n t a n c e
W e w i s h y o u a s u c c e s sa n d a r e s u f e y o u w i l l e n j o yw o r k l n g w i t h y o u r Z E I S Sm i c r o s c o p e .
CABL ZEISS, Microscopy Depariment
6 7 8 -1o
1.This is a microscope l ) E y e p i e c es, i p p e d i n t o u p p e r e n d o f t u b e . T h e e n g r a v e dv a l u e ,e . 9 . , 1 2 . 5 x , l n d i c a i e st h e m a g n i f i c a t i o n o f t h e e y e p j e c e ;m u l t i p l i e db y t h e m a g n i f i c a t i o n of the objective, this gives the tota magnification 2 ) I u b e ( h e r e a n l n c l i n e dt u b e f o r m o n o c ua r o b s e r v a t i o n ) . W h e n t h e s c r e w c o l l a ri s l o o s e n e d a n d t h e t u b e p r e s s e da g a i n s ta s p r l n gi n i t , it can be lifted oui. j ) N o s e p i e c et o . q - i c l e \ c h a - 9 e o - o b e c i ! e s 4) Objecuve designation 40/0.65 1 6 0 / 0 . 1 r7n e a n s : o b j e c u v em a g n l f i c a t i o4n0 x , n u m e r i c aal p e l 1 u r e0 . 6 5 ( s e ee x p l a n a t i o o n n p a g e3 7 ) , c o m p u t e df o r m e c h a n i c at lu b e l e n g t ho f 1 6 0m n a n d 0 . 1 7m m c o v e r g l a s st h i c k n e s s . 5) Specimen siage wiih spring clips. 6 ) C o n d e n s e r( c o r r e c tp o s i t i o ni s a w a y s j u s t b e l o w
STANDAFDKF M croscope
C o n d e n s e frr o n t l e n s s w i n g i n go u t f o r i l l ! m i n a t i o no f l a r g e fo b j e c if i e l d s . 7 l l r i s l e v e r ( c o n d e n s eor r a p e r i u r ed i a p h r a g m ) . 8) Swjng-out filier holder for filters of 32 rnrn diameter. 9) Fine adju€trnentknob. On STANDARD KF Mlcroscopes t h i s a T l o w su n l i m l t e dr o t a t i o n l 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 ( t h e s a m ea p p l i e st o t h e f i n e a d j u s t m e n t ) . 10) Attachable simple substage illuminaior for direct connecuonto the mains (which can be exchangedfor mirror) with rotary switch and ground glass. 1 1 ) B a s ew i i h i h r e e - p o i n si u p p o r t .O n t h e ! n d e r s i d e , iapped holes for screwing down in cabinei as we l as two holes for mounting ihe connecilng brackeis o f s e p a r a t ei l l u m i n a t o r s .
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 i n s t r u c i j o nm a n u a l .l f y o u s h o ud b e u s i n g a m l c r o s c o p ef o r the first ijme, we recommendihai you stari by making youF s e l f f a m i l a r w i t h i t s m o s t e s s e n i i a lc o m p o n e n t s s, u c h a s objective, eyepiece, condenser,coarse and fine adjustments, u s i n g F i g .I a s a g u i d e .
Screwing in ihe objectiv€s
S t a r t b y s c r e w i n gt h e o b j e c t i v e si n t o i h e n o s e p i e c ew, o r k i n g f r o m o w m a g n i f i c a t i o nusp w a r d s P . l e a s ea v o i dt o u c hn g g l a s s 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).
W e n e e d€ p e c i m e n s
Our first acquaintance with a rnicroscope rnay end on a rather cljsappoiniingnoie, because we may not have realized t h e f a c t t h a t a n o r m a lm i c r o s c o p ec a n n o ts i m py b e u s e d f o r magnifying any desired object - such as a fly - (for which thefe are ZEISS stereomicroscopes),but that the oblect we w a n t t o e x a m i n es h o u l da l w a y sb e m o r e o r l e s s t r a n s p a r e n t . he wlng of a fly is a good example. a n d a s f a t a s p o s s i b l eT T h e s e r e q u i r e m e n t si h e r e f o r e u s u a l l y n e c e s s l t a i ei h e s o c a l l e dp r e p a r a t i o no f s p e c i m e n sa, b o u t w h c h d e i a l s c a n b e g a t h e r e df r o m i n t i o d u c t o r yb o o k so n m l c r o i e c h n q!e, seethe secuon on literaiure, page 59. For ihe purpose of the present d e s c r i p i i o nw e m u s t a s s u m el h a t s u i t a be s p e c i m e n s o f w h i c h , i n c i d e n t a l l ya, w l d e c h o l c e i s o f f e r e d b y c o m p a n i e s specializing i n t e a c h i n ga i d s - a r e a v a l a b l e .O u r s p e c i m e n s a r e u s u a l l ym o u n t e do n a s o - c al e d s l d e , a p i e c e o f g a s s a b o u t2 6 x 7 6 m m l n s i z e a n d 1 m m l h i c k ,w h i c hi s c o v e r e db y an exiremely thln cover glass (various sizes; thickness as c l o s e a s p o s c i b l et o 0 . 1 7 m m ) . - B e s u r e t h a t t h e c o v e r g l a s sj s a l w a y so n t o p , f a c i n gt h e o b j e c t i v e
T h e m i c r o s c o p es t a n d so n a i a b l e s o t h a i y o u c a n t o o k i n r o m e e y e p r e c er r o m a c o n v e n i e n p t o s t u r e .M i c r o s c o p e sw i t h o-i a b-ilt-:n oest sei _o - f,onr oI a aTp w t f . l d r g e J f o r n y e n i ! . 9 s u . { " c e . ( W o - < w - n s p e cd l rnicroscope i l u m i n a t o r si s d i s c u s s e do n p a g e 4 5 ) .
ileight of condenser,
T h e c o n d e n s e ri s i n p l a c e a n d t u r n e d o r p L r s h e du p a t h e w a y . T h e c o n d e n s e frr o n r l e n s i s s w u n gi n . T h e e y e p i e c ew b e i n s e d e d l a t e r . A t i h e b e g i n n i n go f a n e x a r n i n a i i o nw, e a r w a y s u s e a o w - p o w e r o b l e c t v e o f a b o u r6 . 3 o . 1 0 x , b e c a u s ei t p e r r n i t sc o v e r a g eo f a w i d e r f i e l d a n d i s t h u s b e s t s u l t e df o r s c a n n i n g .I n a d d i t i o n ,i s d e p t h o f f j e t d i s g r e a t e r ihan thai offered by objedives of higher power, and ii is i h ! s e a s e r t o f i n d t h e p l a n eo f b e s i d e f i n i r i o n _
Whaiis themagnificarion?
T h e p o w e r o f a n o b j e c t i v ei s n d i c a r e db y i t s n i t i a lm a g n i f i caiion. This s ihe f rst one of rhe values engraved on every objective (see page 28). The toral rnaqnificaiionof rhe micro s c o p e s d e t e r m i n e db y m u l i i p l yn g r h e m a g n i f i c a t i o o nf i h e objective with that of the ey--piece,which s engraved on the l a i t e r . A 1 0 x o b j e c t i v eu s e d i n c o n j u n c t o n w i t h a i 2 . 5 x e y e p i e c et h u s y i e d s a t o t a l m a g n i f i c a t i oonf 1 2 5 x .
C a u io n i n s p i t eo f b u [ - i n s l d e p r o t e c u o n l
The specmen has been fxed on ihe stage,and we now c a u t i o u s l ym o v e s p e c i r n e na i d o b j e c t i v ec o s e r t o g e r h e rb y ihe coarse adjustment, uniil they are onty a few rii imoters apart (observe from the s de!). I n Z E I S S m i c r o s c o p € sa, s t o p a n d t h e s p r i n g m o l n t o f r h e o b j e c t i v e sp r e v e n to r d l n a r ys p e c i m e n s( 1 m m s l i d e , 0 . 1 7m r n c o v e r g l a 6 s )f r o m b e i n g d a m a g e db y t h e o b j e c t i v e .O n t y a ' " w s p e ca l p r p o s eo b l e ( t . - . a e e(ep-o^s
T h e o b j e c t i v ei s ' l u m i n a t e d
B e f o r e n s e i r i n gt h e e y e p i e c eo f l o w e s t p o w e . j n t o t h e i u b e , w e o p e n t h e j r s d i a p h r a g mo f t h e c o n d e n s e ra n d t u r n t h e i l l u r n i n a t i nm g i r r o r u n t i l t h e b a c k l e n s o f t h e o b j e c i i v e ,w h i c h i s v i s i b l ea i t h e l o w e r e n d o f t h e t u b e , i s e v e n l y i u m i n a r e d . W h e n i h e a t i a c h a b l es i r n pe s u b s t a g ej l u r n i n a t ofro r d i r e c t c o n n e c t i o ft o t h e m a n s s h o w ni n F t g . 1i s u s e d , t h i so p e r a i i o n i s n o t r e q u i r e d .A f t e r t h e e y e p i e c eh a s b e e n i n s e r t e d ,w e
c o s e t h e c o n d e n s e rl r s a b o u t h a l f w a y ,l o o k i n t o r h e e y e p i e c e a n d i n c r e a s et h e d s t a n c e b e t w e e nt h e o b j e c t i v ea n d t h e s p e c j m e nb y t h e c o a r s e a d j u s t m € n tu n t I d e t a ts o f t h e s p e cm e n c a n b e r e c o g nz e d ,e v e n i f t h e y a r e s t l l b l l r e d .
E x a c t f o c l s i n g i s n o w a c h i e v e dw t h t h e a i d o f t h e f i n e adjustment. n ihe case of spec mens of very low conrrasi, f i n d i n g t h e f o c a p a n e r n a y o c c a s i o n ay b e s o m e w h a r d l f f l c L r l ti;t i s f a c l t a t e d i f d u r i n g o p e r a t i o no f r h e c o a r s e a d j u s i m e n i h e s p e c i n r e ni s s l o w l y d i s p l a c e do n t h e s t a g e , b e c a u s em o v j n gs i r ! c t u r e s c a n b e r e c o g n i z e dm o r e r e a d i l y . C l o s i n gt h e c o n d e n s e irr i s f u r i h e rm a y a s o b e h e l p f ! . W h e n t h e a r e a i o b e e x a m l n e di s i n i h e c e r i e r o f t h e f i e d o f v i e w , i h e o b j e c t i v eo f r e x t h i g h e rr r r a g n i f i c a i i omna y b e m o v e dt n t o p o s i t i o nf,o r w h i c ho n y t h e f i n e c o n t r o li s n e e d e df o r f o c ! s i n g (parfocal objectives see page 29).
D o . o t s i o p d o w nt o o f a r
T h e a d j u s t m e no t f t h e c o n d e n s e ri s a p a r t i c u l a r t yf r e q u e n t s o u r c e o f e r o r . T h e c o n d e n s e ri s c o r r e c t l ya d j u s r e dw h e n i i s i n l t s h i g h e s ip o s l t i o no f d i . e c t l y b e l o w t h i s a n d w h e n i t s l r s l r a s b e e . c o s e d o i l y f a r e n o u g hr o o b i a i n j u s t s u f f c l e n t c o n i r a s tT h e b e g n n e r u s ! a y s l o p s t h e c o n d e n s e r d o w nf a r i o o m u c h N . e v e ru s e t h e c o n d e n s e r s t o d m t h e i m a g e lO i h e r m e a n s , e . g . g r a y f l t e r s , m u s t b e L r s e df o r i h i s p u r p o s e . L o w e r n g i h e c o n d e n s e r" i o j n c r e a s ec o n t r a s t ' w h l c h s p o p ua r w j t h m a n ym c r o s c o p i s t so n l y h a s t h e s a m e e f f e c t a s f u r i h e r c l o s i n gt h e c o n d e n s e ri r j s a n d s t h e r e f o r e l f w t h o b j e c i i v e so f v e r y l o w p o w e r i h e u r n i n a t e df e l d s h o u l db e t o o s m a , s w n g o l t t h e f . o n t e n s o f i h e c o n d e n s e r a n d o p e n i s d j a p h r a g mi u l l y .
ALwayskeep a hand on ihe fineadjustment
T h e d e p t ho f f i e l d l n t h e s p e c i m e nc o r r e s p o n dn g t o a c e r t an s e t t i n go f t h e f i n e a d j u s t m e njts e x t r a o r dn a r y s h a l l o w( m o r e p r o n o u n c e dw j t h h i g h - p o w e r o b j e c t v e s t h a n w i t h l o w e r p o w e r s ,m o r e w j t h h i g h - q u a l i i yo p t c s t h a n w h e n t h e s e a r e l e s s g o o d ) .T h i s i s i h e r e a s o nw h y n m c r o s c o p i cw o r k i h e f i n e a d j u s i m e nits c o n t i n u o u s l yr n o v e du p a n d d o w n n o r d e r t o c o v e ri h e e n t j r ed e p t h o f t h e s p e c i r n e n .
W h e nt h e f n e a d j u s t m e n tf a i l s
lvlost microscopes {excepi theSTANDARD KF) have a I rnited f n e a d j u s i d r e nrta n g e ,a n d i t m a y h a p p e nt h a ! u r e u p p e r o r o w e r s i o p i s r e a c h e dd u r i n g o b s e r v a t i o nA . g t a n c ea r i h e s i d e o t t h e r a c k a n d p n i o nw c o n f i r mt h i s ( F i g . 2 a ) . t n i h i s c a s e ,r o t a t ei h e f i n e a d j u s t m e nkr n o b u i r i t i h e i . d e x I n e i e s r o u g r r l yi n b e i w e e n t h e i w o o r h e r s ( F i g . 2 b ) . T h e n r e f o c u s w l t h i h e c o a . s ea d j u s t r n e nai ,n d y o ! w i t t a g a i nh a v es ! f f i c i e n r L . r p p earn d l o w e r m a r g i nf o r f i n e a d l u s r m e n r .
poinis: Two rnportant
W h e n ! s n g o p t c a l i n s t r u r n e n t ss,u c h a s a m i c r o s c o p eo r a l e l e s c o p e t w o t h i n g sa r e o l g r e a t i m p o r t a n c ea n d s h o ! d b e ' ) r h e e / e ' L o u l d b e e " ' e d w h e n l o o k i n gi n l o t 5 ' e / e o e 2 ) t h e e y e s h o u l d b e k e p t a i a c e r t a i n d i s i a n c ef r o m t h e
r e a x e da c c o m m o d a t i o n B e g l n n e r so f t e nn a k e i h e m i s t a k eo f f o c u s i n gt h e m i c r o s c o p e as if the magnified mageihey want to view were ocated v e r y c l o s et o t h e e y e ,a s c o s e a s a p o s l a g es t a m pw h l c hw e w a n t 1 0 s t u d y i n d e i a f o r i n s t a n c eT. h s n e a r s e t u n go t t h e e y e ( = a c c o r h m o d aoin ) i s h o w e v e r e x t r e m ey t i r i n gl l r n a i n t a i n e d f o r a l o n g e ri i m e . W e t h e r e f o r er e c o m m e n dw o r k i n g w i i h p e r f e c t L yr e a x e d e y e s . A t t h e b e g i n n i n g t h l s i s d o n e m o r e e a s i l yl f w e i m a gn e t h a t w e a r e v i e w i n gt h e m a g e a s "into ihe m croscope i f i t w e r e a t i n f n i t y .T h u s d o n o t l o o k but thro!gh the microscope". l f y o u a r e n o t q u i l e c e r i a j n a s i o w h e t h e ry o u a r e r e a l l y w o r k i n gw i t h r e a x e d e y e s ,m a k ei h e f o o w i n gt e s l : F i r s tf o c u s t h e s p e c m e n .I h e n h o l d a r n i r r o r! n f r o n t o f y o u r s e c o n de y e ,w h i c hi s n o t l o o k i n gi n t o i h e r n i c r o s c o p es,o t h a i y o ! c a n l o o k o l t o f t h e w i n d o w o r a i a n o b j e c ta l t h e o t h e r e r o o l L h er o o n W - e _ y o u L " - I o r q o dt i s e . e " - _ e f a l o b j e c t ,t h e o t h e r e y e m l s t a t t h e s a m e u m e s e e i h e m i c r o s c o pc l m a g ew l i h r n a x i m u md e f n i i i o n .l f t h i s i s t h e c a s e ,y o u are slre that you can work n an absolutely relaxed'siate. A l w a y sr e m e m b e lrh a t t h e m i c r o s c o p ec a n b e r e f o c ! s e dw i t h t h e a d o f t h e f i n e a d j u s i m e nat n d c a n t l r u s r e L e v ey o u r e y e c f the experlenced o f a c c o m m o d a t i o nl t i s c h a r a c t e r i s t i o r nc r o s c o p j s t h a t h e k e e p s p l a y L n gw t h t h e f i n e a d j u s t m e n t . l n i h i 6 w a y h l s e y e s l v l n e v e rg e t t r e d , o r a c i l a y h ! t 1
a n d g o o d c o n i a c t "o f t h e p u p s
T h e s e c o n di m p o r t a n p t o n t i s t h e c o r r e c td l s l a n c eo f t h e e y e f r o r nt h e e y e p l e c eS . h o ud i h e e y e b e b r o u g h ia s c l o s et o t h e a s w t h a r n a g n i f i e rG ? e n e r ay n o t , e y e p i e c ea s p o s s l be b e c a u s et h e c o r r e c t e y e p o i n t a s t h i s d s i a n c e i s c a e d , v a r i e s a c c o r d i n gt o t h e e y e pe c e u s e d .T h e b e s t p r o c e d u r e s t o m o v e i n f r o m a f e w i n c h e s d i s t a n c eu n t i Lt h e f i e l d o f
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'croscope s 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 6ye rrom eyepece
Eyepuprrredu.esrhe fierd.
L e t ! s i o w p a ! s e t o c o n s i d e rw h e r e y o u m a y s l i l l e n c o ! n t e r d i s . u l l . i n . o . r r i - 5 - dq . a i , r c n ( rer . er '.o opi s L a t e r a l lrye v e r s e d
Y o u h a v e ,o f c o l r r s e ,n o i i c e dt h a t t h e m i c r o s c o p i cm a g e s u p s d e d o w n . f y o ! d i s p l a c et h e s p e c r n e n1 c 'o n e s i d e , t h e l m a q e m o v e s i n t h e o p p o s i t ed r e c u o n .Y o u w g e 1 a c c u s t o n r e dt o t h i s s o q u c k l y i h a t y o ! w s o o n f o r g e t t h e f a c t
Y o u c € n n o to b t a i na s h a - pi m a g e
L t m a y h a p p e ni h a t y o u c a n n o t g e t a s h a r p i n r a g e o f t h e s p e c i m e ni n s p i l e o f e v e r y i hn g y o ! d o . n t h s c a s e ,t h e t n e " d . J " _ n e_ L c e r L e r e d e d " s t o o r ' e r e d . - e p r g e 9 l o t r - o b e r . e r o c F e , r h e , o e u i n " n b e ' o _ e" s ' " o - r a g e s o b t a i n e dT . h e n t h e c o v e r g a s s i s e i l h e r f a r l o o t h i c k ,o r y o ! h a v e l n a d v e r t e n l l yp l a c e dt h e s p e c r n e nw r o n g s j d e u p o n i h e s l a g e .W h a t e v e rt h e c a u s em a y b e , l h e s p r n g m o u n t o f y o u r Z E S S o b j e c tv e p r e v e n t sd a m a g et o l h e s p e cm e n .
O v a l f l e l do f v e w ?
I t s n o i o n l y t h e b e g l i n e r w h o s u d d e ny f i n d s t h a t t h e f e l d o f v e w i s n o t r o l n d . I n t h i s c a s e ,e t h e r t h e o b t e c u v eh a s n o t f u y e n g a g e di t s c l c k s i o p p o s t o n o r t h e f i t e r h o d e . b e l o w t h e c o n d e n s e ro r n f r o n l o f t h e I l n r i n a t o ri s n o t s w u n g i n
' M o u c h e s! o a . t e s '
A n o t h e r p h e n o m e n o nm a y a t f i r s t b e n e x p i c a b e t o t h e b e g i n n e r S p e c m e n s w i t h a w i d e c l e a r b a c k g r o u n dm a y s h o w s t r a n g e ,i r r e g u l a rp a t c h e s ,s h a p e d r a t h e r l k e w o r r n s , w h i c hr e m a i ns t a t i o n a r yw h e nt h e e y e l s f x e d , b u t w h i c hs t a r t r n o v i n gw h e n t h e v e w l n gd f e c t i o n s c h a n g e dT. h e s ea r e i h e s h a d o w so f i n h o m o g e n eei ts i n t h e a q u e o u sh u r n o ro f i h e e y e , w h c h b e c o m ev i s i b l e w h e n e v e rt h e e x t p u p i l o f l h e m c f o s c o p e b e c o m e sv e ' . / s m e l l : j f t h e c o n d e n s e ri r i s i s c l o s e d d o w n t o o f a r o r f a h g h - p o w e re y e p e c e s ! s e d . ( l n c d e n t a y t h e s a m es h a d o w sc a n b e s e e n I y o u l o o k a t a u n t o r m s k y . H o w e v e r i,h e s k y m u s i b e b r i g h t b e c a u s eo n y t h e n d o t h e e y e p ! p i l s c l o s ef a r e n o u g h . )
O b l q u e ! u r n i n aot n
A s t r a n g ee f f e c l m a y o c c u r w h e n t h e i m a g e i n t h e m l c r o s c o p e a d o p t s a c e r i a i n r e i e f a p p e a r i n ga L m o s t o h a v e a 'shlft ihe image. t h l r d d i r n e n so n , a n d f o c u s n g s e e m s t o q u e I u m n a to n " . A T h s a w a y s h a p p e n si f y o u h a v e o b g l e n . e i n t o t h e t u b e w t h t h e e y e pe c e r e m o v e dw i i ! s h o w
12
y o u t h a t t h e b a c k l e n s o f t h e o b j e c t v e i s n o t u n i f o r my i l l u mn a t e d .T h s c a n b e c o f f e c t e db y r e a d j u s r i n g rhe minor
K e e pb o t h e y e s o p e n
I y o u a r e n o t u c k ye n o l g h t o h a v ea b n o c u l a rt u b e f r o m t h e v e r y s t a r t ( s o t h a t y o u c a n l o o k i n t o t h e m c r o s c o p ew i i h b o t h e y e s l , o b s e r v a t i o nw i i h o n y o n e e y e w i i l a t f i r s t b e s o m e w h a td f f c ! l t . W e t h e r e f o r e a d v s e € v e r y b o d yw h o i h r o w s r n o r et h a n l u s t a n o c . a s l o n a lg a n c e n t o t h e m c r o s c o o et o k e e p h i s s € c o n de y e o p e n f r o n rt h e v e r y s t a r t .T h e i m a g e s e e n b y t h e s e c o n de y e , w h j c h i s n a t u r a l l ys u p e r i m p o s e d o n t h e m i c . o s c o pc m a g e w i l l b e f o u n d d l s i u r bi g o n y i n t l r e b e g n n n g a n d w I l a t e r b e s u b c o n s co u s y s ! p p r e s s e d .l n o r d e r n o t t o h a f m t h e u n l s e d e y e b y l h i s f o r n r o f d i s c o r n e c t i o n, t h e e y e s s h o ud b e L s e d a l t e r n a t e l y .
A . a c c e s s or : t h e e r e s ( r e e n
Cosing one eye for a longerperiod of lme s very lirjng. Ll you canrot get acclsiomed io leav.g ihe secondeye o p e na n d i f t h e a c q ! i s i o n o f a b i n o c l r l atru b e s n o t p o s s i be a c a r d b o a r ds c r e e n m a y b e s l p p e d o n i o t h e t u b e n s l c h a m a n i e r t h a t t h e s e c o n de y e i s c o v e r e d .S o m e p e o p l e m a k e h " , ro . \ b i . o .p-o1 ( F g . 4 , n o t c o m m e r ca l l y a v a l a b l e ) .
A n y Z E S S m j c r o s c o p ec a n e a s i l yb e c o n v e r i e df o f b n o c u l a r o b s e r v a t i o ns n c e i h s o n l y m e a n sc h a n g i n gt h e l u b e
Wjlhor witho!t eyeglasses?
H e r e a s o t h e q l e s t o n a r i s e sh o w e y e g l a s sw e a r e r ss h o ! d / o r k T t s Fb " . L of e - p e g t o r . - o a n d t o l s e s p e c l a e y e pe c e s f o r e y e g l a s sw e a r e r s .B l b b e r g u a r d sa v o l d s c r a t c h l n go f t h e e y e g l a s s e s .
L ft h i s i s n c t p o s s b e , y o u s h o ! d f i r s r o f a f n d o l t w h e r h e r y o u r e y e ga s s e s h a v e s p h e r i c a o r t o r i c e n s e s .T h i s c a n b e d e t e m n e d v e r y e a s l y . H o d y o u . g l a s s e si n f r o n t o f y o ! a t a r m ' s e n g i h : t h e e n s e sw e r h e f r e d u c eo r m a g n t f ya n o b j e c to n i h e i a b l e .T h e n r o t a i et h e g l a s s e sa b o u rt h e c e n t e r o f t h e e n s t h r o l g h w h c h y o u a f e o o k n s . l f t h e s h a p eo f i h e ob 6.
dob
oi cha od d
r'-
g as
otdteo
. e
6
13
h a s o n y s p l r e r i c apl o w e r t h o w e v e r ,i f l e n g t ha n d w i d t h o f t h e o b j e c t c h a n g ed u r n g r o t a i i o n ,t h e g a s s e s h a v e r o r i c e n s e s ( F i g . 5 ) . l n t h e l a t t e rc a s e y o ! w i l l h a v e i o k e e p t h e g a s s e s o n o r h a v e a u x l i a r y l e n s e s m a d e f r o m y o u r e y e g l a s sp r e s c r i p t i o nw , h i c ha r e t h e n s l i p p e do n r o t h e e y e p i e c e s( e n s u r e correct orentaton!) lf your glasseshave only sphertca enses/o. on p o.e Lher .o ^orl n rl Lhe . o. ope a n d n o o s s o f s h a r p n e s sw i l l b e o b s e r v e d .I n r h s c a s e i h e v j s u a l d e f e c t i s c o m p e n s a t e db y t h e a d j u s t m e n io f r h e
3. A brief glance behind the scenes A f t e r y o u h a v e f o c l s e d t h e s p e c i r n e n sr n a c c o r o a n c ew r t h t h e e x p l a n a t i o nlsn i h e p r e c e d i n gs e c t i o n ,y o u w i k n o w t h a t y o u r m c r o s c o p ed o e s w o r k . H o w e v e r ,y o u w t l o r t y d e r i v e f u l l s a u s f a c t o nf r o m i t f y o L rk n o w a t l e a s ts o m e i h i n ga b o u t h o w i t w o r k s . l t i s n a t u r a lr h a i i h t s " g t a n c e b e h i n d t h e only be a brief one, and lsers s t u d yt h e i h e o r yo l t h e m c r o s c o p ej n m o r e d e r a ta r e r e f e r r e d t o f l r e l t e r a t u r ei n d i c a i e do n p a g e5 9 . A s a n o p U c a i n s t r u m e n rr h e m i c r o s c o p ef o r m s a m a g n j f i e d l m a g eo f a s m € l io b j e c t .
H o w l s a f i r n a g ef o r m e d ?
b r i g h t n e s sa n d € h a r p n e s s
W e m u s tf i r s t o f a l l ! n d e r s r a n dt h € f o r m a u o no f a n i m a g e ,f o r w h i c h w e s h a l c h o o s ea v e r y p r i mt i v e e x a m p l e . f y o u a r e s t a n d i n gi n a d a r k c o r r d o r a n d t h e k e y h o l eo f a d o o r t s o f a p p r o p ra t e s i z e y o u c a n c l e a r l ys e e a p j c i u r eo f t h e w i n d o w i n t h a t r o o m p r o l e c i e do n t h e o p p o s i i ew a l l o f r h e c o r i d o r t p i n h o l e c a n r e r a ) .Y o u i m m e d i a t e l yr e c o g n i z e w h a t s h a p p e n l n ge: v e r y p o i n t o f i h e w l n d o w e m i l s l i g h r r a y s n a l i d r e c t i o n s .W h i l e m o s t o f t h e m e n d o n i h e w a s o r a i t h e door, a few of them pass through the keyhole (Fig.6). C o n s e q u e n i l ya, b r g h t p a r t o f t h e w t n d o wp r o d u c e sa b r i g h t p a t c h o n t h e w a i l , a r d a d a r k p a r t p r o d u c e sa d a r k p a r c h A n d b e c a u s ea l l i g h t p a t h s c a n j u s t a s w e l l b e f o l l o w e di n i h e o p p o s i t ed i r e c to n , w e m a y a l s o s a y t h a i e v e r y p o f t o n t h e w a l i o ft h e c o t r i d o r ( = u n i t oaf r e a l s e e s a m o r e o r r e s s b r i g h ts p o t o f t h e w i n d o wt h r o u g hi h e k e y h o l e( F i g .7 ) .
T h e d r a w b a c ko f s u c h a n m a g e i s a b o v e a l i t s l o w b r i g h i n e s € w h i c hw o u l d i n r m e d i a t e lbye i n c r e a s e di f t h e h o l e w e r e l a r g e r .S o l e t s m a k e i t a r g e r T h r o u g hi h e b i g g e r h o e , a u n i l o f w a l l a r e a n o w s e e s " n o i only ihe dark flower pot, blt aso ihe brightwindow pane b e s i d e t ( F i g .8 1 .A s a r e s l l i , i h i s ! n t i s n o w n e i t h e rd a r k n o r b r l g h t ,b L r tg r a y - t h e d i v i d i n gl i n e b e t w e e nt h e p o t a n d t h e p a n e i s n o w b l u r e d i n i h e i m a g eo n t h e w a l . I n o t h e r w o r d s , t h e t w o p o i n t s a t a d s i a n c ed a r e n o o n g e r r e s o l v e d ' l . e . , i h e y c a n n o l o n g e rb e r e c o g n j z e ds e p a r a t e l yN. a l u r a l l y , i h e b g g e r t h e h o l ei h e m o r e b l u r r e d s t h e l m a g e .T h i s i s d u e to the fact that in a p n-hole camera ihe rectilinear propag a t i o no f l i g h t d o e s n o t m a k e a p o i n t c o r r e s p o n dt o a n o t h e r t5
l l
a
v/t
I
16
I
point, but io an area whjch increases in size, the bigger the opening is (Fig. 9). A l e n si s t h e s o l u t i o n
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).
T h e r e s o n l yo n e p l a n e
A s a n e x a m p l e ,l e t u s c o n s i d e ra c a m e r at a k i n ga p i c t u r eo f a p l a n t .T h e l g h i t r a v e l sf r o r n a n y p o i n i o n t h e p l a n t j n a l d i r e c to n s ( F i g .1 l ) . A p a r t o f t h e l l g h t r a y s i s l n t e r c e p t e db y t h e o b j e c t i v el e n s ,r e f r a c t e da n d r e c o m b i n e di n t h e f i l m p l a n e l f b e h i n dt h e p l a n t t h e r e i s , f o r e x a m pe , a w i n d o w ,t h e n t h e ray6 emanatingfrom there do not recombine exactly in the film planebut in front ol it, aid on the filrna circleof diam e t e r I i s i n a g e d i n s t e a do t t h e p o i n t :t h e w i n d o w i s o u t o t "Focuslng c a n b e a c h i e v e do n l y b y a r e l a i i v e f o c u s( F i g . 1 2 ) . b d i s p l a c e m e n i e t w e e n e n s a n d f i l m p l a n e .T h i s s h o w s t h a t ! n a n i m a g ef o r m l n gs y s t e m a g i v e n o b j e c t p a n e i s a w a v s . o ' r e d t e or o " d . r ' l e p l a n eo ' " F a p I n a g e l
H o w b i g w i l l t h e i m a g eb e ?
Apart from the locaiion of the irnage pLane, however, ihe o b j e c i i m a g e r a t o i s l i k e w i s ef i x e d . T h i s r a i i o i s n o r m a l l y e x a c t l yi d e n t i c a l w l i hi h e r e a t o n s h i pb e t w e e no b j e c t d i s t a n c e (objeci-lensd stance) and lmase distance (lens'image
(Fis.13).Therorrnula *""a ,r'r" -"d, 4-i distance) S l n c ei h e i m a g ed i s t a n c eb i n t h e c a m e r ai s s m a l l e r ,u s u a l l y m u c hs m a l l e rt h a n t h e o b j e c t d i s t a n c ea , t h i s o p t i c a l j n e t r u m e n i o f c o u r s ef ! r n i s h e s r e d u c e dp i c t u r e s .
W e b u i l da m o d e lm i c r o s c o P e
and f rst imageJorming system
18
W e n o w k n o w e n o u g hi o t r y a n d b u i l d a r n o d e m c r o s c o p e frorn the cornponenis we have Learnt to understand - a c o n c r e t em o d e li f p o s s i b l eo, r o t h e M l s e a n i r n a g i n a r yo, 'n e
T h e m i c r o s c o p ei s i o m a g n i f y ;t h e r e f o r et h e c a m e r ag l v j n g r e d u c e d m a g e s i s n o t s u i t a be a s a b a s i s f o r o u r m o d e l microscope. We are ooki.g fo. an opiica inslrurnentwhich p r o v i d e se n l a r g e di m a g e s ,i . e . , n w h i c h t h e i m a g e i s m l c h further away from lhe lens than the object SLrchan instrum e n t i s f o l n d i n t h e f a m i i a r s l i d e p r o j e c t o r( F l g 1 4 r A = object,ihe slidei B = screen imagei L = proiection ensl S l n c e i n t h i s c a s e t h e o b j e c t t h e s l i d e ,d o e s n o t e r n i t L i g h t i t s e l f , i t m l s t b e i l u m i n a t e df r o m b e h i n d L e t u s i h e r e f o r e p l a c e a L a m pb e h i n dl t ( F l S .1 5 ) .H o w e v e r ,t h e s i d e j s m u c h l a r g e rt h a n t h e l a m p f a r n e n ia n d i h e l e r s d i a m e t e r s, o i h a t o n l y t h e i g h i r a y s p a s s i n gt h r o l g h i h e c e n i e r o f t h e s l i d e
r e a c ht h e l e n s . B e s u l t :t h e f e d s n o i f l t l y u m j n a i e d i,h e m a r g i n a zl o n e s r e m a i nd a r k - T h t s c a n b e r e m e d i e db y a ight source which is at leasi as arge as ihe stde. An i l i u m i n a t e dg r o u n d - g l a s s c r e e ni s s u i r a be f o r t h i s p u r p o s e . T h e l g h t s t r i k i n g i h s s c r e e n i s d e f l e c r e di n a d i r e c i t o n s ( F l g .1 6 ) -S u c ha s c r e e na l s o a o w s i h e j g h t r a y s t r a n s m i i t e d b y t h e p e r i p h e r a pl a r t s o f r h e s l i d e t o e n l e r r h e t e n s .H o w e v e r , t h i 6 i s r a t h e ru n e c o n o mc a l b e c a u s et h e m a j o r p a r r o f i h e i g h t r a y s s d e f e c i e d i n d i r e c r i o n si n w h c h w e d o n o i need any light. A better solution is a tens (here called c o n d e n s e rw ) h c h d i r e c t sa l l i n c t d e n tr a y s e x c t u s i v ey t o t h e p o i n tw h e r et h e y a r e r e q u t r e d t: o i h e p r o j e c t i o ne n s ( F i g . j 7 ) . W e t h l s h a v et h e f a c i i h a t i n t h e f i r s i p l a c e t h e l i g h t s o u r c e s i r n a g e db y t h e c o n d e n s e ri n t h e p r o j e c r i o nl e n s ,w h i t e i n t h e s e c o n d p a c e a n r n a g eo f i h e s t i d e i s f o r m e d b y r h e p r o j e c i i o nl e n s o n t h e s c r e e n :a s o c a t l e d n r e r t o c kn g l g h r p a t h s u c h a s w e f i n d i n t h e m i c r o s c o p ew t h t h e s o , c a e d K d h l e rm e t h o do f i l l ! m n a t i o n( s e ep a g e4 5 1 . T h es e c o n d m a q e J o m n g s y s t e m
Let Ls now turn to the enlarged mage on the projection s c r e e n .H o w b i g w e s e e t h i s m a g ed e p e n d sq l i r e d e c i s i v ey o n l h e d s t a n c e f r o m w h i c hw e v e w i r . f w e m o v e h a t f w a y u p t o t h e s c r e e n ,w e s e e t h e i m a g ee x a c t l yr w i c e a s l a r g ea s b e f o r e ,i . e . , w i i h 2 x r n a g n f i c a t o n " . t w e d r a w a l i n e , a s s h o w n i n F r g .1 8 , f r o r no n e e d s e o f t h e p i c t u r e1 ot h e e y e o f i h e o b s e r u e ra n d f r o m t h e r e b a c ki o i h e o t h e r e d s e , a n a n g l e ( c ) i s s u b t e n d e dw , h i c h s t h e a n g l eo f v i e w . T h e r n a g n i f i c a t i oins d e r e r r n i n e d soletyby the magnl t u d e o f t h s a n g l e .F o r n o r m a ls l i d e p r o j e c ro n w e s h a l l n o r m o v e t o o c o s e t o t h e s c r e e n ,b e c a u s et h e u n s h a r p n e s so f i h e i m a s e ( e 9 . , d u e t o s r a i n ) w o ! d b e d i s t u r b i n gF . or our experiments we therefore Lrse a microscop c specimen n s i e a do f a s l i d e ,f o r e x a m pe a f e e , w h i c h ,c o n t r a r yt o t h e s i d e r e v e as e v e r n e w d e t a s e v e n L r n d e rc o n s i d e r a b l e m a g nf l c a u o n . n t h e i f t e r e s t o f h i g h m a g n i f i c a r i ow n e may, o f c o u r s e ,m o v e ! p a s c o s e a s p o s s i b l et o r h e r n a g e ,b u t eventua y ou. own shadow w ll nierfere We therefore look a i t l r e p i c t u r ef r o m t h e b a c ko f t h e l r a n s p a r e npt a p e r s c r e e n . The fact thai ihe picture is thus aierally reversed need not w o r r y u s . T h e n e x t s i e p : w e e x c h a n g et h e p a p e rs c r e e nf o r a g r o u n d - g l a s ss c r e e n a n d , n a d d i t i o n ,u s e a m a g n i f i e rf o r v l e w i n gt l r e p i c t u r e .A m a g n i f i e rc a n i n c r e a s et h e a n g e o f v i e w a n d i h u s t h e m a g n l f l c a t o nc o n s l d e r a b l yA . n object
(Fig. 19) viewed once with the naked eye from a reading distance" of 10 ins. and then through a magnifier is seen under considerably higher magnificationdue to the refraction of rays. But ihe magnifier also magnifies rhe texture of the g r o u n dg l a s ss o t h a t i t b e c o m e sd i s t u r b ' n g( F i g s . 2 0a n d 2 0 a ) , and we therefore investigate whai happens if we simpty remove rheground glass. The image witt 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
(see the corespondinsrLtudraudns
s h a r p n e s sb, u i c o n . r ' o L r e st o b n g h t e n i n gt h e ,rd,g,ra,zones o r r n e n a t d b a c € u s er h e r a y s p a s s i n gt f e s e z u , r e 6 are rrans -nill-ed I n i o t h € e y e ( T , 9 . 2 2 ) .f h e s e t r e r dt e n s e s , a s r!ey are , r h e . e r o . e ca eo. are becer tha- Srourd Srass"s ana ,n @ o r f l o n , i r e y d o n o t . i n l r o d u c ev o - b t e s o m e g . a i n irlo tte rtr€g€ trn otherwords we have here a case which:s very s r n l a r l o r h e o n e n e n r , o n e da b o v e , w n e - e we had a tens p r a c e da s a c o n d e n s e rb e t w e e n, a n p a n d s , i o e_ t i k e w i s e as a ground-gJasssubsiitute). Tle- effrciency ol our noder p.oveo by rhe ." fte ro ow:rs p€se. rhese are 1ol l i -r*.:rlo- :s c o p ; ci T a 9 e s senurne m b . t i n a g e s a c . L € J Jpyr o d u c e db y a 2 r 2 p r o j - c t o ra n d t w o s o e c t a c t et e r s e s .O n e s p F c t a c , er e n sw a s u s a o a s a t i e t d i e r s , l h e o r h e r a s a r e y e t e n s .r l e eye was represented by a reftex camera wjth its jens focused at inf'nity.
i h s s w h a i r h ei t e r m e d r a tm e a q eo o k r k . a.c ob3eiy:d sr.!qE a m.sn fer tF n 20)
remore.r(Fiq ,r)rrra srah i6s d epp*E
o p i n a r e s !t s , r e p r o d r c e d o i y w h e n! 3 n s
24
: r o m t h e n r o d e l t ot h e
V e r y f e w c h a n g e sa r e r e q L r t r efdo r t h e s t e pf r o r nt h j sp r o j e c i o r w t h m a g n i i e . v e w n g t o t h e r n c r o s c o p e .T h u s r h e i m a g e d s t a n c eo l s e v e r a J e e t s i o o a w k w a r d .W e s h o l l d a t s o i k e t o h a v e b o t h o p t c a l s y s i e m s .v : . . i h e o n e c a L sn g r h e f i r s r m a g n t c a t o n ( t h e o b j e c t v e )a n d i h e o n e m a q n f y n g t h i s r m a g ea s e c o n dt m e c o m b n e d n o n e n s ! r ! n e n i . T h e e n t r e s e i u p c a n b e k e p t m l c h s m a e r b y a p p r o p r a r es e e . i i o n o f b ,l . ! . w 6 -e . e - d o , o . - o t. .-o o.op" aroLrnd t h s i g h t p a t ha n d k e e p i n m n d t h a i t h e p a t h o i r a y s s u s ! a l y f c d e d ' f o r g r e a t e cr o n v e n i e i c e a i d w e h a v e a r e a m i c f o s c o p{eF s . 2 3 )
o @
T h e p r i n c i p l ei n a n u t s h e
T h e p r i n c i p l eo f i h e m i c r o s c o p en r a yi h u s b e s u m r n azr e d a s T h e m i c r o s c o p eo b j e c tv e f i r s t f o r r n s n t h e t l b e a m a g nf l e d a e r a l i m a g e( n l e r r n e d i a tiem a g e )o f t h e s p e c i m e ni u m i n a t e d f r o m b e l o w b y t h e l a m p a n d t h e c o n d e n s e r .H o w e v e r ,t h i s i m a g e i s n o t i n t e r c e p t e d ,b u r v i e w e d u n d e r m a g n i f i c a t i o n t h r o u g ha m a g i i f i e r( c € l l e de y e p i e c e )
T h e d e s i g no f i h e l i g h i p a t h
n t h e f o r e g on g p a r a g r a p h sw e h a v e t r t e d t o e x p a n i h e b a s i cf a c t so f t h e m l c r o s c o p ea s c e a r y a s p o s s i b l e ;i n d o i n g s o , h o w e v e r ,w e w o u l d g i v e n o e x a c i d e s c r i p t o n .t n i h e f o t l o w i n g ,i h e r e a d e rw h o s m o r e f a m i l a r w i t h o p t i c a ls u b j e c t s w i l l f i n d i n s t r u c t i o n sf o r t h e d e s i g n o f a r n i c r o s c o p et i g h t p a t h .I n p r j n c i p l et,h s i s r e a y q u t t ee a s y .
F i r s t m a g e j o r m i n gs y s t e m
Fig. 24 shows the objective Ob with rhe two foc F I and F 2. T h e s p e cm e n o f s i z e A B i s i o c a r e ds l t g h t t yo u i s i d et h e f o c a l e n g i h . F r o m A t w o r a y s a r e t r a c e d : o n e p a r a l t e lt o r h e o p i c a l a x i s t h r o l g h i h e f o c u s F 2 , a n d i h e o i h e r i h r o u g ht h e lens center, which is therefore noi refracred.The iniersection o f b o t h r a y s g v e s t h e i r n a g eo f A , A ' j i n t h e s a m e m a n n e r , B ' r e s u l t sa s t h e i m a g e o f B . T h e i m a g eA ' B ' r h u s o b t a i n e d l s e n l a r g e d r e a i ( i . e . , i t c a n b e i n t e r c e p t e da) n o r e v e r s e o . T h i s c o r r e s p o n d si o i h e p a i h o f r a y s i n t h e m c r o s c o p eu p t o theflrst aeria inrage.
. . . a n ds e c o n dm a g nf c a t o n
n a n o t h e rc a s e [ F j s . 2 5 ) w e a r a n s e a l e n s O k n s u c h a m a n n e ri n r e l a i i o r t o a n o b j e c t B ' A ' t h a i t h e l a l r e ri s s h i f t e d s l i g h i l yo f f t h e f o c a Lp o i n t n t h e d i r e c t i o no f t h e l e n s . T h e d e s i g no f t h e p a t ho f r a y s i s a n a l o g o u sA: f t e r b e i n gr e f r a c t e d , t h e p a r a l e l r a y p a s s e si h r o u g ht h e f o c a l p o i n l ,w h l e t h e r a y i h r o u g ht h e e n s c e n i e r t s n o t d e f l e c t e d S . ill, the two rays do nor intersectr only their extensions ro ihe rear have a po nt of intersecuon a i B " o r A " . T h e m a g eB " A " i s l i k e w i s e e n l a r g e db , u t e r e c t a n d v i r t u a l ,i . e . , t c a n n o tb e i n t e r c e p t e d b e c a u s et h e l i g h i r a y s d o n o i r e a l l yi n t e r s e c a t t t h e p o i n t sB " a n d A " . - T h i s i s t h e l i g h t p a t h o f a m a g n i f i e ra n d t h u s a s o ofamcroscopeeyepiece L fw e c o n n e c it h e s et w o l i g h tp a t h si n s u c ha m a n n e rt h a i t h e mage formed by Ob s at the same iime the objeci for Oc, t h e n w e h a v e t h e t w o - s t a g el m a g ef o r m a io n o f a c o m p o u n d m i c r o s c o p e( F i g 2 6 ) .
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 waveoptical 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 t h e r e s t l r e m a i na f e w f a c t s t h e u n d e r s t a n d j n o gfwhich s l ork. o t l m p o r t a n c ef o r p r a c t i c aw Objective Engraving a n d o t h e rc h a r a c t e r i scis
T h e o b j e c i i v e i s e n g r a v e d w t h a v a l u e ( F i g .2 7 ) w h i c h n d i c a t e sf a r m o f e t h a n i n i t i a m a g n l f i c a 1 ! oant o n e . L e r u s r e p e a ti h e e x a m pe o n p a g e 5 40/0.65 I60/0.17 m e a n s , n i l i a l m a g n f c a t i o n 4 0 x ( r n u l ip e d b y e y e p i e c e m a g n l f i c a i i o n= t o t a l n r a g n f c a t i o no f m i c r o s c o p e ) n; u m e r c a a p e r t u r e0 . 6 s ( f o r e x p l a n a o t n, see page 37Jr for use w t h m c r o s c o p e sw i t h a m e c h a nc a l i u b e e n g t h o f 1 6 0 m r n (d*d-.e oe.^ae' ob,.'r - rl".qe cnd e.eo.ce s". 9 f a c e )a n d w t h a c o v e r g a s s o f 0 . 1 7 1 0 0 1 m m t h c k n e s s . n s t e a do f 1 0 . 1 7t h e e n g r a vn g m a y a l s o r e a d / = i n s e n s l t i v e t o a r g e r d e v i a l l o n so f t h e c o v e . g a s s i h i c k n e s s / 0 = c o r r e c t e d f o r " z e r oc o v e r - qa s s t h i c k n e s s . e . f o r u n c o v e r e d O b j e c i i v e sw i t h o ! t a n y f u r t h e . e n g r a vn g a r e i h e w l d e l y ! s e d a c h r o r n a t s .O t h e r e n g r a v e da b b r e v i a i i o n sf r e q u e n t l y P l a n = P a n a c h r o r n ait. e f l a t - f i ed a c h r o m a t Neofuar = high'aperiureobjective (see pase 37) wiih m p r o v e dc o l o r r e n d i t i o nh, l g h c o n t r a s i ,b ! t w t h o u i f a t f i e l d . P l a n a p o= P a n a p o c h r o r n a tor b j e c t j v e o f o p t m ! m c o o r c o r r e c to n , m a x i m l m € p e r t l r e a n d f a t f i e d . L J l t . a f u a r= s p e c i a o b j e c t i v e f o r L rt r a v o l e t r l 1c r o s c o p y c o t r e c t e df o r w a v e e n g t h sb e t w e e n2 3 0 a n d 7 0 0 n r n .
n
E p p a n = f a t - f e l d o b j e c t i v ef o . v e r t i c a b r g h t ' f e d i u E p p l a n H D - f l a t ' f i ed o b j e c t i v ef o r v e r t c a b r g h t f L ed a n d d a r k - fe l d i l i u m i n aot n ( i n c o n j ! n c to . w i t h c o n c e n t rc r e f e c t i n go r . e f . a c t i n gc o n d e n s e r ) . P O L = s t r a i nf r e e o b j e c t i v ef o r p o l a rz e d g h t m c r o s c o p y . P h 2 = p h a s ec o n t r a s i o b j e c i i v e f o r d i a p h r a g m2 o f t h e p n a s ec o n r r a s rc o n o e n s e r s . m . l . = o b j e c t i v ew t h i r i s d i a p h f a g m a s o s u t e d f o r t r a n s r n t t e d d a r k J i ed I u r n i n a t i o n .
Oel .9 Glyz E E
.t
glycerin
5 1 5 )b l a c kr i n g 333)whiie r ng 455) orange rins 7401yel ow rlnq
K o n . - h i s h a p e r t u r e o b l e c t v e w i t h c o r r e c t i o nc o a r i o a l l o wf o r d i f f e r e n ic o v e F ga s s t h i c k n e s s e s( s e e p a s e 4 3 ) . n a d d i t i o n ,c o l o r e d r . g s n e a r t h e k n u r l e d r n g f a c i l i i a t e r e c o g n i i i o no f i h e d i f f e r e n t m a g n i f i c a t j o n sbi l a c k- 1 x , brown 2.5,
F o r m e r l y ,c h a n g l n g o b j e c t i v e s w a s r a t h e r i n c o n v e n i e n i , b e c a u s et h e t u b e h a d t o b e l f t e d b e f o r e i u r n i n g i h e n o s e p e c e , a n d e v e r y o b j e c t i v eh a d i o b e f o c u s e d s e p a r a t e l y . Today, tl.e objectives are parfocal . Th s means thai they a r e o p t j c a l y a n d m e c h a n ca l y d e s g n e d s o t h a t i h e d l s i a n c e b e t w e e n t h d s p e cr n e n a n d t h e a e r i a l m a g e i s a l w a y s c o n s t a n t .S l i g h t r e f o c u s i n gw i t h t h e a i d o f i h e f i n e a d j u s t nrent is therefore s!fficient to resiore critical sharpness of t h e i m a g e a f t e i c h a n g i n gf r o m o n e o b j e c t i v et o a n o t h e r , w h i l e t h e c o a r s e a d j u s t m e n tn e e d n o l o n g e r b e o p e r a t e d . T h i s f e a t u r e i s p a r t i c u l a r yv a l u a b l ew h e n w o r k i n g w i t h o i i i m m e r s i o ni,n w h i c h c a s e t h e d e p t h o f f o c u s i s s o s m a l lt h a t focusing wlthout padoca ized objeciives usually presents c o n s i d e r a b l ed i f f l c u i t y .( O n l y t h e e s p e c i a i l yo n g P l a n a c h r o mat 1 objecuve ls not parfoca ). a n di h e l rp r e r e q u i e l i e s
I t s h o u l d ,h o w e v e r ,b e n o t e d t h a t a f e w c o n du o n s m u s i b e s a t s f i e d t o m a i n t a i nt h e p a r f o c a s e t t i n g o f t h e o b j e c t i v e s : j n t h e f i r s t p l a c e , o n y e y e p i e c e se n g r a v e d" C A R L Z E I S S ' must be used (oiher eyep eces are des gned for a different l o c a i i o no f t h e a e r l a l j m a g e ) ;s e c o n d l y ,t h e o b s e r v e rm ! s t h a v e n o r m a lv s i o n ( o r t h e v i s u a d e f e c t m u s i b e c o t r e c t e d b y e y e ga s s e s o r a f o c u s l n ge y e l e n s ) ia n d f i n a l l y ,c o r e c i t u b e e n g r hi s e s s e n t i a( w i t h b r n o c u l a r i u b e s , i d e n t i cvaa t u e s m l s t b e s e t a t t h e e y e p i e c et u b e s a n d t h e w h e e b e t w e e n t h e e y e p i e c e sS . ee page32.).
N o r m a l m e d i u mt o h g h - p o w e ro b j e c t i v e sh a v e a r e t a t i v e l y s h o r t w o r k i n gd s t a n c ea n d m a y k n o c ka g a i n s t h e s p e c i r n e n u n l e s ss p e ca l c a r e i s t a k e n . n o r d e r t o p r e v e n id a m a g er o 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, p a r i c ! l a r y t h o s e f o r o i l i r n m e r s i o nc, a n b e l o c k e d i n t h e r e t r a c t e dp o s i t o n b y c o c k w i s e r o l a i o n . T h s i s i o a v o i d s o i l i n go f t h e s p e c r n e nw t h o l w h e n i h e n o s e pe c e i s t u r n e d . A very important h ni regarding h gh dry objecrives wt I be f o ! n d o n p a g e3 9 ( c o r r e c t i o nc o l l a r ) .
A s i n g l et y p e f o r a l l o b j e c tv e s
E y e p l e c e fso r s p e c t a c l ew e a r e r s
r0t obledve,3x ayepiec= e
30
Wh le a few year6 ago il was necessary to use differefl types of eyep ece for olv power and high-power objectives ( H u y g e n sa n d c o m p e n s a t i n se y e p i e c e s )Z, E I S S m c r o s c o p e objectives are now generaliy used with compensaung o c u a r s . C o n s e q u e n t y e y e pe c e s n e e d n o l o n g e r b e e x c h a n g e dw h e n c h a f s n s o v e r f r o m o f e o b j e c t i v et o a n o i h e r . l n s p t e o f t h l s , t w o c l a s s e s o f e y e p i e c ea r e s i t b e i n g m a n u f a c t u r e d .W h i l e i h e s m p l e C + y p e e y e p i e c e s a r e p r i m a r ly u s e d l n c o n j ! n c io n w i t h a c h r o m act o b j e c i i v e si h e K p l ( c o m p e n s a t i nfga t f e l d ) e y e pe c e s a f e i n t e n d e df o r t h e o t h e r o b j e c t i v e so f h i g h e r c o r r e c t i o n b l t r n a y ,o f c o u r s e , a l s o b e c o n r b i n e dw i i h a c h r o m a t s .
T h e m o s l w i d e y u s e d e y e p i e c em a g n f c a t o n s a r e 8 x a n d 1 2 . 5 r . F g . 2 8 s h o y T sa l s i o f a l l r e s u l i a n tm a g n l f i c a u o n e w h i c h c a n b e o b l a i n e dw l t h o u r m j c r o s c o p eo b j e c t v e s a n d P a r t i c ua r m e n i i o ns h o u l db e m a d e o f t h e e y e p i e c e s p e c i a l l y d e s i g n e df o r s p e c t a c l ew e a r e r s .A s w a s m e n t i o i e d a r e a d y o n p a g e 1 0 , i h e e x i t p u p o f t h e m i c r o s c o p ea n d t h e e y e p u p i lm u s tc o j n c i d ei f i h e e n t i r ef i e d o f v e w i s i o b e c o v e f e d . W i i h n o r m a l e y e p i e c e s l,h e d i s t a n c eb e t w e e ni h e e d g e o f i h e e y e p i e c ea n d t h e e x i t p u p i l ( e y e p o i n t ) i s s o s h o r t t h a t e y e g l a s s e sc a n n o tb e k e p t o n i f t h e t w o p u p i l sa r e t o c o i n c i d e . T h e h g h e y e p o l n t o f t h e s p e c i e l e y e p i e c e sf o r e y e ga s s w e a r e r sr n a k e st h i s p o s s i b l ee v e n w h e n t h e e y e g l a s s e sa r e n o t r e m o v e d .T h e r u b b e r g u a r d s s u p p l i e dw i t h t h e s e e y e p i e c e sp r e v e n ts c r a t c h i n go f t h e s p e c t a c l el e n s e s -. f t h e s e
(1600) 125 0 1000 600 630 500 100 320 250 200 160 125 t00 60 OJ
50 10 32 25
20 125 t0 0 63 5
s p e ca l t y p e s o t e y e p i e c ea r e u s e d b y o b s e r u e f sn o r w e a r i g g a s s e s ,c a r e m L r 6bt e t a k e n1 o k e e pt h e e y e sa t a n a d e q u a i e d i s t a n c ef r o m t h e e y e p i e c e sl.f n e c e s s a r yf l, n d f 9 s r e c o r r e c r d l s t a n c ec a n b e f a c i l i t a t e db y s t i p p i n g e y e c u p s o n t o r h e
F i e l d - o f -e vw nurnber
A p a r t f r o m t s i n t a l m a g nf r c a to n , a n e y e pe c e s c h a f a c terized by rs fied ofvew nlmber. Wiih the ad of ihs n u m b e r l i i s e a s y i o c a l c ua t e r h e d i a m e t e ro f i h e f i e l d c o v e r e di n t h e s p e c i m e n p l a n e , b e c a u s e r hi si s( i n m i m e r e r s ) eq!a to the fleldof-vtew number djvjded by the ini!al r n a g n i f i c a t i oonf t h e o b j e d i v e .T h e f l e t d - o fv i e w n u m D e r so r i h e m o s tf r e q u e n t l yu s e d e y e pe c e t y p e s a r e g i v e n b e l o w j
K p l w d e - a r g l ee y e pe c e
5 x 6.3r 8 X 8 ) < 10 )< l0 r 12.5.1 12.5' ( 12.5).. 16 I 2 5 X
20 18 16 18 16 t6
18 10 6.3
E x a n r p l eai 1 0 ) . o b j e c t i v ei n c o m b n a t i o nw t h a l 2 . S x e y e p e c e p e r r n i t sa f j e l d o f 1 . 2 5m m d i a m e t e rt o l l r e s p e cm e n p l a n e .
T h e m c r o s c o p ec a n e a s i i yb e e q u i p p e df o r b i n o c u l a ro b s e r v a i i o n b y e x c h a n gn g i h e o r d i n a r yi u b e f o r a b i n o c u l a rt u b e . T h e e y e p l e c es l e e v e so f i h l s i ! b e c a n b e d i s p a c e d i n r e l a r i o n i o e a c h o t h e r o n a s l d e t o a l l o w f o r a d a p t a r i o nt o r h e i n d v d l a l i n t e r p u p a r y d i s t a n c eo f t h e o b s e r v e r .l f y o u k n o w y o u r i n t e r p u p ial r y d s i a n c e ,y o L rm a y s e l i h i s v a l u e o n t h e w h e e j n t h e c e n i e r b y d r a w i n gt h e t u b e s a p a r t . f y o u d o n o t kfow this value,look into the eyepiecesand vary ihe d i s t a n c eb e i w e e nt h e t u b e s u n i i l t h e f i e l d s o f v i e w s e e n b y b o t h e y e s c o n c i d ee x a c ty . T h e v a l u et n d i c a t e do n t h e c e n i r a l 32
wheel rnust then also be set on each of the eyepiece sleeves.This restoresthe co rrect .nechanical tu be lengthwhich has been changed by the inierpupillary disiance setting, at the same time ensuring that the objectjves are padocal (see p a g e2 9 ) .
l f y o u s h o u l dh a v e t h e i m p r e s s i o ni h a t y o u r v i s u a l a c u t y i s different in the two eyepieces, adjusi the eyepiece s eeve o n i h e p o o r s i d e u n t i l b o t he y e s s e e t h e s a m e ,s h a r p l yd e f i n e d
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 m e t h o do f i l l u m i n a i l o n{ s e e p a g e 4 5 ) .
I u r n i n a t i n lga r g ef i e l d s
O b l i q u ei l l u mn a t i o n
I
T h e r e q u i r e r n e n tm s a d e o f a c o n d e n s e ra r e p a r t l y c o n t r a 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 t h a n t h e c o n d e n s e ri l l u m i n a t e st h , e c o n d e n s e lrr i s i s o p e n e d fu y and the front ens swung out. Condensers without a s w i n g - o u l e n s u s u al y a l l o wt h e f r o n t e n s t o b e u n s c r e w e d l i f e v e nt h i s i s i r n p o s s i be , t h e n l t i s n e c e s s a r yt o c h a n g et h e
F o r p h a s e - c o n t r a sj ltl ! m i n a t i o na s p e c i a lc o n d e n s e ri s u s e d , the design and operation of which are described in the per t i n e n t i n s t r u c u o nm a n u a l .T h e s ep h a s e c o n d e n s e r sa r e a l s o s u t e d ' o s o - c a e d o b l i q u ei u - r i n a lo n . a n d f o r d a r k - f e l d i l l u m i n a t o n . A f t e r t h e m i c r o s c o p eh a s b e e n a d j u s t e du n d e r b r l g h t - f e l di l l u m i n a t i o nt ,u r n t h e t u r r e i p l a t e "l" (iris, f r o r n i t s c l i c k - s t o pp o s i t i o n i . e . , b r i g h tf i e l d )t o t h e e f t o r r i g h t , a n d y o u w l l l i m m e d i a i e l yn o t i c e t h e t h r e e d i m e n s i o n ael f f e c i w h i c h i s t y p i c a l o f o b l i q u e j l u m i n a t i o n . Shortly before ihe field of view is completely obscured as y o u k e e p t u r n i n gt h e p a t e , o n e - s i d e dd a r k j i e l d i l l u m i n a t i o n
Nofrna i.e., darkjie d ilumiraton from all sdes can be o b t a n e d w i t h o w a n d m e d u m p o w e r o b j e c t v e sb y c h o o sn s a n n ! l a r d a p h r a g m so f i h e p h a s e ' c o n t f a sct o n d e n s e rw h c h , d ! e i o t h e r s i z e ,r e m a l no u t s i d et h e o b l e c t i v ea p e d l r e t t h i s c a n e a s y b e c h e c k e db y l o o k n g i n t o t h e t l b e a f t e r h a v i n g removeo rne eyep ece. F o r f u d h e r d e t asl o n s p e c i ad a r k J e d c o n d e n s e r sr ,e f e r e n c e s m a d et o t h e b r o c h ! r e G 4 C - 1 6 5 .
5. Care and cleaning of the microscope An instrurneniwhlch has to sat sfy the most exacring requ re, m e n t s r e g a r d l n gr n e c h a n t c aa n d o p t i c a l p r e c s i o n n a t u r a y d e m a n d sa c e r t a i na m o u n to f c a r e .
Enemy No.1: dust
D u s t i s p r a c tc a l l y e v e r y w h e r ea n d m a y b e c o r n ea s o l r c e o f t r o l b e w h e r e v e r - a s n t h e n r i c r o s c o p e- . f e c h a n i c a l p a r t s a r e m o v n g i n p r e c i s i o ng u i d e s .W h e n t h e t r a c k s a r e c lr t y t h e y n e e d w a s h i n g a n d r e g r e a s i n g whlch shoutd p f e f e . a b l yb e d o n e b y t h e m a n u f a c r u r e r . D u s t o n o p ! c a l e l e r n e n t sw i l l d e g r a d et h e i m a g e q u a lt y t o a greater or esser exteni. lt is true rhai a surfaces exposed t o d l s t a r e a l s o e a s i y a c c e s sb e f o r c e a n n g , b u t n o g a s s s u r J a c ei s i r n p r o v e db y ' c l e a n i n g ' ,s o t h a t i h e b e s t a d v i c e i s a l w a y s i o a v o i d e x p o s L r eo f t h e m c r o s c o p et o d u s t b y c o v e r i n gi t u p w i t h a h o o d w h e f n o t n l s e o r b y k e e p i n gi t i n a c a b l n e rS . p e c r a lc a r e s h o u l db e r a k e nr o e n s u r et h a t t h e tlbes of the m croscopeare always cosed ether by an e y e pe c e o r a d u s t p u g .
D r t y o b j e c tv e f r o . i e . s
W h i l e d l s t p a r t l ce s o n t h e e y e p l e c ew o n l y g v e r s e t o p a i c h e s i n i h e i m a g e - w h i c h n o i e v e r y o b s e r v e rf i n d 6 d s t u . b n g - a d i r t y o b j e c t j v ef r o n i l e n s m a y h o p e l e s s l y r e d u c et h e s h a r p n e s so f t h e r n a g e ,o r a t l e a s t i t s c o n t r a s t . D L r et o t h e f a c i t h a t j t l s c l o s e t o t h e s p e c m e n a n d p o s s i b l y t o i h e l m m e r s j o no i l o f i h e c o v e r g l a s sb, u t p a r t c u l a r y t o t h e h a n d o p e r a tn g t h e n o s e p i e c et,h e f r o n t l e n s o f t h e o b j e c t i v e -le is - .p.ri"l d.9e- o" gerr.9 soled. t.Snresf i n g e r p r i n tm a y h a v e g r a v e c o n s e q u e n c e s( F i g s . 2 9 , 3 0 J . B e f o r es i a r i i n gi m p o r t a n tw o r k t s a d v s a b e p a r t i c ua r l y i f t h e m i c r o s c o p el s n o t u s e d b y o n e o b s e r v e ra o n e - t o lnscrew every oblectve and check t carefuy wiih the ad of a magnifierD . r t i s e a s y r e . o g n z e d f t h e o b j e c i i v ei s h e l d s o t h a t t h e i m a g eo f a l g h t s o l r c e i s r e f l e c t e df r o m i t s p l a n es u r f a c e .l n t h e c a s e o f o b j e c t v e s w h i c h h a v a e concave f r o n l e n s , a d l f f e r e n ta p p r o a c hs i n d i c a t e dv, i z . ,i o e x a m j n e i h e s u r f a c eo f t h e f r o n t l e n s f r o m t h e s c r e w t h r e a ds i d e .T h e r e m a i n i n gl e n s e e r n e n t sc a n a s o b e e x a r n i n e de a s l y a n d a n y f a u l s ( c r a c k s ," s t a r t n g o f t h e c e n r e n Od e t e c t e dw i t h -
a.re6n objecrivefroni lens siheftur prerequie 3 f o r b . t i a d m a s a s( r o p l Evena;nsefpl rt oi rhe f.ont Lens w rru n rhe contrud (bordh).
35
H i n t so n r r o u be - s h o o tn g
S h o u l ds t r ! c t u r e sb e f o u f d i n t h e i m a g e w h i c ha r e s u s p e c t e d o f b e l n ge x t r a n e o u st o t h e s p e c l m e nt,h e f a ! l t n a y b e t r a c e d l f t h e t r o u be c a n b e e l l m i n a t e db y s i g h t a d j u s t m e not f t h e c o n d e n s e rt,h e c a u s em u s tb e s o u g h ii n i h e b u l bo f t h e a m p , i h e l a m p c o n d e n s e ro r t h e f l t e r i n f r o n t o f t . F l o w e v e r i,t a d j u s t r n e not f t h e c o n d e n s e rd o e s n o t p r o d u c e a . y r e s u l t , t h e n e x i s t e p s t o t u r n t h e f o c u s i n ga d j l r s t m e nwt ,hi c h s h o u l d e i i r n i n a t ea I t a u l t sd u e i o s o i l i n go f t h e c o n d e n s e fr r o n t e n s o r t h e s p e c i m e nl.f t h l s d o e s n o t l e a d a n y w h e r es, l i g h tv t u r n f r s t t h e o b j e c i i v ea n d t h e n i h e e y e pe c e , a n d y o L rw i l l l m r n e diately notice in which case ihe foreign body lollows ihe D u s t p a r t c e s a r e m o s ! c l e a ry s e e n w h e n t h e a p e r t l r e s t o p h a s b e e n f u L L yc l o s e d , b e c a u s ei n i h i s c a s e l h e d e p t h o f focus is al its greatest,
C l e a n i n gb, u t h o w ?
l n a l r n o s ta l l c a s e s t w i l l b e s u f f i c i e nt o c l e a nt h e o u l e r l e n s faces wlth the a d of a srease'free brush (jf necessarv,wash n e t h e . f i r s t ) o r w l t h a f r e q l e n t v w a s h e d 'a b s o l u t e l vd u s t f f e e i n e n r a g a n d d i s t e d w a t e r ,p r o d u c € de a s i l yb v b r e a t h i n g u p o nt h e s u r f a c et o b e c e a n e d l f a n o r g a nc s o l v e n ic a n n o tb e d l s p e n s e d w i t ht, i s a d v l s a be r o u s e v e r y l i t t l ee t h e ro r b e n z i n ej n s t e a do f w a t e r ,b u t n e v e r alcoho which mght desiroy the cernent between ens E t h e ri s u s u a y p r e f e r r e db e c a u s ei t e v a p o r a t e sr n o s t q u l c k L v a n d a n y h a r r n f u le f f e c t j s t h u s l e s s i k e l y F i n a l y , r e s i d L r e s are aLwaysremoved with water as descrlbed above. a i r b e a v a a b l e f o r c l e a n i n g ,b e s u r e t o S h o u l dc o r n p r e s s e d use a f lter of cotton wool. lf the alr in the work roorn conlin!ousy has a relative h u r n i d i t yo f m o r e t h a n 6 0 0 / 0c e r t an p r e c a u t i o n ss h o u l d b e . o not t a k e nt o a v o l df u n g u sg r o w i h o n t h e o p t i c a le e r n e n t sD k e e p m i c f o s c o p e su n d e rp l a s tc c o v e r s ,d o n o t s i o r e t h e m ! n c a b i n e i s ,b u t e n s u r e g o o d v e n t i l a l i o n ,i n e c e s s a r yw i t h t h e a i d o f a f a n . I n a p a r t j c u l a i yh u m i dc l r n a t e i t s a d v i s a b l et o k e e p o p n c a l p a r i s n p e f e c t l y a r p r o o f c o n t a i n e r sp r o v i d e d w i t h a d e s sc a n t o r l n w h j c h f o r i f s t a n c e ,a a m p a n d a f a n c rcllate a r of 40 +0 o C (100-1 20 o F) - Our agents have s p e ca l i n s t r u . io n s a n d w l l b e p € a s e dt o a s s i s ty o u .
6 . N u m e r i c a la p e r t u r e , useful magnification and limit of resolution
ihegalrinthesde
t o a d i f f r a c u o ng r a i n
T o u n d e r s l a n dt h i s q u e s t i o nb e t t e r , l e i u s n o w t h i n k a g a n oi the prolector and the projecied sl!de described n s e c t j o n3 . A t f i r s t w e o b s e r u ei h e l a n d s c a p eo n i h e s c r e e n f r o m f a r b e h n d t h e p r o j e c t o r . l n t h i s c a s e , m a g n i f i c a t i o jns s t i l l l o w , b e c a u s et h e a n g l e o f v e w i s q u t e s m a l l . A s w e m o v e c l o s e r t o t h e s c r e e n ,r n a g f i f i c a t i o ni n c r e a s e sa n d w e b e g l n t o r e c o g n j z ed e i a j s w h l c h h a d o r i g i n a l l ye s c a p e do u r a i l e n t i o n .B u t f i n a y a p o i n t s r e a c h e dw h e r e h i g h e rm a g n l fication (= movns closer) does noi gve better resuts. l n s t e a do l s i n g e e a v e s o n t h e t r e e s w e t h e n o n l y s e e t h e s k u c t u r eo f i h e c o l o r f i l m , a n a c c ! m ! l a l j o n o f d y e p a r t i c l e s , t h e s o - c a l l e dg r a i n .B y f u r t h e rr e d u cn g i h e v i e w i n gd i s t a n c e w e c o u l d ,o f c o u r s e ,f u r t h e f n c r e a s em a g n i f i c a r i o nb u t t h t s w o u d b e s o c a e d e m p t ym a g n i f i c aot n " , i . e . , r n a g ff i c a t i o n w h i c hd o e s n o i r e v e a a n y n e w d e i a l .
I n t h e m c f o s c o p e ,t h e s t u a i i o n s i h e s a m e . A s w e h a v e s e e n ,t h e p f o l e c t e di r n a g ec o r e s p o n d s t o i h e a e r i a l i m a g e f o r m e d b y t h e m c r o s c o p eo b j e c t v e i n t h e t u b e .i u s t a s w l t h i h e o r o _ e c r d( l d a h a F r - " l o " i T i ! o - . L e p r o _ e . l e o "usef!l aerial niage where r n a g n i f i c a t i o ne n d s a n d " e m p t y m a g n i f i c aot n " b € g n s . t i s t r l e t h a t t h e m i c r o s c o p i ci m a g e w l n o t e x h i b i i a n y a c t u a lg r a i n s t r u c i u r e ,b l t h e r e a L s ow e m a y s p e a k o f g r a i n , b e c a u s et h e r e s n o o p i i c a i m a g e i n w h i c h a i o b j e c t p o i n t l s r e a l l y r e p r o d u c e da s a p o i n i ( i . e w i t h n o r n e a s l r a b l ed a m e t e r ) . D u e t o i h e p h e n o m e n ao f d i f f r a c to n , a n o b j e c i p o i n i l s a w a y s i r a n s f o r m e dj n t o a s m a disk n the mage. i is these so ca ed Airy disks which 'nrage gra f o r r nt h e n". Thjstype of gra n can be found in a n y o p t c a i m a g e f t h e m a g n f c a t i o n i s h i g h e n o u g h .l t l s o b v i o l s t h a t i m a g ed e t a s w i l l n o o n g e r b e c e a r y r e c o g n z e d a s s o o n a s t h e y r e a c ht h e s i z e o f l h e A i r y d l s k - l u s t a s w e w e f e u n a b l et o r e c o g n l z et h e e a v e si n t h e s i d e w h e n t h e y w e r e a s s m a l la s t h e d y e g r a n .
A n o t h e r e x a m p l e : L e t u s e x a m i n ea m o s a i c f r o r n a l o n g d i s t a n c e( i . e . , w i t h l o w r n a g n i f i c a t i o nA) .s w e m o v e c l o s e r w e r e c o g n i z em o r e d e t a i l s .A s s o o n a s w e b e g i n i o r e c o g n i z e i n d i v i d u a le l e m e n i so f i h e m o s a i c ,h o w e v e r ,w e p a s s "u€eful t h e l i m j to f m a g n i f i c a i i o n. l n t h i s c a s e ,t h e e l e m e n t s o f i h e m o s a i c c o r r e s p o n dt o t h e A i r y d l s k s m a k l n g u p t h e a e r i a l! m a g e .
Bewareof pseudostructuresl
lf we vlew a side or a mosaic with higher than lsefu m a g n f i c a u o n ,n o h a r m i s a c t u a l l yd o n e , b e c a l s e w e k n o w very well that the above rneniionedgra n structure does not be ong to the object, for insiance the landscape. Flowever, i n m i c r o s c o p yt h e s i l u a t i o ni s d l f f e r e n t : s o c a e d e m p t y m a g n i f i c a t i ornn a ys h o w s t r u c l u r e si n t h e i m a g ew h l c hd o n o t e x j s t j n t h e s p e c r n e n .B u t s i n c ew e d o n o 1k n o w i h e s p e c i m e n ,t h e r e i s a d a n g e ro f e t r o n e o ! sc o n c l u s i o n sA. n d n f a c t t h e r e a r e e x a r n p l e so f t h n g s b e i n g d l s c u s s e di n s c l e n u f i c Iteraturewhjch did not exjsl ln reality but eveniually revealed t h e n r s e v eiso b e a k l n d o f n r a g eg r a i n , i e t h e r € s u t o t d l f r a c l i o np h e n o m e n a .
W h a t i s t h e h j g h e s ip o w e r
l i f o l l o w sf r o m t h e a b o v e r e m a r k st h a t w e s h o u l d k n o w t h e pefo,na e in.of .henco""ooeob.e .e.^euse.i.' ihe point at whch thelr usefulmagnlfcaion ends. This ls j n d i c a t e db y a v a l u e e n g r a v e do n e v e r y o b j e c t i v ea n d c a l ! e d ' n u m e r c a l a p e d u r e ' . T h e t r e m e n d o l s i m p o r t a n c eo f t h l s characteristicwl rnmediatelybe recognized lt we say that i h e n u m e r l c aal p e r t u f ei s l h e d e c i d i n gf a c t o r\ r y h e nd e i e r . n i n i n g t h e s l z e o f i h e A r y d i s k s l n i h e m c r o s c o p i ci m a g e ,i e , " s c r e e n . T h e r e i s a d e f j nt e r e l a t i o n s hp the size of ihe beiween the nume.ical aperture and ihe s ze of the A ry disk f o ' t h e n o ' r e n t .y o ' e " o o n l y- r o ^ h e ' o l o $ - S : T h e t o l a m a g n i f i c a i i o no f t h e m i c r o s c o p e( i . e . , l h e m a g n l fication of the objective multip ied by ihat of the eyepiece m!ltiplied by any tr.rbe or OPTOVAF factor) should noi e x c e e do n e t h o u s a n di r n e st h e n u m e r i c aal p e r t u r e . Nlicroscop sts who have partc!lafly good vsual acuty s h o ud e v e n u s e f j v e h u n d r e dl i m e s i h e n u m e r i c a al p e r t u r e a s a l i m t . E x c e p to n s t o t h i s r ! ] l e m a y b e a l l o w e . li n o r d e r i o f a c i l i t a t em e a s u r i n ga n d c o u n t i n gw o r k
38
We st owe yo! an expanatoi of th€ term iumercal a p e r i u r e. F g 3 1 g v e s a d l a g r a n r m a t ivce w o f a n o b j e c i i v e f r o n t e n s a n d s p e c m e n . T h e a n g e u ' s u b t e n d e db y r h e o p t i c a l a x s a n d t h e o u t e f r n o s tr a y s s t c o v e r e d b y t h e o b j e c t i v es a m e a s u r eo f t h e a p e d u r eo f t h e o b j e c t v e ; i t i s t h e a p e r t u r ea n g l e .H o w e v e r ,t h e m a g nt u d e o f i h s a n g t ei s n o l n d i c a l e d n c l e g r e e sb u t n t h e f o r m o f a s n e v a l l e , i h a t is a n!mericalval!e. This expa ns the crig n of the ie.m ''n!meri.a a p e d u r e . t s t h e s i e o f r h e h a f a p e r t u r ea n g t e r n ut i p l e d b y i h e f e f r a c t i v e n d e ) in o f r h e m e d i u r nf t i n g t h e s p a c eb e t v r ' e etnh e c o v e r g i a s s a n d t h e f r o n i l e n s . N L r m e i c aa p e r i ! r e = n s i n ! '
s. -"" a rendr o-. o- I - Ta. o€ "q- reo w h e n d r y o b l e c t l v e sa f e u s e d . m m e r s i o no i l , w h i c h i n t h e case of an o imnrersor obtectve f s ihe space between t h e c o v e r q l a s s a n d t h e f r o i t e n s h a s a r e f r a c t i v en d e x o f 1 . 5 1 5 ,a . d i i s o b v o u s t h a t s ! c h a n o b j e c t i v e n r a k e s i t p o s s b e t o a c h l e v e a c o n sd e r a be g a i n i n n u r n e r i c a l *..mpned
! th a dry obF.r L: i ?:lj
ta 39
W h a t j s t h e r e s o l v i n gl i r n i t ?
W i i h i h e a i d o f i h e n u m e r i c aal p e r i u r ew e c a n a l s o c o m p u t e ihe limjt of resolution or maximlm resolving power. By ihis w e u _ d e ' s . a - d l h a s m d l l e 5 t" e p a - d t i o n- d e s g - a . a d o w h i c h t w o s k u c t ! r a l e l e r n e n t s e, . 9 . , t w o a d j a c e n tf l a g e l l a , may have in o.der to be irnaged as two separate elemenis
And howls it determined?
l f n . a . " b j a n d n . a . c o n da r e t h e n l r m e r i c aa p e r t u r e so f o b j e c tve and condensea r n d l - i s i h e , r a v e l e n g r ho f i h e t i g h tu s e d f o r o b s e r v a t i o n( i n 1 / 1 0 0 0m m o r m i c r o n s )t,h e n i h e s r n a e s t resovabLe separaiion between two object poinis (likewjse
n. a. obt + n. a. oond
E x a m p l e : U s i n g a g r e e n f i l t e r , t h e w a v ee n g t h i s 0 - 5 5 p ; a s s u m i n gt h e o b j e c t i v eu s e di o h a v ea n a p e r t u r eo f 1 . 2 5 ,a n d t h e c o n d e n s e ro n e o f 0 - S rt h e n d = 0 . 2 5r r , i . e . , t h e f o u r t h 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?
c o r r e c ta p p l i c a t i o n o f t h e r i g h to i l
lmmersionobjectives mostLy h a v ea s h o r l w o r k i n gd i s t a n c e
O i l i m m e r s l o no b j e c u v e sa r e o b j e c t i v e sw h i c h a r e l s e d w t h immersion oil (instead of alr) between objeciive froni ens a n d c o v e r g l a s s .A s w a s e x p l a i n e di n t h e p r e v i o u ss e c t i o n , t h i s m e t h o d e a d s t o a g a i n i n n u m e r i c a a p e r t u r e .F o r t h e s a m e i m a g e s c a l e , a n o i l i m m e r s i o no b j e c t i v e i s a L w a y s s u p e r i o r t oa c o m p a r a be d r y o b j e c t v e a s r e g a r d s c o r r e c t i o n . T h e r ea r e , o ; c o u r s e ,c a s e sw h i c h d o n o t p e r m i li h e a p p l i c a i o n o f t h e o j l i m m e r s i o nt e c h . q ! e . 1 0 0 x o b j e c t i v e sa f e a ways oil jmrnerslonobjectives.
T h e u s e r s h o u l d ,i f p o s sb e , e r n p l o yo n y t h e t y p e o f i m r n e r s i o n o i l s ! p p l i e d b y t h e o b j e c t i v em a n l f a c t ! r e r ,b e c a u s en o t o n l y t h e r e f r a c t i v ei n d e x m u s t h a v e a c e r t a i nv a l u e ,b u i a l s o t h e d i s p e r s i o no f t h e o i l .T h e i m r n e r s l o o n j Ls l r p p l i e db y Z E l S S s s y n t h e t i c ,n o n - f l u o r e s c i n ga n d n o n - r e s j n i n go i l w i t h a r e l r a c t i v ei n d e x o f 1 . 5 1 5a n d a d i s p e r s i o no f 4 3 . 9 6 T . he oiler sLrpplied b y l r s w i t h e v e r y j m r n e r s r o no b j e c t i v ei s d e s i g n e d i o f a c i l i t a t ea p p l i c a t i o no f t h e o i l t o t h e c o v e r g l a s s w i t h o u i air bubbes: turn oiler upsde down, wait until the air has r i s e n a n d a p p l y a i t t e o i l . T h e o b j e c t i v er n a yb e r n o v e di n t o p o s i t i o na s u s L r a- I n t h l s c a s e , e x c e s s i v eo i l a n d , p o s s l by , b u b be s a r e p u s h e da s i d ed u e t o t h e s h o r tw o r k i n gd s t a n c e . l n a d d i t i o no , b j e c t i v e sw t h a p l a n ef r o n t e n s c a n b e p u s h e d upward in their spring rnount, locked by clockwise rotation a n d l o w e r e dv e l t c a l l y i n t o t h e o i o n l y a f t e r t h e n o s e p i e c e h a s b e e nt u r n e da n d e n g a g e d t h en o t c h .P e r s o n ael x p e r i e n c e w l l F a c l -y o u $ L r ^ h o l F a s e L \ r o n e r h o d s i . o r e _ o n
! f f o c u s i n gt h e s p e c l m e nw l t h o j i r n m e r s i o ns h o u d p r o v e i m p o s s i b i e r e m e m b e rt h e p r e v l o u s l yr n e n t i o n e dp o s s i bl l i y ihat ihe cover gla6€ may be too thick for the shori working d i s t a n c ea v a i l a b l eo r t h a t t h e s p a c e b e t w e e ni h e s p e c i m e n a n d t h e c o v e r g l a s s ,w h i c h i s f i l l e d w i t h r n o u n t i n gm e d i u m , m a y b e l o o w i d e ,w h l c hc o m e si o t h e s a m et h i n g .
alsorequire immersion
lf your condenser has a numerical aperture higher than 0.9, ii is advisable also to immerse the condenser (if not wiih oll, at leasi with water) for use in combinaiion with an oil immersion 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 telescope 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-
yih coreciron carrar Paidchromat63i/0.9o Top: carefullyadlusiedfor coversrassthickness eolibaderiaand.raphylococc {cram s sia n)
42
8. The question of cover glasses " the f rst ens" ofthe objectve
K n o w i n gt h e h i g h p r e cs o n r e q ur e d f o r t h e p r o d u c io n o i m l c r o s c o p eo b j e c t i v e sw h i c h a r e t o g j v e f i r s t - r a t e m a g e s w e s h o ud n o t b e s u r p r l s e di h a t t h e c o v e r g l a s s ,w h i c h s l o c a t e db e t w e e nt h e s p e c i m e na n d t h e o b j e c i i v ef r o n t l e n s n a y n o t b e j l s t a n y c o v e r g l a s s .W e s h o u l d n o i f o r g e t t h a t s r n p l eg a s s p a t e s ,t h a t j s p l a n ep a t e s ,a L s oh a v ea n o p t i c a l power and ihat the op!cal power of ihe cover glass was t a k e n l n t o a c c o u n tl n t h e c o m p u t a t o n a n d d e s i g n o f i h e o b j e c t v e . W i h n o r m a l r n i c r o s c o p eo b j e c t v e s , t l r e c o v e r g l a s s i s p a r i o f t h e l m a g ef o r m i n g s y s t e m l t s a l e n s element (tho!gh wth infnite rad of c!Nature), which is l o c a t e do u t s i d et h e o b j e c t l v em o u n t b u t s t f o r n i s a p a r t o f t h e o p t i c a ls y s t e r n - T h e o p u m l m t h l c k n e s so f a c o v e f
T h e h i g h e r l h ea p e r t u r e t h e . n o r ea c c u r a l e! h e c o v e rg a s sL
S t r i c t l y s p e a k i n g ,i h s a p p l i e s o n l y t o o b j e c t i v e sw h o s e n u m e r l c aal p e r t u r ee x c e e d sa c e r t a i nv a l u e .W t h o b j e c t i v e s h a v i n g a n a p e d u r e n o t e x c e e d l n g0 . 3 s p e c i m e n sm a y b e e x a mn e d b o i h u n c o v e r e do r w t h c o v e rg l a s s e sa s t h i c k a s a m i l l r n e t e r ,a n d t h e s p h e r i c a la b e r r a t i o nw l n o t b e n o t i c e a b l e . W t h a p e r i l r e s b e t w e e n0 3 a n d 0 7 , i h e c o v e r - ga s s t h c k n e s ss h o u l dn o t d e v i a i ef r o m t h e n o m j n avl a u e b v m o r e i h a n 0 . 0 3 m m . W i t h h i g h e r a p e r t u r e s ,e v e n a d e v a t i o n o f 0 . 0 1m r nw i l l c o n sd e r a by i m p a i rt h e q u a i t y o t t h e r n a g e
O b j e c t i v e sw t h a c o t r e c i i o nc o l a r a r e f o r c a r e f u lu s e r so n y
l n o r d e r n o t 1 o b e r e s i r l c t e dt o s p e cf e d c o v e F g l a s st h l c k _ n e s s e sw h e n u s l n g d r y o b j e c t i v e so f p a d i c ua r y h l g h a p e r t u r e , o b j € c t i v e sw e r e d e s i g n e di n w h l c hl h e o p t m u m c o v e r g l a s s t h c k n e s sl s n o t a f i x e d 0 . 1 7 m r n , b u t c a n b e v a r i e d w l t h i n a r a n g eo f 0 . 1 2t o 0 . 2 2m r n ( o b j e c t i v e sw i t h c o r r e c t i o n c o l l a r ) .l f t h e c o v e r - ga s s i h i c k n e s so f t h e s p e c j m e nt o b e e x a m i n e d i s u n k n o w n ,f i r s t s e t t h e c o r r e c u o n c o a r a t 0 . 1 7m m a n d f n d a h j g h - c o n t r a sat r e a l n t h e s p e cm e n .T h e n t r y w h e l h e rt h € c o n l r a s ti s m p r o v e dl f t h e c o r e c i i o n c o a r s s e t f o r g r e a t e ro r e s s e r c o v e r _ g l a stsh l c k n e s sT h i s a s o r e q u i r e sr e f o c u sn g . L t i s o b v o u s t h a t l n i h e h a n d s o f i n e x p e f e n c e d u s e r s w h o u s u a y l e a v et h e c o r r e . t o n . o l l a r a t one end of lts range of a djustmeni, ihese objeci ves rnay give v e r y d i s a p p o i n t i n .ge s u l t s( F g 3 2 J . 43
Oi immersion is lesscriiical
The situaiion is d ffereni with oj mmerslon objecilves, b e c a u s et h e n a r n e du m o f h i g h r e f r a c t v e n d e x ,n a m e y o i l , i s i n f r o n t o f t h e f i r s t l e n s .l n i h i s c a s e ,i h e t h i c k n e s so f t h e c o v e r g l a s si s n o t v e r y c r i l i c a l .S t i l , i t s h o u l db e r e m e m b e r e d t h a t i m m e r s i o n sa r e t o d a y n o l o n g e r" h o m o g e n e o L r se"n o u g h ( i . e - , g l a s s a n d o I o f d e n t c a l r e f r a c l i v ei n d e x )t o a l l o w t h e cover glass io be dispensed with as a matter of course. W h e n t h e c o v e r g l a s s i s o m i t t e d ,f o . v e r y c r i l i c a l w o r k a c o r r e c t i o nl s r e q u l r e de i t h e r b y i n c r e a s i n gi h e m e c h a n l c a l t u b e l e n g t h o r b y l s i n g a s p e c i a l i m m e r s i o no i l ( f o r m o r e d e t a i l s ,s e e f o r i n s t a n c el,V i c h e lV , e n n a 1 9 6 4 ,p a g e 1 7 6 ) .
H o w t o o b t a i ng o o d c o v e rg l a s s e s ?
For pfact cal work, ii ls thus necessary always io use cover 9 l a s s e sw h i c ha r e e x a c i l y0 . 1 7m m t h i c k ,f o r s p e c i m e n sw h i c h a r e t o g i v e o p u m u ml m a g e s e v e n w h e n e x a m i n e dw l t h t h e aid ofhighdry objectives. M crometers,so-called cover glass g a g e s ,a r e a v a l a b l ef o r m e a s u r i n gA. p p r o p r i a t ec o v e r s m a y thus e ther be selected by ihe microscopisi himself, or others u s e dw h i c hh a v eb e e n s p e c j a l l ys e l e c i e db y t h e m a n u f a c t u r e r . It shou d be mentioned ihat in those cases where the cover g l a s s l s n o t i n d i r e c t c o n t a c tw i t h t h e s p e c m e n , i h e i n t e r m e da t e a y e r o f m o u n t i n gm e d i u r nh a s , o f c o u r s e ,t h e s a m e effeci as additional thlckness of cover. lt is therefore advisa b l e i o w e i g h d o w n t h e c o v e r s o f s p e cm e n s d u r i n g d r y l n g .
9. Microscope illumination for more exacting requirements Two important requiremenis: o b j e c t f i e l da n d p u p i l m u s ib e b r i g h i
F o r l e s s c r i t i c a lw o r k , t h e m i c r o s c o p em i r o r m a y b e j u m i preferably d ffused llght, such as nated either by daylight g i v e n b y a w h i t e c l o u d - o r a n o r d i n a r yd e s k l a m p w i t h inside frosted bulb. More convenieni are the attachable s i m p e s u b s i a g e i l l u m i n a i o r sf o r d i r e c i c o n n e c t i o nt o t h e m a i n s f o . t e a c h i n sm l c r o s c o p e s( s e e F i g . 1 ) . I n a l l t h e s e cases optimum cond tions will have io be deiermined by trial and eror. Two things are impodant: the field of view s h o u l db e e v e n l yi l l u mn a t e da n d - w h e n t h e c o n d e n s e ri r i s i s o p e n a n d t h e c o n d e n s e ri n i t s u p p e r r n o spt o s i t i o n- t h e 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.
K d h l e ri l l u m i n a t i o n
For microscopic research work, phoiomlcrography, mjcroprojection, 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 c o n t r o lo f t h e l i g h t p a t h i n t h e i l l u mn a i i n gb e a mo f i h e m l c r o s c o p e ,a n d t h e m e t h o di s i o d a y c a l l e c l K d h l e r l l l m i n a t i o n " ( l o u r n a lo f t h e R o y a lM l c r . S o c . ) .
Use of the Kdhler method depends on ihe availability of a m i c r o s c o p ei l l u mn a t o r w i t h a I a m p c o n d e n s e ra n d a n i r i s d l a p h r a g mi n f r o n t o f i t ( F i g . 3 3 ) . T h e l a m p s u s e d i n s u c h illuminators usually have a sErall,concentraied filameni and operate on 6 or 12 volts; they are therefore called lowvoliage 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 illuminator inco|porated ln the base of !arger m i c r o s c o p e si s , o f c o u r s e a s o s u i t e d f o r a p p l y i n gK i j h l e i s r u l e so f i l l u mn a t i o n .
T h e p r o c e d u r ef o r a p p l y i n gt h i s m e t h o do f i l l ! m l n a t i o ni s a s f o l o w s ( F i g .3 4 ) :
I
46
l . A i g n r n i c r o s c o p ei l l L r mn a t o r w i t h p a n e m t r o r o f t h e rnicroscope a n d a d j u s t t s o t h a t a s m a l l ,b r i g h ts p o r o f i g h t i s p r o j e c t e do n t o t h e m r r o r o r a n i m a g e o f t h e f l a o r e n t f o r m e d ( t h i s a d j l s t r n e n ti s f a c l i t a i e d f a s h e e t o f p a p e r s h e d o v e rt h e s l r f a c e o f t h e m r r o , ' o"ch - _ o po - o r . l F -o rL" o u t o f t h e t u b e . K e e p a d j u s tn g i h e m j r r o r u n t t h e b a c k l e n s o f l h e o b j e c vt e s f e d w i i h g h t . D m r n i n gt h e l g h i b y a . e u t r a l d e n s i t yf t e r w o u d b e a d v l s a be . n s e r tt h e e y e p i e c e a n d f o c L r st h e s p e cm e n w l t h a l 0 ; l o r l 6 : o b j e c u v e . 3. Close ihe iris daphragm of the iilumnator(fied stop) a m o s t c o m pe l e l y e n d m o v e i t s ( ! n s h a f p ) m a g e o i t h e s p e c m e n n t o t h e c e n t e ro f t h e f i e c i o f v l e w b y r o t a tn g a n d 4 . F o c u st h e e d g e o f t h i s d i a p h r a g mi m a g eb y s g h t l y o w e . i n g i h e c o n d e n s e rB . o t h t h e s p e c i m e na n d t h e f e d s t o p a r e n o w s h a r py d e f n e d .T h e no p e nt h e f e l d s i o p l n t I i h e e n l i r e r i e d o f v e w s j u s tc e a r 5 . S l g h t l y . e a d j l s i i h e l a m p s o c k e to r t h e l a m p c o n d e n s e r L r n it t h e f e d o f v i e w l s e v e n l y u m l n a i e d . 6 . A t f i r s t o p e nt h e c o n d e n s e ri . i s ( a p e r t u r es t o p )c o m p L e tye ( i m a g ev e r y b r i g h t l a n d c l o s e i t o n y f a r e n o u g ht o e l m i n a t e g l a r e j n t h e r n o s t l m p o r t a n ti m a g e e e r n e n t sa n d t o r n a k e t h e m a p p e a rw t h s a t s f a c t o r yc o n i r a s t .W i t h s t a n e d s p e c i mens lhis is the case elen al a reatvely wde aperture, w h e f r e s h s p e c m e n s r e q u i r es i g h t y m o r e s i o p p n g d o w n S l o p p n g d o w n f ! r t h e r t h a n s a b s o l u t e l yn e c e s s a r y( f o r exanpe, to redlce ihe brghiness of the mage)is one of t h e b i g g e s tm i s t a k e sa m i c r o s c o ps t c a n m a k e ,s j n c ei t e n t a i l s a o s s o f r e s o l vn g p o w e r .- l f t r e i n t e n s i t yo f t h e g h i s too hlgh red!ce the lamp voltageor insert gr.y or green flte16 ito the filterholder. 7 . l f w j t h l o w p o w e r o b j e c t l v e so n l y p a r t o f t h e f e l d i s l l ! m n a t e d s w i n g o u t t h e c o n d e n s e fr r o n t l e n s a n d o p e n t h e c o n d e n s e rr s f u l y C o n t r a s t s t h e n c o n t r o l l e dw i h t h e a i d o f i h e a m pd a p h r a g m . l n t h e c a s e o l m c r o s c o p e sw i i h a b u l t - i n i l l l m n a t o r s t e p l a n d p a r t y a s o s t e p 2 a r € o m i i t e d .U n d e r 3 , c e n t e r l n g s n o i a c he v e d w t h t h € a i d o f a m l t r o r ,b u t b y t h e c € n t e r i n gs c r e w s o f t h e c o n d e n s e rP . i e a s ec o n s ut t h e o p e r a i i n gi n s i r u c io i s s u p pi e d w t h i h e m l c r o s c o p e
L e t u s n o w e x a mn e t h e p a t h o f r a y s w t h K d h l e ri u r n i n a ot n . t c o n s l s t so f t w o s u p - . r m p o s e db e a n s - F o r g r e a t e rc l a r i t y w e s h a l l s h o w b o t h b e a m ss e p a r a t ey a n d o n y o n o n e s d e ( i n t h e c a s e o f i h e p u p b e a m ,f o r i n s i a n c e ,o n y t h e r a y s o r i g i n a tn g a t o n e e n d o f t h e f i a m e n t ) . F g . 3 5 s h o l v st h e p a i r o f t h e r a y s o r i g i n a t n ga n d m e e u n g n t h e p a n e s w h c h a r e i n s h a r p f o c l s i o g e t h e rw i t h i h e s p e cm e n , i . e t h e s o - c a l l e dw n d o w s .T h e r a y s e m a n a u n g f r o m t h e f i e l d d i a p h r a g mo f t h e a r n p a r e c o r n b i n e dl n t h e s p e c i m e nb y t h e c o n d e n s e r( i n o t h e rw o r d s , a n i m a g eo f t h e f i e l d d i a p h r a g mi s f o . m € d o n t h e s p e c i r n e n )F. f o m t h e r e ,t h e t w o i m a g e sa r e p r o j e c t e do n t o t h e f e l d o f v i e w d i a p h r a g m o f i h e e y e p i e c e( a e r j a i m a g e ) .W h e n t h e a d j u s t r n e n si c o F rect, the magesof the field and field of vew djaphragms a p p e a r s h a r p b o t h a r e c e n t e r e d ,i . e t h e y a r e c o n c e n t rc a n d i h e y a r e o f i d e n t i c as z e f o r t h e a p e d u f es t o p s
Fig. 35a shows the rays which combne at the ap€dure d i a p h r a g mp l a n e s .C o s i n g d o w n t h e s e d i a p h r a g m sw o u d a f f e c tt h e d a m e t e ro f t h e p r e vo l s l y m e n i j o n e de x t p u p i l o f t h e m i c r o s c o p ea b o v et h e e y e p j e c e D . u e t o r e f r a c t i o nb y t h e c o n d e n s e rt,h e r a y s e m a n a t i n gf r o m t h e s o u r c e m e e t n t h e a p e r i u r ed i a p h r a - q m o f t h e c o n d e n s e ra n d a r e t h e n i m a g e d by condensea r n d o b j e c tv e j n t h e e x i t p u p i l o f t h e o b j e c tv e , w h e n c e t h e y a r e i r a n s mi i e d b y t h e e y e p i e c et o t h e e x i l p u pLo f i h e m l c r o s c o p A e .t a t h e s e p o n t s , a nm a g e o f t h e l g h t s o u r c eL i s f o r m e d . r e i h o do f f e r ? W h a t a d v a n t a g ed s o e s t h e K o h L em
o f K d h l e ri l u m i n a to n
1. In spite of srnalfilaments the entire area of the arnp d i a p h r a g mh a s t h e s a r . e l u m l n a n c ea s t h e f i a m e n t , s o i h a t o n l y a o w p o w e r n p u t i s r e q u i r e df o r t h e a m p . 2 . D l e t o i h e a d t u s t m e no t f t h e a n r p d i a p h r a g mt,h e a f e a L L l m l n a i e idn t h e s p e c l m e nc o r r e s p o n c l iso t h e o n e w h c h '
roq-d
@..o d.ncp ^.,
Lhe'edsooof
66160a^e.
T h e s p e c l m e ni s t h u s n o t s u b j e c t e dt o u f n e c e s s a r yi r r a d i a t o n ( e x a m i n a u oonf l i v i n g o r g a n i s n r s la) n d c o n i r a s tr e d u c i n g s t r a y i g h i f r o m s p e c i n r e na r e a s o u t s d e t h e f i e l d o f v i e w i s 3 . T h e a p e d u r ea n d t h u s c o n t r a s ta s w e l l a s d e p t h o f f o c u s a r e c o n t r o e d b y t h e c o n d e n s e irr i s .
4. Viewed from the specimer, the light source is at infinitv (parallel rays in the aperture ray path, Fig 35al Anv irregularity in the source is therefore least effective in ihe 5 . S i n c et h e f i e L ds t o p i s j l s t f u l l y f i l e d w i t h i g h t d u e i o t h e a d j u s t m e not f t h e l a m p d ! a p h r a g mn, o l i g h t w h i c h c o u L dg i v e rise to disturbing flare reaches the inner walls of ihe tube As a res!lt, pariicularly brilliant images are obtained W h e n a d j u s t i n gi h e e q u i p m e n ft o r K i j h L e lrl l u m i n a t i o ni t m a v 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 y e i n s h a r pf o c u s a s s o u r c e .l f t h i s p l a n es h o u l da c c i d e n t a l l b of ihe condenser the heighi io alter necessary it is only well, slighily. W i t h h i g h - a p e r t u r ec o n d e n s e r ss h a r p i m a g i n go f t h e l a m p d i a p h r a ; m i s f r e q u e n t l yp o s s i b l eo n l v l f t h e s p e c i r n e rs l i d e is not thicker than 1 mrn Why aLrxlljarylenses?
WithKdhle. illuniiatron lowennsthe condenserwould lead io on exessive ros ofapsbre(refD.
50
l n m i c r o s c o p e sw i t h b u i l t - i nl l l u m i n a i o rt h e f i e l d d j a p h r a g m s sometimes located very close to the condenser' When i m a g j n gt h i s d i a p h r a g mo n t h e s p e c i m e ni n a c c o r d a n c ew j l h Kcihlerls nrles, ihe condenser has to be lowered to such an js extenl that a considerable Lossof aperiure ensues This tols suppLied which lens avoided by a so-called auxlliarv qether with the microscope and is ocated dlrectlv below the ;ondenser. lts function is illustraied ln Fig 36 lf a separate h i q h p e r f o r m a n c e i l l u m i n a t o ri s u s e d t h e a u x i ! i a r y l e n s letter siill, replaced with ihe longeF sn-ouidbe swunq out or, " a u x i l i a r yl e n s l ! ' . focus
1 0 .L e n g t hm e a s u r e m e n t s u n d e r t h e microscope M e a s ! r e r n e not f r nc r o s c o p r co b j e c r sj s r e a i v e y 5 m p r e ,o u 1
d 6 o 4o
6 . . . t " b t .
o a
o t r 6
o o r
q
m e a s u r i n ge y e p l e c e € , y e pe c e m c r o r n e t e ra n u s r a l j e m c r o m e t e r( F i g . 3 7 ) . A rolnd gass dsk wih a scae -ihe mcro,,ereo r sK c a n b e i n s e r i e dt n t h e m e a s u rn g e y e pe c e . a n d i h e f o c ! € i n g e y e e n s e n s u r e st h a t e v e n o b s e r v e r sw i i h d e f e c ,v e e y e sg n r c a n s e e a s h a r p m a g eo f r h e s c a e _ T h e s t a g en r i c r o m e r e r i s a s c a e g r a d u a r e di n u i i r s o f 1 1 1 0 0n r m o n a c o i v e n i i o n a l
.q
3. , l
\
o'
I
l
ll lll llti il lil l /,1r11 ll,t ,1, ,lllrl{ (\
jlnIlrlllirl Ill'rlrl l
\t
tat
(o
N
51
T h e p r o c e d u r ei s a s f o o w s :
4
1 P o st o n t h e f o ! n d e y e pe c e m c r o m e t e r n t h e m e a s u r i n g e y e p e c e . F o r t h i s p l r p o s e u n s c r e wt h e L o w e rp a r i o f t h e e y e p e c e r e r n o v et h e r i n g a b o v e t h e e y e p i e c ed a p h r a g m a n d - e p l a c ei t a f t e r h a v n g i n s e r t e d t h e m c r o n r e t e rd j s k { s c a l e l p w a r d s ) . T h e n s c r e w t h e e y e Pe c e t o g e t h e ra s a i n 2 . F o c u st h e e y e e n s i n s u c ha m a n n e rt h a t t h e s c a e a p p e a f s w t h n r a x i m ! ms h a r p n e s s ti h e n l n s e r t t h e e y e p l e c e n t o t f r e
5
3 . P l a c e t h e s t a g e n - r c r o m e t eor n t h e s t a g e a n d f o c u s i h e microscopeon the sca1e.Both scaes will then appear shafpy d€fined. By t!rning the eyeplece tlrev are iaid p a f al e i o e a c ho t h e r . 4 . F l n d o l t h o w r n a n yd l v i so n s o f t h e e y e p l e c em c r o n - r e t e r c o r r e s p o n dt o a c e r t a i n d i s t a n c €o n t h e s i a g e m i c r o m e t e r a n d c a l . u a i e t h e l e n g i h w h c h c o t r e s p o n d st o o n e d v s l o n o f t h e e y e pe c e m c r o m e t e r . E x a m p l e ( F i g . 3 8 )8 0 d i v s i o n s c o n e s p o n dt o I 3 s n r m 0.069_.' o, o n oc . i . o so
S.. e.oitias: n.
om*"f
0 1
5 T h e i r c r o m e t e rv a u e t h l s d e t e f r n l n e da p p i e s o n y r o r t h e o b l e c tv e w i l h w h c h t h e c a i b r a to n w a s r n a c r e S u b s e q u e n i l yi t, s o n l y n e c e s s a r vt o m ! u p v t h e n l m b e r o f d v s j o n s c o v e r e d b y a n i n t e r e s tn g d e t a l n i h e s p e c f ' e f b v t h e m l c r o m e l e vr a t e i n o r d e r t o d e t e r mn e i h e l e n g t ho i t n l " a " t n l ' n t t - , "o f j " " t p l a n e ( F o r s e n a l m € a s u r e m e n i st h e r n i c r o m e i evr a L u en e e d o i l y b e s e t o n c e o n t h e s l i d e r ! e \ l h e r e u p o nt h e d l f f e r e n tl e n g t h sc a n s i r n pv b e r e a d o f f ) S l n c et h e i r c r o m e t e rv a u e a l s o d e p e n d so n t h e r . e c h a n l c a i u b e e i q t h . i i s a d v s a b l et o k e e p a n e v e o n t h e s e t t n g o f i h e e y e pe c e s l e € v e so f t h e b i n o c u l a rt u b e w h c h ' n u s t n o t L ft h e n r e a s l r e . n e n t es x t e n do v e r a o n g e r p e r o d o f t r n e o r f t h e s p e cm e n st h e m s ev e s e x h b l t a f i n e l n e a ri e x t u r e w e - e c o m m e r dl h a t ? c o n i r a s t , l n e _ c o n t r a sotr n e t m i c r o m e i e r d s k b e ! s e d n s t e a d o f t h e n o f . n a e v e p i e c em l c r o m e t e r { Fs . 3 9 a l . A m € a s ! r i g e y e p i e c ec a n . o f c o u r s e b e u s e d L nt T r es a m e m a i n e r a s a n y o r d i n a r ye y e pe c e f t h e m c r o m e t e rd i s k i s l a k e n o l t L nt h i s c a s e a n o b s e r u e rw h o h a s n o r m a lv i s o n s h o l l d s e t t h e z e r o f . a r k o n t h e e v e l e n ss c a l e a t t h e b l a c k .stead of the red lne.
52
'll. The I n e c e s s a r yi ,t c a n b e s m p l e
camera on the microscope
T h e s m p l e s i w a y o f t a k i n g p h o t o mc r o g r a p h si s t o m o u n t a c a m e r aw i t h t s l e n s s e l a t t n f i n i i y a s c l o s e y a o o v e r n e m c r o s c o p ee y e p i e c ea s p o s s b l e a n d t o o p e n t h e c a m e r a stop fully. f the nricroscopewas fi.si foclsed w rh re axed a c c o r n r n o d a t o nt ,h e n t h e p h o l o n r t c r o g r a pw h be sharp, b u i l s l a l l y v g n e t t e d .T h s s d u e t o t h e f a c t r h a i i n c o n r b i n a t o i w l t h a c a m e r al e n s o f n o r m a lf o c a l t e n g t h{ 5 0 m m f o r 2 4 x 3 6 m m , 7 5 t o 8 0 m r nf o r 6 ) . 6 c n r t 0 5 r i m f o r 6 : i 9 c m ) o n l y w d e - a n ge m i c r o s c o p ee y e p i e c e sc o v e r t h e e n r i r ef i e d o f v i e w . - A n y d o u b t s r e g a r d n g c r i t c a t i o c L r sc a n b e e m n a t e di f a s l n g l e - l e n sr e f l e xc a r n e r as e m p o y e d ( F j g . 4 0 ) .
F o c l s n g b e a f l s p i l l e f sa r e d e a l
Snce a poriion of ihe lghl s lost bythetime t reaches .o mal ,efe^ c a r n e r a{sg r o u n d - ga s s s c r e e n s, p l t - l m a g er a . s e f i n d e rm , crop r s m f o c u s i n gr i n g e t c . l a r e n o t v e r y c o n v e n i e n lC . onsdera b l y r n o r ea c c u r a t ea n d b r g h l e r a r e s p e c a f o c u s i r g b e a m ' i . . es ^ o" "i,. 'o fo -.irg oa I W i l h c a m e r a sw t h f o c a l p a n e s h u t t e r s u c h a s C o n t a r e x C o n l a x e t c . t i s a d v i s a b l er o r e m o v ei h € l e n s a n d a i i a c ht h e c a m e r ab o d yw i t h a s p e c a l a d e p i e ft o a s o - c a e d a i t a c h m e n t c a m e r aw h i c h i s a v a la b e e t h e r w i h o l t ( F i g .4 1 a ) o r w i t h p h o t o e l e c icr e x p o s ! r e d e v c e ( F i g .4 1 b ) .
W h a t i m a g es c a l el s o b t a n e d w i t h a p h o t o m i c r o g r a p hci ca r n e r a ?
T h e i m a g es c a l e o n i h e f i l m d i f f e r s b y a c e d a i n f a c r o rf r o m t h e r n a g . i t j c a t i o ng i v e n b y t h e m i c r o s c o p ed u r i n g v i s u a l w o r k . T h j s f a c t o r i s f o u n d b y d i v i d i n gi h e f o c a e n g t h o f t h e c a m e r al e n s ( i n l n i l l r n e t e r s b ) y 2 s 0 m m . ( E x a m p l eA : Conraf l e x c a m e f aa s s h o w ni n F i g .4 0 h a s a f o c a l e n g r ho f 5 0 m r n . Consequenl y, the factor is 1/5. Objecrive magnification )< e y e pe c e m a g n j u c a t i o nx 1 / 5 t h u s g i v e s t h e i m a g es c a e o n the 35 mrn negative.)The specia ZE SS attachmentcamera s h o w n i n F i g . 4 1 h a s c a m e r af a c t o r s d e p e n d e n o t n the indi
D e i e r mn a t i o no f i h e c o r r e c i € x p o s u r ef o r p h o t o m i c r o g r a p h y i s v e r y c o n v e n i e nitf s p e c i a p h o t o ee c k i c e x p o s u r ed e v c e s a r e u s e d ( F i g . 4 1b ) A n o t h e rs i m p l ea n d a c c u r a t er n e r h o di s d i r e c t m e a s u r e r n e n t of the finder irnage of a reflex camera, preferab y by means o f a p h o t o e l e c tcr e x p o s u r em e t e rw h i c h h a s a s m a t ta c c e p t a n c e a n g l e a n d m e a s u r i n gs u r f a c e( e . 9 . , i h e Z E T S SI K O N ''IKOPHOT C D ) . N o t e v e n c a l i b r a u o np h o i o s a r e r e q l i r e d , i f , f o r i n s t a n c et h e f o o w i n g p r o c e d u r ei s u s e o : P o i n t c a m e r aw i t h l e n s s e t a t f / 2 . 8 ,f o r e x a r n p t ea i a b r g h t w a l l .T a k e a m e t e r r e a d i n go f t h e w a l l , w t h , s a y , f 1 2 . 8 , 1 1 5 0 s e c .T h e ni a k e a r e a d t n gw i r h t h e n r e t e r p o i n t i nagt r h e g r o u n d g a s s o r i h e f i f d e r i n r a g ea n d r e a d t h e f s i o p c o r r e s p o n d j n g t o l / 5 0 s e c , e . g . , f / 1 . 4 .W h e n r a k i n gt h e p h o t o m i c r o g r a p h s , a f e a d i n g ss h o u l dt h e n b e t a k e n w i t h t h e m e t e rp o i n r i n ga t t h e f i n d e ri m a g e it h e e x p o s u r et i r n ei s t h e n t h e v a l u e c o r e s , l f w o r k i n g c o n d i i i o n sa r e a w a y s i d e n t i c a l( o b l e c r v e , e y e p i e c e , p o s i t i o n o f c o n d e n s e ri r i s , l a m p , f i l r n s p e e d ) , t h e n 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 o r e n e r g e t i cd e v e l o p e r l
56
W i t h n o r m a l d e v e l o p m e n t ,h g h - c o n i r a s t f l m s h o ! l d b e employedb , e c a u s ec o n t r a s ti n p h o t o m i c r o g r a p his ! s u a l l y l o w e r t h a n i n o u t d o o rp h o t o g r a p h y-. F i l r n so f " p a r t i c ua r l y h i g h r e s o i v i n gp o w e l ' a r e n o t n e c e s s a r yb, e c a u s ee s p e c t a l l y
i n h i g h - p o w e$r , o r kt h e r e s ou t o n o f e v e n t h e e a s t s u t a b l e f l r n s i s s t i l l h i g h e rt h a n i h a t w h i c h s p h y s l c ay p o s s i b l ei n t h e r r r i c r o s c o p eT.h e s m p l e s t a n d o n e o f t h e b e s t m e t h o d s s t o u s e o r d n a r y a l - r o u n df l m a n d t o d e v e l o p t i a p a p e f
W h e n i s 3 5 n r mt o o s m a l l ?
t h a s b e e nf o l n d i n p r a c i i c a lw o r k t h a t n e g a t i v es z e s a f g e r t h a n 2 4 x 3 6 m m a r e o f a d v a n t a g eo n l y f s m a Lsl c a l e p h o t o s ,r e d o m i m i c r o g r a p h sa n d p a r i i c u l a r l yp h o t o r n a c r o g r a p h p nate. n addition, arger sjres should be used if coor photos have io be taken fof printng purposes.ZELSS s u p p l e s s p e ca l p h o i o m l c r o g r a p h iact t a c h m e nct a m e r a sf o r 2 r " / r " ) < s r l "( 6 x 9 c r n J , 2 r l i " > . 2 r L' ( 6 t B c r i ) , 3 t l a ' t 4 t l . " ( 9 ) . 1 2c m ) a n d 4 " x s " ( P o a f o d l .
12. Micro.proiection l l i c r o - p r o j e c t i o ni s a v a l u a b l et e c h n i q u ei f s e v e r a l p e o p l e w a n t t o o b s e r v e a m c r o s c o p i ci n r a g es m u t a n e o l s l y . F o r s m a l l e r g r o u p s a v i e w i n g s c r e e n i s e r n p i o y e d ,w h i c h s s m p l y e x c h a n g e df o r t h e o b s e r v a o i n t l b e a n d w h i c ho f f e r s a n i m a g es c a e o f 1 0 : i h e o b j e c t i v em a g n i f i c a t i o (nF t g .4 2 ) . A l e s s e x p e n s i v es o u t i o n i s t o p r o j e c t t h em i c r o s c o p t ci m a g e o n i o a w h t e s l d a c e w t h t h e a j d o f a d e v l a tn g p r i s r na t t a c h e d -o
h6 e.eo,o.a
wL F, .hp ob.pn6.
..ano< oqsioe ha aya
p l e c e ,t h e m a g n i f i c a t i o ins i h e s a m e a s i n t h e m i c r o s c o p e .f t h e v i e w i n gd i s t a n c es h a l v e d ,m a g nf c a t o n s d o l b l e d e t c . 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 w h i c ha b o v ea l l t h e s p e c i a le q u i p m e nfto r m c r o p r o j e c t i o ni s p . o v i d e d .F o r f u r t h e r d e i a i l s ,r e f e r e n c e s m a d e i o t h e c o t r r e s p o n d i n gZ E S S i t e r a t u r e . 5T
53
Annex
Bibtiography F o r m o r e c o m p r e h e n s i v en f o r m a i i o no n t h e f i e d o f m c r o s c o p y w e g i v e b e t o w t h e t j t e s o t a n u r n b e ro f w o r k s . ''Using t h e t V i c r o s c o p eb , ,y A . L . E . B a r r o n C h a m p m a na H a l , L o n d o i , 1 9 6 5 'Microscopy by W. c. Harfley E n g l i s hU n i v e r s t e s p r e s s L r d . ,L o n d o n ,1 9 6 2 A n r e r i c a ne d i t i o ne d i t e db y j o h nJ .L e ea n dB e r n a . dF r i e d m a n T h e N a t u r a tH i s r o r yp r e s s ,C a r d e nC i i y , N e w y o r k , 1 9 6 4 L e c r ! r e n o t e so n t h e u s e o f t h e r n i c r o s c o p b ey R . B a r e r B l a c k w e S c i e n t i f i cp u b t c a r o n s ,O x f o r d ,1 9 5 3 a n d F . A . D a v i e sC o . , p h t a d e l p ha , p a . ''An lntroductjon i o t h e p r i n c t pe s o f t h e M c r o s c o p e a . d i r s A p p t c a i i o nt o t h e p r a c t i c eo f p h o i o mc r o g r a p h y . . b y F . S c h e n ka n d c . K s i l e r C h a p m a n& H a t t ,L o n d o n ,1 9 6 2 ''phoiomicrography by R. M. A en , e w l e r s e y ,j 9 5 B D . V a n N o s r r a n dC o . , I n c . p r t n c e r o nN "Ana ytica Cytotogy E d i t e db y F . C . M e o r s 2 n d E d i i i o n 1 9 5 9 ,M c c r a w N i l t ,L o n d o n "Progress i n M i c r o s c o p y 'b y M F r a n c o n p e r g a m o np r e s s ,L o n d o n ,1 9 6 1 ''Handbook of Chemica Microscopy, b y E M . C h a m o ta r d C W . w a s o n r o h n W l e y a r d S o n s ,I n c . N e w y o r k , N . y . 1 9 S 8 Comprehensiveworks in cerman and Ffench: ''Die C r u n da g e n d e r T h e o r i ed e s M i k r o s k o p s ,b y K . M t c h e l 2 n d e d i t i o n iW i s s e n s c hV. e r a g s ge s e s c h . ,S i u t t g a r t ,1 9 6 4 D i e M j k r o p h o t o g r a peh, , b y K . M c h e 2 n d e d t i o n r S p r i n g e rV , enna, 1964
s n o m o u r r v e n s Av t - * . p "
'Tratte de Microscopie" b y A . P o l i c a r dM , . B e s s u s a n d l v l .L o c q l i n M a s s o ne t C i e P a r s , 1 9 5 7 59
ZEISS literature T h e M i c r o s c o p yD e p a r t m e n h t a s d i s t r i b u t e da w h o l e 6 e r i e 6 o f c a i a l o g sw h i c hg i v e s a g o o d i d e a o f a l m o s tt h e e n t i r el l n e o f e q u i p m e n st u p p l i e db y t h e r n ;t h e r n o s ti m p o d a n to f t h e s e
Routineand BesearchlVicroscopes S T A N D A R DK F M l c r o s c o p e s STANDARD UNIVERSALM l V i c r o s c o p feo r V e r i i c a l l u m i n a t i o n Phase Contrast and InierferenceContrasi nve.ted lvlicroscope l v l i c r o s c o pIe u m i n a i o r sa n d L i g h iF i i e r s I n s i r u m e n i sf o r t h e P h o t o g r a p h i cG, r a p h i c and Projectjve Reproduciion
ULTFAPHOT II ResearchMeta ograph ULTFAPHOTll lV I n s t r u m e n tfso r P o l a r l z l n gM i c r o s c o p y ntederence lvlicroscopyw th Transmitied L ghi Epi-Microscope S T A N D A R DM e t a l u r g i c a M l icroscope
4A-120 40 130 40 143 40 160 40-215 40-254 4A-344
40-400 40-430 4A-452 40-453 40-550 40-560 40-655 40-660 40-700
ZEISSreprints ev a i R e p r i n l so f p a r t i c ! l a r l yi n t e r e s t i n ga n l c e s a r e L i k e w i s a able. However, ihe stock of such maierial is llm ied, and very o f t e nt h e a r t i c l e s w e r er e p r l n t e do n l y i n t h e a n g u a g ei n w h i c l r t h e y o r l g i n a l l ya p p e a r e d . o i r e q u e s t ,s c h o os , i n s t l t u t e se, t c . ,m a y r e c e i v ea n l n s t r u c tionaldiagram l n t h e f o r m o f r o e r p o s t e r ss, h o w i n g t h ec r o s s s e c t i o n o f a m i c r o s c o p ea n d t h e c o r r e s p o n d i n gl g h t p a t h (Cat. No. 40-020). 60
Additional information The staff of our guesi laboratory at the Oberkochen rnain f a c t o r y a s w e l l a s o u r s u b s l d i a r j e so r r e p r e s e n t a t i v eas l l o v e r t h e w o n d a r e e n t i r e l ya t y o u r s e r v c e f o r c o n s u l t a i i o n o n y o u r p a r t i c u L aprr o b l e m s . M a y w e a l s o c a l l y o u r a t t e n u o nt o i h e f a c t t h a t a n y o n e w h o u n d e r s t a n d sG e r m a n a n d i s i n t e r e s t e di n p h o t o m l c r o gfaphy rnay participate in a five-day introductory course w h i c hl s h e l d e v e r yy e a r l n M a r c h o r A p r i l a t C d i i i n g e n .F o r a p p l i c a t i o n sp, l e a s ew r i t e t o o L r rZ E I S S a g e n t o r d j r e c t l yi o t h e M i c r o s c o p yD e p a r t m e n tC, A R LZ E I S S ,7 0 8 2O b e r k o c h e n Wuertt. - West Gerrnany.
Delivery programmefor ZEISS l,/lcroscopes and SupplementaryAttachments R o u t i n ea n d F e s e a r c hM j c r o s c o p e s M i c r o s c o p e sw t h b u f t - i na u t o m a l i cc a m e r a A t t a c h r n e nct a r n e r a sw t h m e c h a n i c aal n d a u t o m a t c s h u t l e r for photornic.ography Microc ne cameras P o l a r i z i n gr n i c r o s c o p eas n d a c c e s s o r i e s E q u i p m e nfto r l n t e r f e r e n c er n i c r o s c o pw y t h l r a n s r n i t t e dl l g h t P h a s e - c o n t r a rsnt i c r o s c o p e s Fluorescencm e l c r o s c o p e sa n d f l L r o r e s c e n cI el u r n i n a t o r s S p e c i a l. n i c r o s c o p efso r r n e t a l l o g r a p h ei cx a r n i n a t i o n s E p i M l c r o s c o p e sE, p l T e c h n o s c o p e s M i c r o s c o p e sf o r d e p t h m e a s u r e m e n t s (rnicroscopesfor measuringthe depth of eich ns we s) Dlfferent types of Stereomicroscopes M i c r o s c o p e sf o r p l a n k t o na n d t s s u e e x a m i n a t i o n s C y t o p h e r o m e t ef ro r e l e c t r o p h o r e i iecx a m i n a t i o nos f c e l l s U n v e r s a ll V i c r o - S p e c t r o p h o t o r n eUt eMr S P I M i c . o s c o p eP h o t o m e i e IrV P M Micro projection devices M i c r o s c o p el l u n r i n a i o r a s nd jght f ters Tubes for conneciing the micfoscope !o the TV camera D r a w i n gi n s t r u . n e n tfso r r i i c r o s c o p e sh, e a i i n gs t a g e s a n d o l h e r a c c e s s o re s D i f f r a c to n a p p a r a t u sf o r m c r o s c o p i cd e m o n s t r aot n s 6l
ZEISSand the art of microscopy The origln of your ZEISS miffoscope l e s n t h e w o r k s h o p so f t h e f i r r n o f C A R L Z E I S S . n 1 8 4 6 t h e u n i v e r s i t yn r e c h a n i o c f l h a t n a m ee s t a b l i s h e d a workshop f o r o p i c a m e c h a n j c sa t J e n a ,w h l c h g a i n e d w o r d r e n o w n a f t e r D r . E r n s tA b b e h a d j o i n e d h i m € s s c i e n tf c c o l l a b o r a i o r i n 1 8 6 6 .E r n s t A b b e , w h ow a s a l s o p r o f e s s o ro f r n a t h e m a t i c a l p h y s i c sa t l e n a L J nv e r s i t y , a d t h e s c i e n t i f i cb a s s f o r t h e e n t i r ef e l d o f m c r o s c o p y .A f t e r t h e d e a t h o f C a d Z e j s s h e s e t u p t h e C A R L Z E I S S F o u n d a t i o n( 1 8 8 9 1t ,r a n s f e r r e dh i s e n t i r e f o r t u n e t o i i a n d p l t n t o p r a c t i c es o c i a l i d e a s w h c h a t t h a t u m e w e r e s e n s a to n a ( e . g . , p e n s l o nr g h t s a n d p r o f t s h a r i n g )E . v e r s l n c e Z E S S h a s b e e n w o r l d f a m o u sf o r t o p q u a lt y o p i i c sf o r u s e i n i h e f e l d so f m c f o s c o p yp, h o t o g r a p h y a s t r o n o r n ye, t c A f t e r W o r d W a r l l t h e f j r m o f C A B L Z E I S S w a s d i s m a n t e d a n d e x p r o p r i a t e d .T h e M a n a g e m e n ta n d a b o u t 1 0 0 l e a d i n gp e r s o n n e o f t h e c o m p a n yb e g a nt o b l i l d a new ZEISS factory at Oberkochen (Wueirtemberg/ West Cer ".)l $f. " 1". ^..- q dd . y Jo edb. nanr.pe.i.l s t s f r o m t h e o l d w o r k s . l n i h e n e w p r o d u c t i o np r o g r a m , w h c h r a n g e sf r o m s p e c t a ce e n s e s t o p l a n e t a r l aa n d f r o m m a g n f j e r s t o e e c i r o n r n i c r o s c o p e s i, g h t m t c r o s c o p e sa r e c o r n n r e r c i a l lt yh e m o s t i m p o r r a n tt e o r .T h e y a r e b e i n g b u t n a n e f f o d t o c a r r y o n t h e t r a d i i i o no f a g r e a t n a m e .
ZEISS makes microscope h;story
I h e Z E I S S w o r k s h a v e m a d e d e c s l v e c o n t r i b u t i o n st o i h e d e v e i o p r n e not f t h e m o d e r nm i c r o s c o p eF . r o r nt h e l o n g i s i o f i n n o v a i i o n sj n t r o d u c e db y t h i s c o m p a n y , w e h a v es e e c t e d t h o s e p o n i s w h j c hm a y b e o f p a r t i c Lar r j n t e r e s t1 o y o u .
1812
F o r t h e f i r s t t r n e i n t h e h i s i o r yo f t h e m c r o s c o p eo b j e c t i v e s w e r e o f f e r e dw h i c h h a d n o t b e e n n r a d es i m p y b y " t r y l n g l t o t ' t b l i o n t h e b a s l s o f r n a t h e m a t c aal n d o p l i c a l f e s e a r c l r . T h e C A R L Z E I S S i s t N o . 1 9 ( 1 8 7 2 )o p e n sw t h t h e s e n t e n c e : "The m c r o s c o p es y s t e m s b e l o w a r e e x c u s i v ey b a s e d o n theoretica complrtaiions carried out by Professor Abbe at
Ai the same time, the bis Abbe illuminator appeared for rhe "combjned first time i n a l l i t s p a r t s e x c l u s i v e l yo n r h e b a s i s of theorerical considerations _ 1886
In the ZEISS works, Ernsi Abbe succeeded in making inicroscope objectives of unusually high correction,wh ich he called Apochromais- At the same iime, rhe corresponding c o m p e n s a t i n ge y e p i e c e sw e r e i n t r o d u c e d .
1893
P r o f e s s o rA . K d h l e r , w h o j o i n e d Z E I S S i n 1 9 0 0 ,p u b l i s h e d l L e m . - h o d o " i l l u T i n a t o - a l e c a l l a od h e h i r n .
1896
ZEISS built the first Greenoughstereomicroscope.
1904
Professor K0hler published his work on ihe uliraviolei
I911
F o l l o w i n ga s u g g e s t i o nb y K o h l e r , Z E I S S i n t r o d u c e dp a F focallzed microscope objectives.
1933
z L S : i e . o r u l o i / e d r n e c o r s t . u c t j o n o l T i c r o s c o p e sb y deeigning ihe L stand (not iiltable, stage always horizontaT,
r935
F o r m l l i t a r yr e a s o n s ,t h e a n t i - r e f l e c t i ocno a t i n go f l e n s s u r f a c e s ( ' b l o o m l n g ) i n v e n t e db y Z E I S S h a d t o b e c l a s s i f i e d
r936
ZEISS built a firsi prototype of the phase contrast microscope 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 phasecontrasi microscope (Dr. K. Michel). Wjth this examination method, cell research entered a new phase. 63
1944
Z E I S S b u l l i t h e f i r s t s t e r e o m i c r o s c o pw e ith magnificauon c h a n g e r{ e l i m i n ant g e x c h a n g eo f o b j e c t i v e s ,o f f e r l n s c o n stant workjng di6tance).
1950
The ZELSSSTANDARD Microscope ntroduced coaxrar controls and spring- oaded objectives (specirnenprotection) i n t o m i c r o s c o p ec o n s t r u c t i o n .
1950
Z E I S S o b i a i n e da p a i e n t f o r a m a g n i f i c a t i o cnh a n g e rd e s i g nated as OPTOVAF.
1955
T h e f i r s t p h o t o m i c r o g r a p h idce v j c e w i i h f ! y a u t o r n a t i ce x p o s u r ec o n i r o lw a s s o l d ( Z E I S SP h o t o r n i c r o s c o p e ) .
1959
W i l h t h e i r U l t r a f u a r s ,Z E I S S s u c c e e d e di n m a k i n g r e f r a c t i n g o b j e c u v e ss u i l a be f o r b o i h u t r a v o l e t a n d v i s i b l e l l g h t (imporlantfor m crospectrophotomeiry and cancer researchl.
T h l s b o o k i s c o p y r g h i . l i m a y n o t b e r e p r o d u c e do r i r a n s ' l a t e d b y a n y r n e a n sl n w h o l e o r i n p a r t w i t h o u t p e r m i s so n . A p p l i c a l i o n s w l t hr e g a r d t oc o p y r i g h ts h o r . r lbde a d d r e s s e d t o ; CARL ZEISS, Oberkochen/West Germany
