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Ziess Lang Zeiss Information 70 114 120 1968 PDF
Ziess Lang Zeiss Information 70 114 120 1968 PDF
Ziess Lang Zeiss Information 70 114 120 1968 PDF
interference-contrast
microscopy
by Walter Lang
microscopes, there is another distinguishing a principal section 3. The so-calied ordinary microscope, be cause , after re combination,
characterlstlc, that of splitting and recombin wave ts transmitted by the crystal without the two coherent bundles can only interfere
ing the beam. In the interference micro any deflection. The so-ca lled extraordinary and produce an interference image, lf the
scope of the MicheJson type a beam-split wave is deflected to one side, in spite of vibration d irections of the two plane-po
ting prism is used . (For further details, see its vertical (90 0 ) incidence on the plane larized waves lie in one and the same
11,13,16,17,28,35,36,37,40 and 41). parallel plate. It emerges from the crystal plane 4.
In the lamin-Lebedeff and Nomarski double parallel to the axls of the ordinary wave.
Similar considerations also apply to the
beam interference microscopes, on the other If the direction in which the crystal is cut
Nomarski differential interference micro
hand, the beams are split and recomb ined is known, the separation between the two
sco pe. They are of decisive importance not
by birefringent crystals. Birefringent or waves is a function of the th ickness of the
only for the formation, but also for the
doubly refracting crystals (3, 4, 23, 34) are crystal plate. In the case of the ZEISS
crystals which spl it an incident light wave lamin-Lebedeff transmitted-l ight interference
into two components which are plane equipment it amounts to minimum 0.05 mm 2 The optlc axls of a blr ef rlngent cryslal Indi cal es
polarized and whose vibration planes run (Achromat Pollnt., 100 x, 1.0 N. A ., oil) and the direct lon In which the ord lnary and exl raor dlnary
wa v es co inclde. Thls dlr ect lon, In whlch lhe cryst al
perpend icular to each other. Fig. 2 may max imum 0.5 mm (Achromat Pollnt., 10 x, behaves Ilke an isotrop lc , i. e., not blrefrlngent
serve as an explanation. It also illustrates 0.22 N. A.). As is evident from Fig. 2, a medium, ls als o called the Isotroplc axl s. In lhe ca se
of calcite and qua rlz 11 ts Idenl lcal wilh the oplic
the principle of wave splitting in the lamin birefringent plate ls characterized by an axis of the cryslal. Cryst al s whlch hav e only a single
Lebedeff microscope. Let us assume that a other feature in addition to lateral beam opti c axts, e. g. , calcite or quarlz , are calied unlax ial
light wave, of which the diagram shows crysl als .
spl itting. The plane-polarized light wave
only the axis, hits a plane-parallel calcite wh ich hits the crystal and whose vibration
3 Any plane through the c ryslallograph lc axls Is a
plate perpendicularly. Furthermore, we as direction is offset by 45 to the plane of principal sec n on of a crystal ,
sume that the inc ident wave is plane-po the diagram, ts split into two plane-polarized
larized and its vibration plane is inclined by components. The vibration direction of the (Fo ll ow lng general parlance, lhe terrn "beam" laken
45 0 to the plane of the diagram. The optic from geom etrlcal oplics Is here and In lhe followlng
ord inary wave is perpendicular to the plane
frequently used Instead of lhe more approprlate
axls 2 of the ca lc ite plate - marked by the of the diagram, while the vibration direction term s " bundle of reys" or "light wav e" .)
double arrow - runs parallel to the plane of the extraordinary wave co inc ides with
of the diagram. As the beam enters the the plane of the diagram. This la a fact The lerm "plane of polarlzallon" (plane perp en
dlcular 10 lhe vibration dlrectlon of the electrlc
crystal, the wave ls split into two parts, the which must be taken into account in the vector) used In earlier Iilerature can be dlspensed
axes of wh ich are entered in the diagram as des ign of the lamin-Lebedeff interference wlth and la nol used In thts paper.
Fig . 2: Beam path In the principal section 01 a birelrlngent plate.
Fig . 3: Sp l itt ing the veet or E into er and ez, the las t
(auerordentlicher Strahl = Extraordinary ray , ord entlicher Strahl = Ordinary ray, Richtung der optischen
two at ri ght angles to eaeh other .
Achse = Direction of optic axis, parallel zur BIldebene = parallel to Image plane, Schwingungsrichtung des
line ar polarisierten Lichtes ~ Vibration direetion 01 piane-polarized light , um 45 geneigt ~ inellned by 45,
/
auerordent- ordentlicher /
licher Strahl Strahl Richtung der optischen Achse /
/
- - parallel zur Bildebene /
/
/
SchWingungsrichtung des linear /
polarisierten Lichtes
/ - - - - um 45 gene igt
I
I
- senkrecht
l +t+H parallel
tI zur Bildebene
I
2 3
interpretation of the interference image. It summarizes the most essential results of angle of 45 with the incident wave (see
Therefore, the splitting of a plane-polarized Figs. 3 and 4. Let W in (a) be a plane Figs. 3 and 5). At the cemented surface of
wave into two plane-polarized components polarized wave normal to the plane of the the Wallaston prism the two component
of different vibration directions w ill be dis diagram. Following the scheme of Fig . 3, waves are deflected in two directions at a
cussed In greater detail (see also 6, 21, 22, certain, relatively small angle. The ord l
this wave with the vectors E and E' (b) can
34). The opposite process, namely the nary wave 5 ls deflected towards the base
be split into the tho components w, and W2
recombining of two plane-polarized corn of the upper prisrn, wh ile the extraordinary
ponents into a plane-polarized wave, will with the vectors el, el' and e2' e2" In (c) wave 5 is deviated towards the edge of the
be dealt with in connection with the inter the vibration plane of the wave W coincides upper prism. The two component beams
pretation of the differential-interference with the plane of the diagram. encounter different indices of refraction so
image. that the wavefronts travel with different
As was mentioned above, a strong lateral speed.
The vibrat ion direction of a plane-polarized beam-spl itting effect is achieved in the
light wave is identical with the plane in Jamin-Lebedeff interference microscope with
which the vector of the electric field strength the aid of a birefringent plane-parallel plate. 3. Double-beam interference-contrast
(Questions of the beam recombination and microscope with two Wollaston prisms
E vibrates. In accordance with the rules
compensation cannot be discussed here.
go verning the ca lcu lus of vectors, E can On the basis of the explanatory remarks in
For more information interested readers are
the previous section, the design of a double
be split up into two vectors, e, and e2, which referred to the pertinent literature : 9, 10, 14,
beam interf erence-co ntrast rnlcroscope can
20, 23. For numerous references, see 25).
are at right angles to each other and each
Flg . 5 a): Plane-polarlzed wave W wlth vtbra tlcn pisne, at rlght angles to plene ot drawlng ; b) Splitt ing 01 the
wave W Into !wo plane-polarlzed waves w, and W2; c) Same as b), but travellng dlrectlon 01 wave W Identlcal
wlth plane ot diagram.
Flg. 6: Angular splitting 01 a plane-polarlzed light wave wlth the ald 01 a Wollaston prlsm. (For greater clarlty,
the angular splitting haa been exaggerated . The term "Image plane" stands tor the plane ot the diagram and
not Ihe Intermediale Image plane 01 the mlc ro scope .)
(auerordentlicher Strahl - Extraordinary ray , ordentlicher St rahl = Ordlnary ray , Richtung der optischen
Achse = Dlrection ot optl c ax ls, parallel = para llel , senkrecht zur BIldebene = perpendlcular to Image p lane ,
Schw ingungsebene des Lichtes = Vibrat ion dlrectlon 01 light , unter 45 geneigt = Incl lned by 45, senkrecht
= pe rpend icular, parallel zur Bildebene = parallel to image pl ane)
4
above, angular beam splitting occurs at the
cemented surface of th is prism. (For greater
clarity, one of the prism segments is dotted) .
If the center plane of the Wollaston prism
lies in the iamp-side focal plane of the
condenser, the two beams will travel along
parallel path s, slightly separated laterally
in relati on to each other, after they emerge
from the condenser. They pass through the
specimen and the microscope objective and
converge in the image-side focal plane of
the microscope objective. This is where a
second Wallaston prism is located, wh ich
has the same dimensions and optical proper
ties as the first one . Together with the
microscope objective the second prism re
combines the two bearns . After emerging
from the second Wallaston prism, the two
components once more form a single beam,
but they are still plane-polarized and their
vibration planes are at right angles to each
other. In order to enable the two waves to 5
produce the desired interference effect in
the intermed iate image plane, their vibration
planes must coincide. This can be achleved a) b) c)
by inserting an analyzer Into the light path.
The diagram in Fig. 5 shows the two com W W E W
ponents now recombined to form a single,
plane-polarized wave that Is capable of in ej e2
terference. The resulting interference inter
mediate image can be viewed through the
eyepiece in the conventional manner.
e; ei
While Fig. 7 merely shows the principle of E'
beam splitting and recombination, diagram E'
Fig. 8 also indi cates the vibration directions
of the polarized components at different
points along thelr path . Fig . 8 shows only
the axes of the light beams . The polarizer 6
ls oriented In such a way that the natural .----- - -- - -- - -- - -- - -- - -- - -- - - - - -- - -- -- - - -- ,
light emerging from the fteld diaphragm
auero rdent ordentlicher
Intermed iate
----,--- I image
Analyzer I
I
I
Analyzer
(135 0 )
Second Wolla
ston prism
Second Wolla
ston prism
Analyzer
Objective
Objective
Half-silvered
Specimen mirror
Specimen
Wollaston
Condenser Condenser prism
First Wolla
ston prlsm
First Wollaston
prlsm /
e I I I
Objective
Specimen
I
I
I
I
Polarizer Polarlzer
Field diaphragm
I (45 0 )
7 8 9
the microscope objectlve and the second cancelled out when it passes the prlsm the the conventlonal manner In the Intermediate
Wol/aston prism. It becomes obvious that second time. In other words, when the beam image plane (not shown) and viewed through
the condenser with the first Wol/a ston prism travels the first time through the prlsm the an eyeplece.
and the objective w ith the second Wol/aston reflected-lIght objectlve and the Wol/aston
As regards the diagram in Fig. 9, It may be
prism are funct iona lly correl ated. The ana prism act in the same manner as the con
Iyzer is oriented so as to form an angle of mentioned that the beam passing through
denser and the first Wol/aston prlsm of the
45 w ith the vibrat ion plane of each of the the polarlzer Is inclined by 45 0 to the plane
transmitted-Ilght setup illustrated In Figs . 7
entering waves. In accordance with Figs. 4 of the diagram. The polarlzer should there
and 8, whlle when It trave ls through the
and 5 this ensures that both beam com fore be Imagined as Iying below the plane
prism the second time, these components
ponents act with the same Intensity. What of the drawlng. For thls reason - and be
correspond to the objectlve and the second
happens when the analyzer forms an angle cause of the perspective view - the vibration
Wol/aston prism In those figures . Thls Is
other th an 45 with the two beam cornpo indicated by the diagram In Fig. 9: the beam directions of the polarized components have
not been indicated. In principle, however,
nents will be d iscussed in detail in connec that is plane-polarlzed by the polarizer is
tlon with the interpretation of the differential deflected to the Wol/aston prism by a half conditlons are similar to those shown in
Figs. 7 and 8 for transmitted light.
interference-contrast Image. silvered mlrror. The two beam components
emerge from the reflected-l ight object lve
In the equipment for transmitted light. two parallel to each other and wlth a sllght
4. The Nomarskl double-beam Interference
Wol/aston prlsms are needed for beam lateral separation. The beams rellected by
contrast microscope
splitting and recomblnlng , whereas only one the specimen are then recomb lned with the
Wollaston prism is requ ired for a reflected aid of the objective and the Wol/aston prlsrn, The preceding remarks were primarily de
light setup, slnce the light passes through pass through the half-silvered mirror and, voted to the design and operation of the
the prism twice In opposlte directions (30. by means of an analyzer, are brought to birefringent components used to split and
31, 32). The spl itt ing of the beam when it vi brate in one plane . The Interference-con recombine the beams, while nothing was
passes through the pr ism the first time is trast Image of the specimen is formed in said about the effect of the specimen on the
beams . This will be discussed together with side focal plane of the objective, although Fig . 10: Nomarakl prlsm with dlrection of optic axes
and position of Interference plane Indlcaled.
the formation and interpretation of the inter the Nomarski prism itself is located at a
ference-contrast image in the second paper relatively long distance from the objective.
to be published on this subject in this This is of considerable importance for the
journal. We shall now examine what require des ign of a differential interference-contrast
ments have to be fulfilled by the Image microscope : since the parfocal distance of
forming optical system. the objectives, i. e. the distance between the
object plane and the objective seating face,
When dlscusslng the double-beam inter is given and constant, the Nomarski prism
ference-contrast microscope with two Wol/a can Iikewise be mounted at a given distance
ston prisms for transmitted light, we as from the objective, e. g. in an interference
sumed that the lamp-slde focal plane of the contrast silde. Moreover, lf the position of 10
condenser and the eyepiece-side focal plane the eyepiece-side focal plane of the objec
of the objective are located in the central tlves used only sllghtly deviates axially from
planes of the Wol/aston prisms (8, 15, 30, a certa in mean position, a single Nomarski plement and should therefore be briefly
31, 32). In addition, we assumed the inter pr ism will suffice to recombine the beams discussed: first, imagine all optical corn
ference planes of the Wol/aston prisms, with all available transmitted-light objectives. ponents such as the condenser (with second
which coincide with their central planes With the ZEISS interference-contrast equip ary prism), specimen, objective and prln
(parallel to the upper and lower surfaces of ment for transmitted light, one and the same clpal prism between the polarizer and the
the prism) to be exactly superimposed. The Nomarski prism, i. e. the same interference analyzer removed from the light path. The
first of these requirements presents funda contrast sl ide 6, is used for objectives of field of view will then be completely dark. If
mental difficulties: with high-power objec 16 x, 40 x and 100 x. For this reason - and the principal prism, that is a birefringent
tives, the image-side focal plane lies in the for other reasons which cannot be explained component, is now inserted diagonally be
objective, below the thread seat: a Wol/a in detail here - this is called the principal tween polarizer and analyzer, the field will
ston prism can therefore not be located in prism, to distinguish it from the so-called be partly illuminated; interference fringes
that plane. This is equally applicable to secondary prisms accommodated in the will be visible parallel to the wedge sldes of
transmitted-Iight and reflected-Iight objec substage condenser. In the ZEISS transmit the component prlsms; the fringes are black
tives. But neither do transmitted-light con ted-light equipment, there are three second or colored, depending on whether mono
densers of high numerical aperture and cor ary prisms whieh are contained in the chromatic or polychromatic light is used for
respondingly small focal length allow a achromatic-aplanatic condenser (type VZ) illumination (only the fringe or zero path
Wol/aston prism to be located in their front for interference contrast, phase contrast and difference will always be black). If the
focal plane (i. e. on the side of the light bright field and designated as I, 1I and 111. secondary prism is then moved into the light
source). As a result, differential interference Like the different sizes of annular condenser path tagether wlth the image-forming optics,
contrast microscopy with the aid of Wol/a diaphragms for phase contrast, the second the interference fringes of the secondary
ston prisms is limited to very low magnifi ary prisms in the condenser are adapted to prism can be made to cancel out the fringes
cations (30). the numerical aperture (and the initial magni of the principal prism in the objective pupil,
fication) of the objectives to be used. provided that the two prlsms are sultably
A very neat and technically feasible solution dimensloned and oriented : the field is once
to these problems was the introduction of The terms "prtncipal prism " and "secondary more completely dark. This corresponds to
Nomarski's modified Wol/aston prism which or compensating prism" are used in con interference contrast explained above in
in the following will be briefly called "No nection w ith an entirely different way of connection with the interference micro
merski prism". This prism (Fig . 10) consists describing the differential interference-con scope, which exists in the case of infinite
of two cemented components of a uniaxial. trast microscope (8, 26, 30, 32). In the pre fringe spacing.
birefringent crystal, such as calcite or sent paper, the design of the Nomarski
quartz. The optic axls of one of these differential interference-contrast microscope While in the transmitted-light setup the
prisms (the lower one in Fig. 10) is parallel has for didactic reasons been described as principal and secondary prisms are separate,
to the wedge side, as in the Wol/aston that of a double-beam instrument. The afore the principal prism of the reflected-Iight set
prism. The optlc axis of the other prism , mentloned second approach 15 useful sup up also acts as secondary prism . It is
(the upper one in Fig. 10), however, is contained in the interference-contrast equ ip
inclined at a certain angle to the upper ment into which the proper reflected-light
bounding face. As a result, the interference The fact that the Interference-contrast allde for the objectives are screwed. The prisms are
blg STANDARD UNIVERSAL, PHOTOMICROSCOPE
plane is outside the compound prlsm and ULTRAPHOT 11 la different from that of the adapted to the numerical aperture of the
(dashed line in Fig. 10). By suitable orienta STANDARD mlcroacopes of the WL, RA and KL objectives. - For further teehnical details
serles Is due to the different dlstance of the Inter
tion of the Nomarski prism its interference ference-contrast alldea from the objectlvea (and other
and practical hints consult the literature
plane can be made to lie in the eyepiece tube optlcs). Iisted overleaf (1, 2, 8, 24, 26, 29).
References: [21] Michel. K.: Die Grundzge der Theorie des
Mikroskops In elementarer Darsteliung, 2nd ed.,
Voi. I of the "Physik und Technik" series, edited
[1] Boe/cke. C.: STANDARD RA Routine and Re by F. Gssler, Wissenschaftliche Verlagsgesell
search Microscope, Operating Instructlons G 41 schaft mbH, Stuttgart 1964.
120. CARl ZEISS, Oberkochen.
[22] Michel. K: Die Mikrophotographie, 3rd ed.,
(2) Boelcke. C.: Vertlcal Illumination on UNIVER Vol. X of the senes "Die wissenschaftliche und
SAL mlcroscopes, PHOTOMICROSCOPE and angewandte Photographie", edited by J. Stper,
ULTRAPHOT 11. Operating Instructions G 41-655. Springer-Verlag, Vienna-New York 1967.
CARl ZEISS, Oberkochen.
[23] Mtze, K, L. Foitzlk, W. Krug. and G. Schreiber
[3) Born, M .. and E. Wolf: Principles of Optics , (editors): ABC der Optik, leipzig edition, Verlag
third editlon. Pergamon-Press Oxford-london Werner Dauslen, Hanau/Main 1961.
Edinburgh-New York-Paris-Frankfurt 1965.
[24) Neupert, H.: Nomarskl Interference-contrast
[4] Buchwald. E.: Einfhrung in die Krlstalioptik, equipment (CNRS Ileence). ZEISS Information 65
3rd ed .. Sammlung Gschen, leipzig 1937. (1967), 96.
[5] Flgge, J.: Das Phasenkontrastverfahren nach [25] Author not named: Transmitted-light interference
Zernike und Erklrung des Interferenzkontrasts equipment, leaflet 40-560/1, CARl ZEISS. Ober
nach Nomarskl. lecture glven at 1967 Course of kochen.
Photomicrography heid by CARl ZEISS. Ober
kochen. at Gttingen. [26] Author not named: Phase contrast and interfer
ence contrast, leaflet 41-210. CARl ZEISS, Ober
[6] Frencon, M.: Interlerences, Diffraction et Polari kochen.
sation, Handbuch der Physik XXiV (edlted by
S. Flgge), Sprlnger-Veriag, Berlin-Gttingen [27] Author not named: Wild Interference Attachments
Heidelberg 1956. for Incldent Light for the Wild M 20 Microscope.
leaflet M 1 421 e - VI. 67, Wild Heerbrugg LId.,
[7] Franr;on, M.: Einfhrung In die neueren Metho Switzerland.
den der Llchtrnlkroskopre , translated by L. Ai
bert, Verlag G. Braun, Karlsruhe 1967. [28] Author not named: Interference Microscope.
leaflet 64-60211 Ii , CARl ZEISS, Oberkochen.
[8] Gabler, F.. and F. Herzog: Eine neue Interferenz
kontrasteinrichtung fr Arbeiten Im Durchlicht. [29] Author not named: Grand microscope auto-eclai
leaflet SD Interf. Kontr. Dl D 8/66, C. Relchert, ra nt Nachet 300, Notlee 1500, leaflet of Messrs.
Optische Werke AG, Vlenna. Nachet, Paris.
[9) Gahm, J.: Polarlzed-light microscopy and double
[30) Nomarskl, G.: Mlcrolnterferornetre dlfferentielie
beam Interference microscopy by transmitted
a ondes pclertsees. J. Phys. Radium 16 (1955), 9.
light, ZEISS Information 61 (1966), 89.
[10] Gahm,J.: Durchlicht-Interferenzelnrlchtungen nach [31J Nottierskl, G.. and Mme. A. R. Well/: Sur I'obser
Jamin-lebedeff, ZEISS-Mltteilungen 2 (1962), 389. vatlon des figures de croissance des cristaux
par les methodes interferentlelles a deux ondes.
[11] Hansen, G.: Die Sichtbarkeit der Interferenzen Bull. Soc. franc. Mlner. Crist. 77 (1954), 840.
beim Micheison und Twyman-Interferometer.
ZEISS-Nachrichten, Vol. 4, No. 5 (1942), 109. [32J Nomarskl, G., and Mme. A R. WeilI: Application
a la metalloqraphie des methodes lnterferenttel
[12] Hansen, G., and W. Kinder: Abhngigkeit des les a deux ondes polartsees, Rev. de Metallurqte
Kontrastes der Flzeau-Streifen Im Michelson 52 (1955). 121.
Interferometer vom Durchmesser der Apertur
blende. Optik 15 (1958), 560. [33J Plller, H.: Durchllcht-Interferenzmikroskopie nach
dem Jamin-lebedeff-Prlnzip, ZEISS-Mitteilungen
[13] III/g, W: Oberflchenprfung mit dem Interfe 2 (1962), 309.
renz-Mikroskop. Metalioberflche 7 (1953), 97.
[34) Poh/, R. W.: Optik und Atomphysik, 10th ed ..
[14] Jemin, M. J.: Sur un refracteur differentlei pour Springer-Verlag, Beriin .. Gttingen - Heldelberg
la lumlere polartsee construit par l.utz , C. R. 1958.
Acad. Sei. Paris 67 (1668). 814.
[35] Rntsch, K.: Oberflchen prfung durch LichtInter
[15J Jeglitsch, F.. and R. MItsehe: Die Anwendung fer enz , Feinmechanik und Przision 52 (1944), 75.
optischer Kontrastmethoden In der Metallogra
phie. Radex-Rundschau, No. 3/4 (1967). 587. [36) Rntsch, K: Optische Verfahren zur Oberflchen
prfung, ZEISS-Nachrlchten, Vol. 5. 6 (1945). 189.
[16] Kinder, W.: Ein Mikro-Interferometer nach W. Lin
nik, ZEISS-Nachrlchten, Vol. 2 (1937), No. 3. p. 1. [37] Rntsch, K.: Grundstzliches zur Interferenz
[17] Kinder, W.: The Interference Flatness Tester and 42 (1952), 434.
[20] Lebedeff, A A: lnterferometre polarteatlon et [41] Uhl/g, M.: The ZEISS Interference Mlcroscope in
ses appllcations. Revue d'Optlque 9 (1930), 385. Practice, ZEISS Werkzeitschrift 30 (1958), p. 70.