Nomarski

Differential Interference-Contrast Microscopy

Part IV : Applications

By W alter l ang

l aborato ry for Mi cr oscopy

CARl ZEISS. überkochen

In the comp re hen si ve de scription of Nomar­ ski differ ent ia l int erfe rence-co nt ra st (DIC) microscopy, th e fundam entals and ex peri­ me ntal designs (Part I) and the fo rm ation of the interference imag e (Part 11) were dis­ cu ss ed and a comparison made with the phase-con tr ast techn ique (Pa rt 111) [26. 27. 28]. The pres ent Part IV will now give a review of t he app lic at ions of Nom arski DIC mic roscopy to get her w ith a list of refer­ en ces . N o c lai m is made wi t h regard to the completene ss of the b ibl iographic data in v iew of t he rap idl y increas ing number of appl ications an d pu blicat io ns on the subject. The bibliograp hy is th ere fo re on ly intended to se rv e as a gu ide to the potential uses of th e No marski DIC method in the different field s o f micro scopy o f organi c and ino r­ gan ic objects. A. Microscopy of organic objects An e ssential advantage of Nomarski DIC microscopy ls the fa ct that - like pha se contra st, for example - it allows the ex­ ami natio n of uns ta ined specimens. A new and extreme ly useful ai d has th us been created, ab ov e all for exam in ing liv ing spec l­ men s unde r t he opti cal mic roscope . Cytology Us ing a liv ing cell of Haemanthus katherinae in the pr ocess of div ision as an example, Bajer and All en [5] demonstrate the superi­ ority of the DIC image over phase-contrast re pr esentation: wh ile in phase contrast the halo effe ct makes lt impossible to recognize detarls, the spi ndi e fibers can be clearly seen by the Nomars k i d ifferentia l interf er­ ence-cont rast met hod . DIC micrographs of heia cells in a nutrient so lution and denatured w ith 96 % alcohol we re publi sh ed by Gabler and Herzog [18 .19] .

Wun derer and W itt e [47] publ ished a com­ pa rison o f photom ic rog raphs of cells and group s of cells from the mucuous membrane of the hum an stomach, tak en by ph ase con­ tra st and by Nomarski differential interfer­ enc e contrast. Th ese examp les prove that th e tw o met hods co mplement each other very nicely. However, in the case of a group of gland ula r cells of t he gastric mucosa , the int erference method offers the advantage of improved detail defin it ion. G ivin g many pr actica l exarnples, Padawer [39] expla ins the cha racteri sti cs and advan­ tages of D IC microscopy. In one particu lar exam pl e nuclei and vac uol es appear in the DIC image as depressions, wh ile highly refractive structu re s such as eos inophile gra nula or fatty inclusio ns seem consider­ ably ele vate d . A ls o the vacuoles of macro­ p hages appear as depress lons, wh ile the nuclear memb rane shows up as a bulge. The author shows that phase structures located

22

outs ide the f oca l pla ne cannot always be neg lected in th e interp reta tio n of DIC im­ ages. W hen erythrocytes are v iewed by phase cont rast, the f ormatio n of haloes is rather troublesom e. In th is case, th e DIC image is unm istakab ly supe rio r. The si tu­ ation is simila r w ith epi the lia l ce lls of the mucous membrane of the human mouth. Duitschaever [14] uses th e DIC method fo r micro scop ic investi g ati ons on somatic cells in cow's milk and other body flu ids. Engel s and Ribbe rt [15] also use Nomarsk i d iffer­ ent ial interference con trast fo r the exami­ nation of nucleoli in M usca d ome st ica. Rib ­ bert and Bier [41] mak e use of th e Nomars ki meth od for stu dy ing inse ct ovaries . StoII · and Gundla ch [43] compare the pha se-contr ast im age w ith the DIC ima ge of a cell sm ear in a sa line so lut ion . Th e l iv ing trich omonad beside an ep ithel ial cell and erythrocyt es shows mo re detail in interf er­ en ce co ntra st. Thi s ho lds tru e above all fo r the marg inal portions of the trichomonad wh ich in ph ase con trast reve al consi derable fl ar e du e to ha lati on . A s im ilar situ at ion is encountered in the cell smear in a sal i ne solu t ion. Th is examp le also shows t hat it is eas ier to d istinguish superimposed stru c­ tures in the DIC image than in phase con­ trast. The au thors prove that in such a cas e it is frequent ly imposs ib le to re cog nize the borders of t he ce ll in p hase contrast du e to halatlon. Bo tany The ph ase -contrast technique ls weil suited for examin ing sm all particles - especia lly organelles - in protoplasm [44]. However, the great depth of f le ld of th is me thod is a dis advantage in botany . As a re sult, pha se structures in the light path will impair th e phase image even if they are located ou t­ side t he fo ca l plane [44] . Accord ing to Ur' and Gabler [44]. the shall ower depth of field of Nomarski DIC microscopy opens up a cons iderably wider field of app lication f or light microscopy in botany. These authors show, amo ng others, DIC micrographs of the ins ide and outside epidermis of Al lium cepa, cells of Closterium lunu la and - in a comparison w ith phase contrast - M icraste­ rias denticulata and Closterium lunula . In the case of Allium cepa , mltochondrta, the Golgi comp lex, leucoplasts, the nucleolus and large and sma ll spherosomes stand ou t in high contrast, the latter due precisely to the great difference between their own re­ fractive index and that of the surround ing areas, Accord ing to Padawer [39], observation of plant material offers considerable difficulty, be it in ph ase contr ast or differential inter­ ference contrast. In the one case , the pro­

nou nced differe nce o f refra ct iv e index give s ris e to heav y ha lat ion , in the ot he r b ir e­ fr ingen t compon ent s disturb the imag e. This ha s be en proved for ex ample in the ease of dried poll en, such as Sa lix d isco lor and ab ove all Coreopsi s. S im ilar cond it ion s are encoun tered w ith freshwater Chlorophyceae. M aguir e [29] inv estigates subch rom at id stru c­ tu res in corn w ith th e aid of the No mars ki method and, for compa ri son , in phase con­ trast. Us ing the African blo od li ly , Ha emanth us k atha ri nae, as an example, Allen , Dev id and No tnerski [3] sh ow tha t the spin die fibers of a liv ing cell during div is ion sta nd out clearl y in the DIC imag e (see also [5] and [6]) , wh ereas they are hardly v isible by any other microsc opic techn iq ues. Baum [8] uses DIC micro sc opy to sh ow na tu ral hyb rids of Av ena sat iva and Aven a fatu a in th e cu lti­ vat ed oa t. Histo logy

Gab/er and Herzog [18, 19] show the thyroid gl and of a mous e in po sit ive and negati ve pha se co ntr ast as w eil as in Noma rski dif­ ferent ia l inte rference con trast. Nomarski DIC is also su ited for ampl itude stain ing of stained speci mens . as shown by Allen , Dav id and Notnerski [3] on large chromo­ somes of Drosophila melanogaster. Even hu man chrom osome s ca n ea sily be examined by the DIC meth od using amp litude sta in ing. Very th ick bone sections gen eral ly used, for exarnple, for examination by inci dent f1uores cent ill urninati on, re sult in a notice­ abl e de cre ase in contr ast in the D IC lrna pe, same as in ph ase contrast. Th is is due to the f act t hat pr oper imag ing of t he contrast­ p roduc ing components , such as annular dia­ phragm on ph ase p late or auxiliary pr ism on princ ipa l p rism , is no lo ng er gu aran teed. This is d emo nstrated by Lang [28] both for ph ase contrast and Nomars ki differential interfer enc e contras t on th e ex am ple o f an excessively thick transparent specimen (pol ished bone sec t ion) and a th in , well ­ sui ted transparent spe cime n (r at' s to ngue, unstained) . Despite th is qu allficat lon , t he No mars ki DIC rnet hod , owin g to its high useful aperture and the consequent small depth of fie ld, ls the idea l method fo r the observation of so -calied op t ic al sections . Th is may be ve rlf ied by an ex ample from zo ology: Fig . 1 sho w s photo microg rap hs of Ma cronyssus b acoti in br igh t-f ield (a) . ph ase contrast (b) and Nomarski differential inter­ ference contrast (c) all with the same fo cal • See the monograph meanwh i le pub ll shed by Peter StoII : Gyne c olog lca l V ital Cytoloq y, Spr inger-Verlag Berl tn, Heldelberg, New York 1969. whic h contal ns numerous pracllcal examples of DIC mtcroscopy, often with comparatl ve phase-contrast mtcrc qraphs .

plane. Figs. d and e also show the DIC image of the same object with two other focal plane settings. The Nomarski DIC method makes it con­ siderably easier to analyse the structure of thin seetions as is shown by the contrast with the bright-field view in Fig. 2. For this type of work the DIC method can also be successfully applied with stained specimens. The colour distortion caused by the No­ marski method in such cases remains within reasonable limits. It should also be remem­ bered that the transition to bright-field ob­ servation for comparison purposes, for instance by removing the interference con­ trast siide from the light path, is swift and convenient. Hematology

With the aid of Nomarski DIC microscopy, unstained erythrocytes can be rendered visible with excellent results (Gabler and Herzog [18, 19]). According to the authors, the DIC image of a crystal in the blood Iymph of an eel is superior to the corre­ sponding phase-contrast image that ls im­ paired by halation. Padawer [39] discusses differences between phase-contrast and differential interference­ contrast observation of the hemolysis of frog erythrocytes. The photomicrographs taken under identical condltlons show non­ hemolyzed cells, spherocytes and completely hemolyzed cells. The author shows that with normal cells the nucleus in the DIC image stands out more c1early from the cytoplasm than in phase contrast. The nucleus becomes more elevated from the cytoplasm all the more clearly the more water the cell ab­ sorbs and the more hemoglobin it loses. With completely hemolyzed cells the cyto­ plasm will show up only weakly due to the loss of hemoglobin, while the nucleus stands out in good contrast. In another case, viz. a fresh blood smear, the coiling makes phase-contrast observation impossible due to heavy halation. In the DIC image, how­ ever, sufficient detail can be recognized in spite of the stratification. In phase contrast, fibrin fibers may appear dark or bright, depending on whether or not they Iie in the focal plane. This complication does not exlst in differential interference contrast. Neurology

Neuhoff [31] uses Nomarski DIC microscopy to render human ganglion cells visible and especially for examin ing cells in which an appendix leads back to the same cell, so­ called feedback neurons. Bacteriology

With bacteria specimens, the disturbing halation known from phase-contrast images presents an advantage in Nomarski DIC microscopy; as an example, Gabler and Her­ zog [18 , 19] show a smear of Klebsiella. Hydrobiology

Quite a number of authors have published DIC photomicrographs of dlatorns which show up 3-dimensionally in the DIC image. Due to the excellent resolution of the No­

marski DIC method, minute detail can be recognized in the diatoms. Gab/er and Her­ zog [18, 19] show the DIC image of Auliscus sculptus. The use of Nomarski DIC micro­ scopy in micropaleontology is described by Barbieri and MazzoJa [7] . Padawer [39] compares phase-contrast and differential interference-contrast images of various diatoms. In this comparison , the superiority of the DIC image is very evident. In a very comprehensive paper, Allen, David, Hirsch and Watters [2, 13] cover the subject of image interpretation in transmitted -light polarizing interference microscopes both of the image-duplication and the differential type . In addition to an extensive comparative discussion of theoretical and experimental principles, the differences are illustrated impressively by a number of practical ex­ amples. Under Nomarski even large path differences of up to 2 'I. ). between Stauro­ neis acuta diatoms and the mounting medium give images that are rich in detail. The Surirella robusta diatom can be reproduced with good contrast even with an i1luminating aperture of 1.25. As a result, object details that are invisible at a smaller numerical aperture of, for example , 0.6, can be clearly distinguished. Allen, David and Nomarski [3] report on the fundamentals, design, function and charac­ teristics of ZEISS differential interference­ contrast equipment. A number of practical examples explaining the special features of the equipment concern diatoms: Staureonis acuta diatoms in the DIC image as com­ pared to the interference-contrast image (photographed with the ZEISS Jamin-Lebe­ deff system) show that particularly pro­ nounced gradients of optical thickness in the specimen are reproduced very clearly in the DIC image. The azimuth effect of the technique can be demonstrated very impres­ sively in the Hantzschia amphioxys diatom. Radial structures such as Anachnodicus ehrenbergii diatoms also reveal the azimuth effect. Using the example of the Surirella robusta diatom, the authors explain the ad­ vantage of DIC equipment over bright-field and phase-contrast observation, in that ex­ cellent contrast is obtained even at high aperture. In other words, the Nomarski DIC method has the effect of a filter that arnpli­ fies high spatial frequencies and subdues low ones . The advantage of the shallow depth of field of the DIC method as corn­ pared to phase contrast is i1lustrated by a Triceratlum favus diatom. B. Microscopy of inorganic objects Metallography As early as 1954, Nomarski and Mme WeiJl [32] pointed out the advantages of differ­ ent ial interference-contrast microscopy in the field of metallography (e, g., electro­ polished cobalt). A second publicatlon by the same authors [33] dealt exclusively with metallographic applications. Among other things, it was devoted to a detailed study of various growth spirals of silicon carbide (SiC). Nomarski and Mme WeiJl were able to prove that growth steps of 440 A ± 30 A

for example, can be resolved without dlf­ ficulty. Under certain conditions, arelief of the order of a few Angsträm units can be recognized in the DIC image. Slipbands in cobalt subjected to a tension of 440 g/mm 2 for aperiod of five minutes can be repro­ duced with excellent contrast. The photo­ micrographs show various patterns of slip­ bands which, with bright-field illumination, for instance, can be recogn ized only with difficulty or not at all. Measuring thin films of an order of magni­ tude of 2000 A with the aid of yellow sodium light (589 ,u m) and Nomarski differential interference equipment, Le Mehaule [24] ob ­ tains an accuracy of ± 1.5 %. The author comes to the conclusion that the DIC method is superior to bright-field and par­ ticularly to phase-contrast observation for testing highly polished surfaces, exam ining thin films on glass substrates (vacuum­ deposited films), checking quenched steel for undesirable phases such as ferrites, austenites, etc. , and finally for the testing of diffusion processes by phase changes, the creation of new phases, recrystallization or other defects such as porosity due to different rates of diffuston between two elements, and lastly checking for dis­ locations and displacement of grain bound­ aries. As practical examples Le Mehaule publishes photomicrographs of quenched Cr-Ni-Mo steel, cold-worked bronze and sintered iron . Bertocci and Noggle [9] use a differential interference-contrast system for the quanti­ tative examination of small etched copper surfaces down to a mean size of 6.um . De­ pending on the magnification of the objec­ tive used , they attain an accuracy of be­ tween ± 5' and ± 30' . The superiority of Nomarski DIC micros­ copy over bright-field observation is illus­ trated by Gahm [20] who compares photo­ micrographs of unalloyed quenched and tempered steel taken by both techniques. The deformation of the surrounding area produced by a microhardness indenter, of which no trace ts visible in bright field, can be c1early dislinguished in the DIC image. At the same time, the series of micrographs shows the azimuth effect characteristic of the Nomarski DIC method, which can here be observed along a linear grinding trace. leg/itsch and Mitsehe [23] use Nomarski to investigate the metallographic structure of Vacutherm sampies (Widmannstätten struc­ tures, ferrite after yla-conversion, pearlite containing 0.85 % C), of low-temperature martensite, white cast iron (hypereutectic and hardened) and fused high-speed steel. On the left, Fig . 3 shows a martensite needle magnified 800 x in bright fleld, on the right, the same needle in Nomarski dif­ ferential interference contrast. Crystallography

In 1954 Nomarski and Mme WeilJ [32] re­ ported on numerous practical examples and illustrated the use of Nomarski DIC micros­ copy in crystallography, e. g. growth spirals on silicon carbide (SiC) with triangular

23

la

Ib

lc

ld

1e

Fig. 1: Ma cr onyssus bacoti i n bri ght field (a), in phase co ntrast (b), and different ial int erference co ntr ast (c , d , e). Figs . d and e w ere taken with different focal plane se tt ings. ZEI SS U ltr aphot 11, Piana ch rom at 40 x , 0.65 N .A., magnifi catlon 320 x .

24

2a

2b

5a

3a

3b

5b

4a

4b Fig . 2: C at mus eie in bright field (a) and in Nom arski DIC (b). ZEISS Ultraphot 11, Pl anach r omat 16 x, 0.35 N . A ., mag nifi cati on 144 x . Fig . 3: M artensite ne edl e, bright field (a), Nom arski D IC (b). ZEISS Ultraphot 11, Ep iplan 100 x, 1.25 N . A., oi I, magnificati on 800 x . Fig . 4: Wafer: bright field (Iett) and Nomarski DIC mic rogr aph (right). Z EISS Ultraphot 11 , Epiplan 8 x , 0.2 N . A ., mag nificat i on 72 x . Fig . 5: Crcss-aec t ton o f an inte gr ated c irc uit that failed d ue to ov er lo ad , i n bright field (a) and in Nomarsk i DIC (b). ZEISS Ultraphot 11 , Epiplan 8 x, 0.2 N . A ., magnifi cati on 72.

25

symmetry; principal spirals with secondary recrystallization on SiC; star-shaped growth spirals etc . Using the example of microhardness Inden­ tations in cleavage surfaces of sodium­ chloride crystals and potassium-chloride crystals, Gahm [20] shows that DIC micros­ copy can be used to advantage for quanti­ tative investigations. Slipbands that cannot be recognized under bright-field illumination stand out with extraordinary c1arity in the DIC image. The superiority of the Nomarski DIC method over phase contrast is clearly evident from the replica of a calcite cleavage surface [18, 19].

Padawer [39] compares sodium-chloride crystals in bright field , phase contrast and differential interference centrast. In bright field practically only the contours of the crystals will become visible, even if the illuminating aperture ls reduced , and in the phase-contrast image, large parts of the ob­ j ect will be ve iled by halation . For the optical staining and examination of the surface of germanium and sili con oxide, Franeon [17] takes recourse to reflected­ light differential interference centrast. How­ ever, ammonium-alum crystals can be repro­ duced with a wealth of detail by optical stain ing eve n when transmitted light is used.

Besides a comprehensive and easily under­ standable introduction to Nomarski DIC microscopy, Franr;on [17] publishes a num­ ber of photomicrographs of a germanium surface , a microcircuit and a cadmium­ telluride f ilm on silicon dioxide. Owing to its high resolution , the Nomarski method is weil suited for the examination of silicon monocrystals, as has been shown by Vieweg-Gutberlet [45] . If the specimens are properly etched, inhomogeneities in the doping concentrations such as strtattons, the microstructures of strlatlons. stacking faults in concentrations etc. can be made visible. Fig . 4 shows a wafer in bright field (left) and in Nomarski differential interfer­ ence contrast (right). Fig . 5 shows a cross-section of an integrated ci rcuit that failed due to overload, once in bright fleld (a) and on ce in DIC (b). Differen­ tial interference contrast above all repro­ duces the conductors with greater detail.

Glass technology Minute detalls in a glass surface are re­ produced with high contrast in the DIC image . As is proved by Gabler and Herzog [18, 19], pronounced differences of refrac­

tive inde x between object and mounting

med ium wh ich, in phase contrast, result in

extrem ely disturblng haloes, do not have the

same unfavorable effect in the DIC image.

Mineralogy

Gahm [20] has suc cessfully used Nomarski DIC mi croscopy to make microhardness impressions in varl ous minerals (e. g. covel­ lite, boul angerite) visible. Cracks, scab­ biness and bulges around a microhardness indentation in a periclase cleavage surface can be made optimally vis ibl e by color con­ trast. Von Gehl en and Piller [21] have shown that Nomarski DIC microscopy is an ideal means for exam in ing polished specimens or ore minerals for hardness differences. This method has pro ved to be superior to the conv entional "Schneiderhöhn llne" , More­ over, the Nomarski method is a valuable aid in te sting the quality of polished surfaces whose reflec tivi ty is to be measured by microphotometry . Finally, with sub-stage il­ lumination the Nomarski method offers many advantages for assessing the morphographic properties of fine-grain minerals and iden­ tifying clay minerals , as Correns and Piller [11] have show n. Semiconductor technoiogy In the introduction to his paper, Le Mehaute [24] describes th e fundamentals and charac­ teristics of the Nomarski differential inter­ feren ce- cont ras t method and conti nues by giving a summary of its advantages over bright-field and phas e-co nt rast obs erv ation, particularly for metallographi c uses. In the semicondu ctor f ield the DIC technique may be used to advantage for observing struc­ tural chang es such as phase transit ions, the formation of new phases, recrystallization processes, et c . Le M ehaute shows that th e DIC image of a transistor reveals far more deta il than wou/d a bright-field image. 26

Wh en examining oriented linear phase struc­

tures, their orientation relat ive to the split­

ting direction of the Nomarski prisms must

be taken into account [28]. The phase-con­

trast techn ique has no inherent azimuth

effect. However, it has the disadvantage of

greater depth of field so that phase ob jects

Iying outside the focal plane will appear in

the image, for example, as disturbing dlf­

fra ction fringes.

Plastics

Like clear transparent glas s, cle ar trans­

parent plastlos are ideal phase objects.

Ac cording to Gahm [20], microhardness

impressions of extremely minute irregu­

larities that are invisible in br ight field can

be emphasized by suitable setting of the

background , above all in color contrast.

A comparison between the opt ical staining

of spherical plastic parts in dark field and

differential interference contrast is made by

Padawer [39] . This comparison proves the

advantages of the DIC method by which

even very small particles are clearly repro­

duced beside larger objects. With the aid

of polystyrene spheres with a mean diameter

of 1.3 /lm , mounted in glycerin , Padawer

demonstrates the dependence of the DIC

image on the illuminating aperture. The

optimum illuminating aperture depends both

on the object and the illum inating aperture.

In the spec lal ca se under discussion, a set­

ting of 75 % of the maximum aperture has

proved particularly favorable. In addition ,

with the aid of two photomicrographs, the

author explains the effect of defocusing on

the DIC image. A number of micrographs of

plastic spheres shows that diffusion proces­ ses within the particles, inv olv ing a variation of optical thickness, can be clearly repro­ du ced by differential interference contrast.

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Proc . R. rnlcr. So c. 1 (1966) 141

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w ls s. M lkr. 69 (1969) 193

[4] Bai er , A.: 1. Cell. Bio l. 27 (1965) 7A [5] Beter , A, and R. D . Allen : Science, N. Y. 151

(1966) 572

[6J Baier, A., and R. D. Allen: J. Cell. Scl. 1 (1966)

455

[7] Barbieri , F., and S . M azzola : Acta Naturali a IV

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[11] Correns , C. W., and H. Piller: Hdb. d. Mikro­ skopie in der Te chn ik , Va l . 4, Part 1, Ed. H. Fre und . 2nd editio n (In prep .) , Urns chau-Verl aq Frankfurt [12] Davld. G. B ., R. D . Allen, L. F. Hir sh Ir .. and C . D . Watters: Pro c . R. mi cr . Soc . 1 (1966) 142

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[43] Sta ll , P.. and H. Gund/a eh: Per son al cornmuni­ eali on. Phot om i cr oqr aph s kin dly prov ided fo r inc lus lon In (28). S ince pub l ished: Peter St oII : Gyn eeoloqleal Vit al Cy to lc qv, Sprtnqer-Vertaq New York 1969. [44J Ur! , W ., and F. Gabler: M ikroskopie 22 (1967) 121

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