196 

 References 



G. LION, C. MAERTENS AND G. VANDERMEERSSCHE 



1. De Robertis, E., /. Biophys. Biochem. Cytol. 2. 319 



(1956). 



2. De Robertis, E. and Franchi, C. M., /. Biophys. Biochem. 



Cytol. 2, 307 (1956). 



3. Sjostrand, F. S., /. Cell. Comp. Physiol. 33, 383 (1949). 



4. — ibid. 42, 15 (1953). 



5. — ibid. 42, 45 (1953). 



6. — /. Appl. Phys. 24. 1422 (1953). 



7. — unpublished. 



8. SvAETiCHiN, G., Acta Physiol. Scand. 39, Suppl. 134. 



17^6 (1956). 



Submicroscopic Morphology of the Retinal Pigment Epithelium 

 G. Lion, C. Maertens and G. Vandermeerssche 



Centre de Micioscopie Electronique, Medical, Industrie! et Agricole, Briissels-Uccle 



The aim of the present work is to bring out the 

 possibilities and Hmitations of the electron micro- 

 scope in a study of the fine structures of the eye. 

 One of the present authors has done some work 

 with the Philips electron microscope Type EM 100 

 (1); this work was done in collaboration with the 

 Chnique Ophtalmologique of the University of 

 Ghent and was solely concerned with suspension 

 and replica techniques. A comprehensive study of 

 the different ocular tissues, but this time by means 

 of the thin-sections technique, has been undertaken 

 by the present authors. 



Two different types of electron microscopes are 

 being used: the Philips EM 75 and the new type EM 

 100. The work done with the first electron micro- 

 scope will be presented here while the rest of this 

 work will be published elsewhere. 



Material has kindly been provided by the Institut 

 d' Hygiene et d'Epidemiologie of the Ministry of 

 Public Health, Brussels. The eyes of the following 

 animals have been studied: monkey (M. Cynoiuol- 

 gus), guinea pig and pig. It has thus been possible 

 to bring out differences in structural details of the 

 eyes of these animals and those studied by several 

 other workers (1-4, 9-12) in this field (man, guinea 

 pig, carp, perch, ox, frog). 



Our investigations were carried out on individual 

 pigments which were fixed and embedded after isolation 

 and on tissue fragments which were treated by the usual 

 fixation and staining techniques. The isolation of the 

 pigments has been described earlier (2, 3). They have 

 been washed by repeated centrifugation and fixed in buf- 

 fered 1 "^'o OSO4 solution (7). As embedding medium (6) a 

 6-to-4 mixture of butyl and methyl methacrylate has been 

 used. This mixture seems to give the best results with an 

 extremely hard material such as the pigments. Extreme 

 care had to be taken during the preparation of the retinal 

 pigment epithelium itself: it is almost impossible to avoid 

 retinal detachment. The only way which has given rea- 

 sonably good results is to fix the posterior hemisphere as 

 a whole after the eye has been opened at the front end, 

 without removing the complete content of the vitrous 

 body. The orientation of the tissue blocks is made possible 

 by a technique described in a paper by Ruska, Stuart, 

 Winsser (8), and which consists mainly in keeping the 

 embedded material during polymerisation in an atmos- 

 phere of CO2 inside a test tube in the desired position. 

 The fixation technique described above does not give 

 complete satisfaction in so far as the exact fine structure 



of the rods is concerned, because of the presence of the 

 remaining vitrous body (showing up the fixation of the 

 retina), but it allows at least the observation of the distri- 

 bution of the pigments in relation to the rods. The ave- 

 rage thickness of the sections obtained with a Porter- 

 Blum microtome was about 250 A. 



The pigments of the eyes of the monkey, pig and 

 guinea pig show in sections the same typical elonga- 

 ted forms with rounded extremities as those which 

 have been observed in suspension preparations (1-4) 

 (figs. 1-2). So far, only the pigments of carp-eyes 

 have shown a marked difference in shape and size. 



The pigments of the eyes investigated here are 

 usually grouped around some cellular residues but 

 in a much smaller quantity than what has been 

 observed in ox-eyes (1-4). Also the mosaic distribu- 

 tion described in the ox-eye has not been observed 

 here. 



Although the pigments are extremely hard to cut 

 with glass-knives, good thin sections have been 

 obtained and they seem to confirm the hypothesis 

 of Frangois and co-workers that no internal struc- 

 ture (2, 3) is present in the pigments. The parallel 

 striations which can be seen in some of the micro- 

 graphs are an artefact due to the hardness of the 

 material. 



The sections of the tissue blocks show the arrange- 



Fig. 1. Pigments from the retinal epithelium of a pig-eye. 

 Pd. -shadowed. Magnification 8000. 



Fig. 2. Thin section of pigments from the retinal epithelium 

 of a pig-eye. The pigments are still attached to cellular 

 residues. Magnification 6000. 



