OTHER METHODS OF STUDYING VISUAL PIGMENTS 



When such preparations were examined microscopically in the 

 usual way, two kinds of rods could be seen. The great majority were 

 pink and owed their colour to visual purple. A small proportion, 

 however, were grass green in colour, an observation first made by 

 BOLL (1876) and confirmed by kuhne (1878). 



The two kinds of rods are well shown in the photomicrographs 

 (Plate 7.1 (a), (b), (c), (d)) of the same part of one frog's retina. 

 Plate 7. 1 (a) was taken in green light (520 mju) before the preparation 

 was bleached. The dark circles are an end-on view of the numerous 

 *visual purple' rods, which absorb green light very strongly. The 

 light circles are the green rods which do not absorb this light. In 

 blue light (420 m/bi) this picture is reversed : the visual purple rods 

 are almost transparent while the green rods absorb strongly and 

 appear as dark circles. Plates 7.1 (c) and 7.1 (d) were taken in the 

 green and blue light, respectively, after the preparation had been 

 bleached by exposing it to intense white fight. 



METHODS 



By careful standardization of their techniques, denton and 

 WYLLIE were able to make a number of important quantitative 

 observations. In all their experiments a | in. microscope objective 

 of N.A. 0-28 was used and the photographs were taken on Ilford 

 HP3 35 mm roU film with a constant exposure of 10 sec. The 

 magnification from retina to film was 36 diameters. The film was 

 developed in I.D. 11. The source of Hght was a 48 W, 8 V tungsten 

 lamp, underrun at 7-5 V. 



The principle of their quantitative methods was as follows. A 

 suitable colour filter, to isolate the required spectral band, was 

 inserted in front of the light source. This was then switched on and 

 three photographs of the unbleached preparation were taken. These 

 differed only in that neutral filters of densities D + 0-3, D + 0-2 

 and D + 0- 1 had been placed in turn beneath the substage condenser. 

 The neutral and colour filters were then both removed thus exposing 

 the preparation to intense white light. Bleaching was completed in a 

 few seconds but the exposure was continued for 2 min. Ten minutes 

 later the bleached preparation was again photographed a number of 

 times with the colour filter and various neutral filters in position. It 

 was then possible to ascertain what change in neutral filter exactly 

 matched the density change :;aused by bleaching. For example, if a 

 photograph of the unbleached retina through neutral filter D + 0- 1 



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