547 



R. A. Olson 



viewed from the facial aspect show slight or no detectable**tH*BUig«-^!n absorption 

 regardless of the direction of the electric vector of transmitted light. This ob- 

 servation indicates no preferential orientation of oscillators within the plane of 

 the lamellae. 



No dichroism could be observed in the chloroplasts of non-intact or dam- 

 aged cells. While chloroplasts extruded from the cell into conventional aqueous 

 media lose their dichroism, chloroplasts extruded into viscous serum albumen 

 retain this property, although gradually attenuated, for several hours. 



In order to estimate the ratio of oriented chlorophyll to the unoriented pig- 

 ment it is essential to obtain quantitative measurements of the above dichroic 

 observations. In addition, a quantitative determination of the spectral depend- 

 ence of dichroism should be executed in order to compare the spectral properties 

 of oriented chlorophyll with those of other known pigments in vivo . The spectral 

 dependence of dichroism is usually represented by the ratio at successive wave 

 lengths of the absorbance parallel to the plane of the electric vector of the light 

 maximally absorbed versus the absorbance perpendicular to that plane. Meas- 

 urements of this type in vivo impose stringent requirements involving unchanging 

 optical path length and a limited chloroplast area of measurement. Unchanging 

 optical path length is assured by effecting a maximum degree of immobilization 

 of the organism such that chloroplasts do not change in shape or position during 

 the absorbance measurements at successive wave lengths. The chloroplast 

 area selected for absorbance measurements must be very small since Euglena 

 chloroplasts are somewhat curved and twisted and thereby show local irregular- 

 ities of lamellar direction. It is therefore necessary to limit the measurement 

 to an area small enough to exclude gross spurious effects from lamellae which 

 lie in planes other than that selected for measurement. In our experiments this 

 area was of the order of one square micron. The minimal radiant power trans- 

 mitted from such a small specimen area when restricted to a narrow spectral 

 band requires the measurement of an extremely low light intensity which results 

 in an unfavorable photomultiplier signal to noise ratio. Attenuation of noise by 

 filter circuitry is limited by time-constant characteristics imposed by the re- 

 quirement of sufficiently rapid spectral scanning to minimize geometrical 

 changes in chloroplasts caused by cell mobility. 



The data are presented in terms of absorbance in Fig. 1. The upper 

 dashed curve shows the enhanced absorbance in the lamellar direction at the 

 longer wave lengths while the lower dashed curve shows the absorption of the 

 unoriented chlorophyll. The ratio of the upper curve to the lower curve, the 

 dichroic ratio, is shown by the solid curve through the triangles. In this par- 

 ticular specimen the maximum ratio of about Z . 2 is observed near 705 m^. 

 Higher dichroic ratios, approaching 4.0, were obtained in other cells and in 

 other preparations but the data offered in Fig. 1 are considered typical. Sim- 

 ilarly, departures from the wave length of the maximum ratio in Fig. 1 were 

 observed in other cells but the average of twelve chloroplasts measured was 

 704 ±5 m^. 



A similar plot of the dichroic spectral properties of the chloroplast of 

 Mougeotia is shown in Fig. 2. While the effect in this specimen is not as pro- 

 nounced as in Euglena the maximum of the dichroic ratio is located in the same 



