549 



R. A. Olson 



region near 705 m/j. Similar effects are observed in chloroplasts of the fila- 

 mentous algae Spirogyra, Syragonium and in the unicellular alga Mesotaenium . 

 Dichroism is also observed in the chloroplasts of Spinach and other grana bear- 

 ing chloroplasts but the scattering depolarization and the small size of the grana 

 in multicellular preparations make quantitative measurements impractical. 



Bifluorescence 



The bifluorescence of Euglena in the far-red region is an even more 

 striking phenomenon than dichroism. Observation of the polarized emission is 

 most conveniently accomplished using the following arrangement: a Nicol prism 

 (immediately behind the objective) that can be rapidly rotated between two ortho- 

 gonal positions, a suitable filter to exclude the exciting illumination and an in- 

 frared image converter which renders visible the broad banded emission in the 

 720 mfi region. The observer can thus readily detect the profound changes of 

 fluorescence intensity occuring in the edgewise chloroplasts lying in one or the 

 other of the orthogonal planes of polarization. The infrared image converter 

 when used with aspherical accessory viewing optics is very satisfactory for ob- 

 servations of these kinds and shows very little pin cushion distortion in the cen- 

 ter of the field. However, photography of the phosphor screen requires the 

 removal of these correcting optics. Under these conditions the demonstration 

 of the azimuthal designation of bifluorescence in chloroplasts in a field of cells 

 is complicated by off-axis distortion which can give erroneous results. In con- 

 sideration of this effect and of the limit of resolution imposed by the granularity 

 of the phosphor screen, direct infrared photography was used for these studies 

 and the image converter was reserved for rapid, cursory examinations required 

 for selection of suitable chloroplasts. 



Plate I C and D are typical infrared photographs of a group of live Euglena 

 cells showing the characteristics of bifluorescence. The exciting light was un- 

 polarized and consisted of the 436 m/i mercury line and nearby continuum. This, 

 along with the emission in the visual region, was excluded from the photographic 

 film by a Schott RG-9 filter. The open arrows in the figure indicate typical edge- 

 viewed chloroplasts which lie parallel to one of the two orthogonal planes of po- 

 larization of the analyzer. The polarized emission is clearly indicated by an 

 increased brightness when the chloroplast lies parallel to a polarization plane 

 and by an attenuated brightness when perpendicular to that plane. Chloroplasts 

 viewed on edge that are positioned with their lamellae at 45° to the polarization 

 planes of the analyzer show identical brightness when observed with the analyzer 

 in either position. Face-viewed chloroplasts on the other hand appear isotropic 

 and show no marked change in brightness at any position of the analyzer. Simi- 

 lar exannples of this phenomemon may be found in appropriate chloroplasts within 

 other cells in the figure. The characteristics of chloroplast bifluorescence cor- 

 respond with those of chloroplast dichroisrn. Polarized emission as well as 

 polarized absorption is maximal in the direction of the lamellae and indicate a 

 chromophore lying in the lamellar plane. Furthermore, with respect to this 

 chromophore, both types of observation in face-viewed chloroplasts indicate a 

 uniaxial chloroplast structure with the optical axis perpendicular to the lamellae. 



