550 



R. A. Olson 



The environmental factors affecting bifluorescence are similar to those 

 affecting dichroism. Chloroplasts in damaged cells and chloroplasts extruded 

 into aqueous media do not show bifluorescence. Likewise, in chloroplasts sub- 

 jected to chlorophyll photooxidative bleaching by the exciting light, the resulting 

 yellow fluorescence does not exhibit polarized emission. A dependence of the 

 polarization phenomena on the integrity of the chloroplast structure at the mo- 

 lecular level is indicated. 



Chloroplast bifluorescence is indifferent to the polarization plane of the 

 exciting light used. Regardless of the vibration plane of the exciting light the 

 direction and intensity of polarized emission of each chloroplast in the specimen 

 field remains unchanged. This confirms that the oscillators of absorption are 

 not identical to those of emission and that, during the excited state, energy is 

 transferred by inductive resonance from unoriented absorbing chromophores to 

 oriented emitting chromophores. A similar energy transfer has been shown to 

 occur between chlorophyll and accessory pigments and a far-red absorbing form 

 of chlorophylla in vivo . (^^' 



Spectral measurements of bifluorescence in Euglena chloroplasts involve 

 even more rigid technical requirements than those of dichroism. Since the quan- 

 tum efficiency of chlorophyll fluorescence in vivo is very low, an intense exciting 

 light in 436 m^ region of high absorption for chlorophyll is required to produce 

 sufficient emission for measurement from a small chloroplast area. A scanning 

 microspectrophotofluorimeter can be used to advantage for spectral measure- 

 ments of chloroplast bifluorescence.'^'*' The device requires no other modifi- 

 cation than (1) a means of observing the position of the chloroplast relative to 

 the entrance slit of the monochromator and (2) a provision for measuring emis- 

 sion spectra polarized in each of two orthogonal planes. Chloroplasts which 

 showed a maximum difference of emission in the two orthogonal planes of polar- 

 ization were chosen for measurement. Typical oscillograph traces for the ennis- 

 sion form an edge-viewed Euglena chloroplast are shown in Plate 11 A. The 

 upper trace records the spectral distribution of emission transmitted by the Po- 

 laroid with its plane of polarization parallel to the lamellar plane, while the lower 

 trace gives that perpendicular to the lamellar plane. Plate 11 B shows the results 

 of similar optical measurements of the fluorescence emitted from an adjacent 

 face-viewed chloroplast. Quantitative comparison of the two measurements is 

 denied by differences in the actual area of chloroplasts observed. It is clear, 

 however, that in edge-viewed chloroplasts, the maximum polarized emission 

 with the electric vector parallel to the lamellar plane is nearly 50% greater than 

 that with the electric vector perpendicular to the lamellar plane. In face-viewed 

 chloroplasts the effect is insignificant. It is also clear that the maximum bifluo- 

 rescence occurs in the 720 mfx spectral region. The data from Plate 11 A are 

 plotted in Fig. 3. Bifluorescence is plotted as a difference, at each wave length, 

 of the emission in the two orthogonal planes of polarization. No correction has 

 been made for the spectral sensitivity of the S-1 photocathode. Such a correc- 

 tion would enhance the values for emission in the shorter wave lengths. Similar 

 measurements were carried out for edge-viewed Mougeotia chloroplasts. Fig. 4. 

 The effect, while somewhat reduced, shows similar spectral characteristics 

 with maximum bifluorescence near 720 m/i. The dichroism and bifluorescence 



