553 



R. A. Olson 



"depolarization of fluorescence" to chloroplasts has been effected by Arnold 



,\, , (15,16) 

 and Meek. * 



Difficulty in excluding scattered exciting light limited their measurements 

 to the spectral region below 630 m/n region. The low polarization of fluores- 

 cence from the chloroplasts of living cells was attributed to the fact that those 

 chlorophyll molecules which emit fluorescence did not themselves absorb the 

 exciting light. The energy transfer thus demonstrated is identical to that indi- 

 cated by our study, namely: the polarized infrared emission of chloroplasts is 

 independent of the plane of polarization of the exciting light. 



The depolarization of fluorescence of a dilute Euglena suspension was 

 measured using the polarized ruby laser as the excitation source. Typical re- 

 sults from such measurements are shown in Plate II C. The upper oscillograph 

 trace shows the time course of In during the laser flash; that of I_l_ is indicated 

 by the lower trace (horizontal oscillograph deflection 100 microsec/cm) . A 

 preliminary calculation of the degree of polarization of fluorescence for the 

 curve maxima indicates a high degree of retention of the sense of polarization 

 of the exciting light. It further indicates that the absorbing oscillators (694 m^) 

 and the emitting oscillators (720 m/i) are coincident. 



DISCUSSION 



The evidence presented indicates an oriented far-red pigment which is 

 assumed to be an energy trapping form of chlorophyll a. The red oscillator 

 (-705 m/i), located in the plane of the porphyrin head, appears to be highly 

 aligned parallel to the chloroplast lamellae. This oscillator emits fluorescence 

 (72 mfi) highly polarized parallel to the lamellar plane. If this pigment is a 

 chlorophyll, the absence of dichroism in the blue oscillator indicates that the 

 plane of the porphyrin head does not lie parallel to the lamellar plane. It would 

 also be of value to determine the molar concentration of this pigment in vivo in 

 relation to other pigments involved in order to consider turnover rates, funda- 

 mental unit size, and other factors at the site of light transformation. 



While the spectral region defined by the maximum dichroic effect indicates 

 in general the absorption characteristics of the oriented chlorophyll it should 

 be noted that actual absorption spectrum is not clearly defined. The absorbing 

 system as a whole in chloroplasts is composed of both oriented and unoriented 

 components each with dissimilar absorption characteristics which spectrally 

 overlap. All of the absorption, for example, at 705 mfx is not due to the oriented 

 pigment. Likewise, within the limits defined by high resolution polarization 

 microscopy the absolute degree of orientation of oscillators cannot be determined- 

 Higher dichroic ratios would have been obtained in an ideal optical system devoid 

 of depolarization effects. However, the approximate definition of the absorption 

 and emission characteristics of oriented chlorophyll by spectral measurements 

 of dichroism and bifluorescence may relate this pigment with C^q^ (Butler), 

 P7OO (Kok), and Ca^gg (Brown and French). (^0' ^'^ • ^8) The demonstrated abil- 

 ity of oriented chlorophyll to accept excitation energy from other unoriented 



