552 



R. A. Olson 



measurements offered in this presentation indicate orientation of a far-red ab- 

 sorbing form of chlorophyll. In addition they suggest strongly the radiationless 

 transfer of energy from randomly oriented oscillators absorbing at shorter 

 wave lengths . 



Confirmation of this energy transfer could be obtained if direct excitation 

 of the oriented oscillators by polarized light demonstrated a dependence of the 

 polarized emission on the plane of polarization of the exciting light. In this way 

 the transfer of excitation energy from successive random oscillators absorbing 

 at shorter wave lengths would be by-passed. In this regard we would like to 

 present briefly some preliminary data to support a concept of energy transfer 

 to oriented chlorophyll. It was pointed out above that, since the quantum effi- 

 ciency of fluorescence in vivo is very low, a high intensity source of exciting 

 light is required for fluorescence microscopy of chloroplasts . No convenient 

 high pressure arc source is available with its output concentrated in the absorb- 

 ing region of oriented chlorophyll, other means of excitation of its oscillators 

 must be sought. The pulsed ruby laser meets these requirements most appro- 

 priately. It has almost unlimited power and an exceedingly narrow band pass 

 at 694.3 mji, a wave length very close to the region of maximum absorption of 

 oriented chlorophyll. The narrow band pass of the ruby laser exciting light fa- 

 cilitates its exclusion from observation at the fluorescence maximum near 720 

 m/i of oriented chlorophyll. The high intensity of the parallel laser beam elim- 

 inates the need of a condenser in fluorescence polarization microscopy and thus 

 avoids depolarization effects introduced by this optical element. Ruby crystals 

 with 90 orientation provide a polarized output thus eliminating the attenuation 

 introduced by the usual polarizer. Joining the laser to the fluorescence micro- 

 scope required a massive modular optical bench apparatus to be described else- 

 where. Observations can be recorded by infrared photography and the laser 

 exciting light removed by an appropriate interference filter having a minimum 

 of "toe" transmission. 



Plate I E and F show typical results of laser polarized fluorescence photo- 

 micrography with Euglena chloroplasts in vivo. An unconcentrated laser beam 

 provided the exciting light. The ruby was oriented with the electric vector of 

 the linearly polarized beam as indicated by the arrow labelled "E." It is clear 

 from the photomicrographs that maximum polarized near infrared emission in 

 edge-viewed chloroplasts has its electric vector parallel to that of the polarized 

 exciting light. The direct excitation by polarized light of the 694 mfj, absorbing 

 oscillators oriented in the lamellar plane results in a fluorescence polarized in 

 the same direction as the exciting light. The emitted light suffers little depolar- 

 ization, and the oscillators of absorption appear to be identical to the oscillators 

 of emission. 



While observations like those in Plate I E and F serve to localize and des- 

 ignate this polarization property with respect to the chloroplast lamellar plane, 

 they are not appropriate for quantitative purposes. Quantitative measurements 

 of the degree to which the polarized fluorescence retains the sense of polariza- 

 tion of the exciting light can be made more conveniently in cell suspensions. 

 Application of the classic experiments of Perrin and others on the so-called 



