72 J. W. COLEMAN, A. S. HOLT, E. I. RABINOWITCH 



changes in fluorescence yield may occur when you add the cross illumination? 

 Fluorescence caused by the measuring light is modulated, and any fluctuation 

 in it will be picked up by your phase-sensitive detector. If the sense of the 

 change is proper, you will get in this way something that could be interpreted 

 as a decrease in transmission, 



Rabinowitch: Unless the actinic light caused changes in the spectral composi- 

 tion of fluorescence and not only in its intensity, how could you get in this way 

 both positive and negative effects? However, the commonly made assumption that 

 the spectrum of fluorescence does not change when changes in fluorescence in- 

 tensity are observed in vivo is in need of experimental confirmation. If this happens, 

 the meaning of many data in the literature becomes questionable. 



Strehler: You are passing through an absorption band here (660 to 720 m/x) 

 going from wavelengths that do excite chlorophyll fluorescence into a spectral 

 region where chlorophyll does not absorb. I am not saying that your effect in the 

 red is necessarily due to this, but this possibility worried us when we measured 

 our 648-m;u band. I believe we can rule it out in our case, because we put a red 

 filter in front of the photomultiplier, which transmitted only the fluorescence, 

 and with this filter in place we didn't observe any significant signals upon croes 

 illuminating. 



Rabinowitch: One probably has to consider this point more carefully than we 

 did, but it still seems to me that you cannot get in this way both positive and 

 negative effects. 



Chance: This difficulty would be minimized in the double-beam apparatus 

 we use. If j^ou could change your apparatus corresponding!}^, you may get an ade- 

 quate control. 



Strehler: Provided the excitation of fluorescence by the two different wave- 

 lengths is the same. Actually, by placing another monochromator after your ab- 

 sorption cell, you can remove most of the fluorescence, since it forms a broad 

 band. 



Chance: Yes, but you may be unable to get enough light into the analyzing 

 monochromator. 



Rabinowitch : Of course, when he measured at 515 mju, Holt did use a filter to 

 cut off fluorescence. 



Strehler: The changes at 515 m^u cannot be due to fluorescence. 



Rabinowitch: When we measured at 515 m^u, the fluorescence-removing filter 

 did not change the results. This means that at 515 m/i, the effect of modulated 

 fluorescence was insignificant compared to the absorption effect. If the total ob- 

 served effect were much stronger at 515 m^i than at 680 m^, one could suggest 

 that fluorescence was much more significant in the latter region; but, as I said 

 before, Coleman's impression is that the effects in the two regions are about the 

 same; and, if this is so, one can infer that the role of modulated fluorescence is in- 

 significant in the red, too. 



Linschitz : If we accept your data at their face value, there would still be a prob- 

 lem of showing that the changes at 680 and 515 m/x are correlated. We have found 

 that, in vitro, these two bands may change at different rates. There are probably 

 two different products which cause the change. 



