REVERSIBLE BLEACHING OF (MILOlU)l'IIYLL ill I'ii'O 73 



(Remarks added in manuscript) 



Coleman : The possible influence of changes in modulated fluorescence, pointed 

 out by Dr. Strehler, was taken into account by Dr. Holt and myself, although we 

 did not include the discussion of this point in our report. Since it has been raised 

 by Dr. Strehler, here is a brief summary of the reasons why we considered this in- 

 fluence negligible. 



In contrast to the constant actinic light, the modulated scanning light does 

 produce a 60-cycle modulated fluorescence to which the photomultiplier can re- 

 spond. In the apparatus as we used it, this modulated fluorescence is compen- 

 sated when the actinic light is off ("darkness"); but, from general knowledge of 

 the fluorescence phenomena in vivo, we must consider it possible — even likel}^ — 

 that switching on the actinic light — changing from "darkness" to "light" — will 

 change the intrinsic capacity of the cells to fluoresce and thus affect (probably, 

 increase) the intensity of the modulated fluorescence. The apparatus will react 

 to such a change in modulated fluorescence as if it were a change (decrease) in 

 absorption in the fluorescence vessel. We thus have : 



AA (true) = AA (measured) — AA^ 



where AA^ is the change in the modulated fluorescence reaching the detector, 

 caused by switching on the actinic light. 



To determine the order of magnitude of AA^, the scanning beam was rerouted, 

 so that it traversed the suspension orthogonally to its usual path. In this wa,y, 

 only a small scattered fraction of the exciting light entered the photomultiplier, 

 instead of the full transmitted beam, as in the usual arrangement. Fluorescence, 

 on the other hand, was collected from the same volume, subtending the same 

 angle at the photomultiplier. 



First, heat-killed cells were illuminated with the rerouted beam; the scattered 

 scanning light was compensated, and the deflection di (corresponding to 1% 

 change in the beam intensity) was recorded, as usual, with the help of a cali- 

 brated neutral filter. Although di changed slightly when the actinic light was 

 turned on (probably because of scattered actinic light modulated by line ripple 

 voltage), this change was negligible. The suspension of dead cells was then re- 

 placed by one of live cells, having the same optical density, and the procedure 

 was repeated, giving the deflections ^2 in darkness and di when exposed to actinic 

 light. We can expect di to be the same as di, and this was in fact the case within 

 a few per cent. The value of AA^, the increase in fluorescence caused by the actinic 

 light, was calculated from the equation 



^, _ K [2ri3 - (dr + d,)] 



^^' dTTT, 



where if is a correction factor (c^l.4) for the loss of intensity of scanning beam 

 caused by additional reflections required for its rerouting. 



The resulting correction curve, AA^ = /(X), is shown by the dashed line in 

 Fig. 1. At X = 680 mX, AA^ contributes about 7% of A A (measured). Where 

 A A (measured) is smaller, AA^. contributes proportionally more, but in no case 

 is the error large enough to alter the shape of the AA-curve. 



