34 PHOTOCHEMICAL PRINCIPLES 



sample, the absorption for various wavelengths with that for a fixed 

 wavelength. Difference spectrophotometry has been used by Duysens, 

 Chance, Kok, Witt, Lundegardh, and Rabinowitch (reviewed by 

 French, 1959) for studies of changes in the absorption of photosyn- 

 thetic organisms when they are illuminated. 



FLUORESCENCE SPECTROPHOTOMETRY OF in vivo PIGMENTS 



By measuring the spectral energy distribution of fluorescent light 

 from living material, it is possible to identify pigments that happen to 

 be fluorescent. A very pale leaf containing extremely small amounts 

 of chlorophyll gives a spectrum similar in shape to that of chlorophyll 

 a in solution but shifted in wavelength, as are the corresponding ab- 

 sorption spectra. However, chlorophyll has a very strong absorption 

 band which overlaps its fluorescence peak. Therefore, if the fluores- 

 cent light generated in a sample containing chlorophyll has to pass 

 through an appreciable amount of this pigment before emerging, those 

 wavelengths strongly absorbed by chlorophyll will be weak or lacking 

 in the observed fluorescence spectrum. A green leaf containing many 

 air spaces scatters the light around within the leaf thus increasing the 

 chances of reabsorption of the fluorescent wavelengths that fall within 

 the absorption band. Illuminating such a leaf with blue light at 436 

 m/x produces a two-peaked spectrum recognizable as that of chloro- 

 phyll but greatly distorted by reabsorption of light in the neighbor- 

 hood of the main peak (French, 1955). In this leaf the far-red chloro- 

 phyll a fluorescence peak at 725 mti is higher than the characteristic 

 685-m//. chlorophyll peak. Blue light is very strongly absorbed by 

 chlorophyll. Green light, on the other hand, is absorbed weakly by 

 chlorophyll so that a large amount of the fluorescence excited by 

 green, 546 m/i., comes from deep within the leaf. On its way out the 

 fluorescent light is absorbed strongly at wavelengths where the chloro- 

 phyll a absorption band is high. For green incident light the 685-m/x 

 peak, characteristic of chlorophyll a fluorescence, disappears almost 

 completely in a dark leaf, leaving only the long-wavelength secondary 

 maximum of fluorescence. Virgin (1954) has found the distortion to 

 be greatly reduced by infiltrating the leaves with water to decrease 

 the internal scattering. It would be very easy to consider a distorted 



