132 



NA TURE 



[July 28, 1923 



the resolving power. In Fig. 4 let O and /' l)e points 

 in the focal plane. If the surrounding medium is air 

 the optical length of the rays from O and P differ by 

 OP sin 0. If c) and P are covered by a uniform layer 

 of a medium the refractive index of which is m, the 

 same rays in the medium make an angle 0' with the 

 axis, where »' sin 0' - sin ^/m. The difference of optical 

 length is then OSti. sin d' = (OSn sin 0)ln^OS sin 0, as 

 it was in air. 



This independence of n does not extend to the case 

 where one of the points O or P is slightly above or 



below the focal plane. 

 If h is the elevation 

 or depression in cjues- 

 tion, the difference of 

 the optical lengths 

 is h{fji cos t)' -cos 0), so 

 that the difference in- 

 creases both with M 

 and h. The quantity 

 o-j\ suggested above as 

 a limit to the resolving 

 power of microscopes 

 with respect to thin ob- 

 jects in the focal plane is a guess rather than an 

 actual measurement. With the maximum angular 

 aperture the radius of the first dark ring is a little 

 greater than o-4\, which would indicate that objects 

 must be separated by o-8\ before a really dark space 

 appeared between their images ; but the intensity of 

 the light in the neighbourhood of the ring is very 

 small, and doubtless the objects would seem as double 

 at a less distance. 



The experience, however, which I have had with 

 fine lines ruled on thin films would induce me to 

 place the limit at more, rather than less, than o-yX. 



A. Mallock. 

 9 Baring Crescent, Exeter, 

 May 19. 



The Fluorescence of certain Lower Plants. 



It will, I venture to believe, interest some of the 

 readers of Nature to know that the Cyanophyceae 

 (Schizophyceae) or blue-green algae, the diatoms and>- 

 some at least of the true green algae among, or closely 

 related to, the Pleurococcaceae, are visibly strongly 

 fluorescent when viewed ultramicroscopically, if the 

 proper optical conditions are achieved. A much 

 wider claim for the usefulness of the method might 

 be made, but it will be unnecessary ; for the moment, 

 if any one who may be interested tries it, he will 

 appreciate at once its many possibilities. 



The optimum conditions are these : a dark-field 

 condenser, preferably of the cardioid type, thin 

 glass object-slides (o-8 mm. thick or less), preferably 

 thin covers and a dry objective of any magnification. 

 Water between the upper lens of the condenser and 

 the slide answers every purpose, and is much more 

 comfortable in extensive ultramicroscopy. The best 

 light source, when one is studying colour, and this 

 becomes of prime importance in this connexion, is 

 a small arc ; but generally speaking, a condensed 

 filament, 400-watt lamp with a suitable condenser, 

 answers every purpose. The thin object-slide and 

 the glass-air interface of the cover are the essential 

 features, since one is then able to focus the reflected 

 hollow beam of light from the upper surface of the 

 cover glass upon the object. It will be seen that in 

 using the dark-field condenser in this manner we 

 are reviving the idea embodied in the " spot lens " 

 of Thomas Ross, in Wenham's air paraboloid (1850) 

 with a similar spot lens, and in his glass paraboloid 

 condenser of 1856 (Siedentopf, H., " Die Vorgeschichte 



NO. 2804, VOL. 112] 



der Spiegelkondensoren," Zeitschr. /. xviss. Mikro- 

 skopie, 24: 382-395, 1907). As these could be usf' 

 only with a dry objective, the later effort was aim. 

 at the result achieved in our present apparatus. I 

 is evident from current published directions 

 manufacturers for work with the dark-field illuminat< 

 that the use of the reflected light cone is not con 

 templated. I refer especially to the specification - 

 as to object-.slide thickness. While not having tl 

 advantage of the magnification afforded by tl.' 

 oil-immersion, we gain very greatly in m uin features 

 of the object-picture afforded. 



One of the most important of these is that the 

 blue-green algs, when seen at the apex of the ligln 

 cone inverted by reflection, afford their fluorc 

 colours. Some species are more readily reco 

 to fluoresce than others, but if the material \>v 

 mounted in strong glycerin, thus obviating the 

 scattering of light by internal surfaces either in 

 the organism or in the surrounding mucilage, the 

 cells are then seen to glow with a fervid light, orange 

 or crimson according to the organism. Indeed, 

 when glycerin is used, the fluorescence can be seen 

 without making use of the inverted light cone, just 

 as, according to Siedentopf, bacteria lightly coloured 

 with a fluorescent stain {" tiber Beobachtungen bei 

 Dunkelfeldbeleuchtung," Zeitschr. /. wiss. Mikro- 

 skopie, 25 : 273-282) may be seen with oil-immersion 

 objectives. The object-picture then afforded has 

 advantages of its own which need not be detailed 

 here. Without glycerin, the fluorescence, even of 

 those species which are most readily observed in 

 this respect, is scarcely visible with' oil-immersion 

 objectives. 



When seen mounted in glycerin, then, some 

 species of Oscillatoria are crimson, as also are Cylindro- 

 spermum, Anabcena Azollce, some species of Nostoc 

 and of Chroococcus, Rivularia, and others ; while 

 other species of Oscillatoria and Nostoc are golden 

 orange. Chroococcus refracius (I do not assert 

 positive identification) appears a dull yellow, all 

 these forms affording a most striking contrast to 

 their appearance by transmitted light, that with 

 which students of them are familiar. 



I have found evidence that the pigment is in 

 solution in minute vesicles (supporting in part 

 Wager's conclusions, Proc. R.S., 72 : 401, 1903). 

 With death, it becomes adsorbed by the cytoplasm 

 and the cells then appear blue {e.g. Nostoc). On 

 examining material of Nostoc commune from China, 

 which I have had in my possession some twenty 

 years, I found the cells as strongly fluorescent 

 as if fresh. The stiff gelatinous sheath appears 

 light blue, perhaps also from adsorbed phycocyanin. 

 When freshly mounted in glycerin, blue-greens hold 

 their fluorescence for some time. I have an Oscil- 

 latoria kept thus for twenty days without loss of 

 fluorescence. The old Nostoc commune lost its 

 fluorescence in less than twenty-four hours, perhaps 

 because it was already dead. 



I have shown that the fluorescence is due to 

 phycocyanin, rather than to chlorophyll, which, 

 because of the " colloidal condition " in which it 

 occurs in the living cell, has not been found visibly 

 fluorescent. E. Raehlmann (" Neue ultramikro- 

 skopische Untersuchungen iiber Eiweiss," etc., 

 Pfliiger's Archiv ges. Physiologic, 112 : 128-171, 

 1906), it is true, thought that he could see chlorophyll 

 fluorescence with the ultramicroscope, but he used 

 suspensions or emulsions, and with these I have 

 obtained similar results. With suitable means, the 

 fluorescence microscope, for example, the fluorescence 

 of the living chloroplast can be observed, but one 

 can scarcely persuade oneself that it can be seen 



