George Gabriel Stokes as a biologist

1. Derivation of the word ‘fluorescence'

The word ‘fluorescence’ was coined by Stokes in a footnote on page 479 of his magnum opus entitled ‘On the change in refrangibility of light' published in 1852 in the journal Philosophical Transactions [1]. ‘Refrangibility’ is defined as ‘capable of being refracted' [2]. As discussed in a review of the history of fluorescence [3], the derivation and use of the term fluorescence itself poses some issues. Fluorescence is one form of photoluminescence whereby matter emits light following absorption of a photon, the other form being phosphorescence. The distinction between the two forms of photoluminescence is one of time. Following photon absorption, the lifetime, or half-life, of fluorescence emission is rapid, in a matter of picoseconds (10⁻¹⁰ sec.) to hundreds of nanoseconds (10⁻⁷ sec.). For phosphorescence, this is generally much slower, from thousandths of a second (10⁻³ sec) to days or years; however, for metal complexes, phosphorescence can be rapid, in the order of tens of nanoseconds. Clearly, at the time Stokes was investigating fluorescence, such short time intervals could not be measured and it had to wait for the development at the beginning of the twentieth century of the quantum theory of atomic structure and the studies of Perrin in 1929 [4] and Jabloński in 1935 [5] for photoluminescence to be explained at the atomic level. Indeed, these studies allowed the distinction to be made between phosphorescence and fluorescence with the former but not the latter involving a metastable intermediate state. Nevertheless, realizing that fluorescence and phosphorescence differed in the speed of light emission, in 1858 Becquerel built a phosphoroscope with which to begin measuring the speed of photoluminescence [6]. As outlined by Valeur and Berberan-Santos, [3] historically, Stokes was certainly not the first to observe the phenomenon of fluorescence, but nevertheless in 1852 his article was by far the most thorough examination of the phenomenon.

2. Stokes' magnum opus

In reading Stokes' article [1], one is immediately impressed by his writing style. Indeed he was a prolific writer of scientific papers and corresponded with other scientists as well as family members. In his introduction to papers, he begins by acknowledging the work of others; by this, Stokes demonstrates his profound scientific integrity. First, he acknowledges two papers by Sir John Herschel published in 1845 in which Herschel describes the phenomenon of fluorescence at the surface of a quinine sulphate solution [7] and in the second [8], Herschel calls this phenomenon ‘Epipolic dispersion' of light; epipolic meaning ‘at the surface'. Based on the detailed observations in his paper [1], Stokes did not like Herschel's term nor the term ‘dispersive reflexion' he used in the text. He therefore stated in a footnote on page 479: ‘I confess I do not like this term (dispersive reflexion). I am almost inclined to coin a word, and call the appearance fluorescence from fluor-spar, as the analogous term opalescence is derived from the name of the mineral (opal)’. This is the first use of the term ‘fluorescence’. Thus the ‘fluor’ part of fluorescence is derived from fluor-spar, or fluorite, which is the mineral calcium fluoride. Small deposits of fluor-spar were found in Derbyshire and were available to Stokes. The presence of various impurities in fluor-spar results in the variable colour of its fluorescence.

Second, in an Appendix, Stokes further acknowledges the observations of Edmond Becquerel [9] who 9 years previously (1843) had described fluorescence and indeed was the first to show that the wavelength of emitted light was longer than that of incident light, a phenomenon we now call the ‘Stokes shift'. As outlined by Valeur and Berberan-Santos [3], Becquerel was considerably angered by Stokes' paper to the extent that he made a priority claim for his own observations. He even proposed abandonment of the term fluorescence since he believed that fluorescence and phosphorescence were merely different forms of the same phenomenon, fluorescence being faster. It must be remembered that in the mid-nineteenth century, scientific observations were communicated at a much slower pace than today and indeed English was not the universal language of scientific communications.

Lastly, in Stokes' second paper on fluorescence [10] in which the term fluorescence is used throughout, he also discusses the work of Sir David Brewster [11,12] who had suggested that fluorescence of solutions was due to light scattering by particles in suspension. This is an important discussion ([10] page 387), since it shows how Stokes politely disagreed with Brewster's conclusions and also viewed the phenomenon of fluorescence as being different from phosphorescence.

It is so often a pleasure to read the original texts of the ‘classical' scientific literature, such as Stokes' papers. Furthermore, the scientific writing of the nineteenth century is far more literary in style and contains far less of the scientific jargon used today; such papers should be compulsory reading for graduate students in science. Stokes' first paper on fluorescence [1] is 100 pages long, but it is worth noting his main conclusions and the means by which these were obtained because it opens a window on his experimental approach, his profound understanding of the materials used, his biological knowledge as well as how it prepared him for the later analysis of blood. Interestingly, however, in his papers, there is no acknowledgement of any technical assistance; indeed one wonders how he could have single-handedly carried out all the experiments he describes.

In order to investigate the phenomenon of fluorescence, Stokes had built an experimental apparatus of surprising simplicity. Thus, in a room darkened by drawing down the window blinds, a small hole in a blind allowed sunlight through. Immediately underneath the hole, a shelf carried the optical equipment. Generally, the sun's rays passed through a lens and a wide range of coloured glass filters between the beam of sunlight and the sample under investigation or between the sample and the eye. Thus, he could observe differences in colour between incident and emitted light. A key addition to this arrangement was to pass the sunbeam first through three Munich prisms in series, thereby creating a wide spectrum of incident light. By moving the sample in an arc through this spectrum, he could vary the colour of incident light striking the tube. Multiple variations in this optical arrangement are described also, especially varying the light source to include alcohol flames, spirit lamps, a hydrogen flame and importantly, electric arcs either produced by a generator or from lightning. Brewster [11] and Herschel [7] had investigated and discussed the question of whether the emitted light of fluorescence was polarized and this issue was also discussed by Stokes.

3. Additional investigations

As had been initially described by Becquerel [9] and Herschel [7], Stokes also carried out most experiments with a tube containing a quinine sulphate solution. However, in this paper [1], Stokes also investigated and reported on many other materials, including extracts of plants, seaweeds, dyes, metals, minerals, including uranium salts, wool, a selection of different glass and water. Interestingly, he noted that unboiled water scattered light because of its content of minute air bubbles whereas following boiling, where the small air bubbles had coalesced and been released from the water, light scattering by water disappeared. This is important since Brewster [11] had argued that particulate matter in the solution was responsible for the dispersion of light. Therefore, systematically, Stokes boiled all water used in experiments in order to eliminate this potential artefact.

The length of time Stokes took to investigate the optical properties of the eye lens perhaps best exemplifies his interest in biology. A report by M. Brücke [13] had appeared in which he suggested that the different parts of the eye, and especially the crystalline lens, are far from transparent with respect to the rays of high refrangibility. Stokes notes: ‘The experiments of M. Brücke necessarily occupied a considerable time, and it may be doubted whether the eye, especially after dissection, might not have changed in the interval, and whether the results so obtained are applicable to the eye as it exists in the living animal’.

His experiments with glass from various sources were also of great importance, since he remarked on the relatively poor quality of the prisms and the chromatic aberration of glass lenses he initially used. He states that he managed to borrow some samples of quartz glass from a Dr Faraday, but in the longer term, Stokes repeated many of the experiments reported with more expensive quartz prisms and lenses ordered for himself. In the nineteenth century, Ireland was a major contributor to astronomy and the Grubb family in Dublin had an international reputation for constructing optical instruments, including telescopes for the renowned Armagh Observatory, the Royal Greenwich Observatory and the Great Melbourne Telescope [14]. On his frequent visits to his native land, Stokes very willingly shared with Irish astronomers his expertise in optics and his experiences with glass and quartz, advising Grubb about the manufacture of achromatic lenses. Stokes also advised the astronomer Thomas Romney Robinson who had helped construct William Parsons' (the third Earl of Rosse) giant telescope at Birr castle in County Offaly and indeed Stokes married Robinson's daughter. The telescope at Birr was built in 1845 and for 75 years was the world's largest.

His experiments with electrical arcs and lightening resulted in important conclusions regarding the distinction between fluorescence and phosphorescence. Thus: ‘But by far the most striking point of contrast between the two phenomena, consists in the apparently instantaneous commencement and cessation of the illumination, in the case of internal dispersion (fluorescence), when the active light is admitted and cut off. There is nothing to create the least suspicion of any appreciable duration in the effect’. Thus, Stokes concluded that fluorescence was always an instantaneous phenomenon whereas phosphorescence could be slower. He also speculated, decades before the development of quantum theory and the work of Perrin [4] and Jabloński [5], as to the molecular nature of fluorescence: ‘If the dispersed light really arises from molecular disturbances, and for my own part I think it almost beyond a question that it does, it follows that in these cases light is absorbed in consequence of its being used up in producing molecular disturbances’. The atomic energy diagram depicting the difference in electronic states between fluorescence and phosphorescence is generally called the Jabloński diagram but, as Valeur and Berberan-Perrin [3] argued, this should be called the Perrin-Jabloński diagram.

4. The Eureka moment

What does come across from reading Stokes' account is the tangible excitement of the main observations made. It is worth quoting his description of passing a tube of quinine sulphate through the spectrum of sunlight generated by his apparatus. Thus:

The above is a wonderful description of what can only be described as a ‘Eureka' moment in scientific research! Those who have experienced such moments can surely recall their own excitement. The description also clearly emphasizes his finding that fluorescence could be initiated by a broad spectrum of visible blue incident light, reaching a maximum and then continuing beyond the visible violet end of the spectrum. The emitted light remained in the visible spectrum as blue light, thereby having a longer wavelength than the incident light that could be in the ultraviolet. This describes what we know today as the Stokes shift of fluorescence. In reality, the spectrum of both excitation and emission frequencies are broad with the peak of emission generally of lower frequency than that used for excitation. However, if using monochromatic light for excitation with a frequency above the maximum for excitation, the maximum frequency of emitted light can indeed be of higher frequency than that used for excitation; this is the anti-Stokes shift.

The main conclusions of this paper [1] are that fluorescence is a rapid phenomenon whereby incident light, regardless of colour, and emitted light are of different colours with the latter being of longer wavelength and that emitted light is emanated in all directions and is not polarized. However, Stokes also notes that upon examination of diverse, especially organic, substances that the phenomenon was extremely common. Therefore, he proposed that the property of fluorescence could be used for the detection of ultraviolet light, to identify fluorescent organic substances in a mixture and to use this property for their purification. In terms of biology, Stokes devoted considerable space to an investigation of the nature of the fluorescent material in extracts of red seaweeds. This work preceded by over a century the purification of the phycobiliproteins, in particular phycoerythrin, a very large protein with extremely bright fluorescence and a very long Stokes shift that is frequently used as a fluorophore in biology.

5. Investigations of blood

Having built and used his novel equipment to investigate fluorescence, in a subsequent paper [15] published in 1864, he showed that chlorophyll extracted from plants using alcohol, ether or chloroform had distinctive absorption bands and showed strong fluorescence whereas biliverdin, a green pigment in bile derived from haemoglobin degradation, was not fluorescent. He thereby resolved a controversy current at the time that the two substances were identical. This study [15] was published in the same issue of the Proceedings of the Royal Society of London just prior to his important paper on blood [16].

The paper on blood entitled ‘On the reduction and oxidation of the colouring matter of the blood' constitutes another major scientific milestone. At the time, it was known that the colour of arterial and venous blood was different, and that oxygen entered the blood upon passing through the lungs. However, it was unclear if the oxygen was merely dissolved in the blood or was combined with a substance in the blood. He begins his introduction by quoting Hoppe's 1862 publication [17] entitled (in German) ‘On the behaviour of the blood pigment in the spectrum of sunlight'. With his apparatus, Stokes repeats these observations with diluted blood remarking: ‘the extreme sharpness and beauty of the absorption bands of blood excited a lively interest in my mind and I proceeded to try the effect of various reagents'. Just as had been previously used, Stokes proceeds to use various acids and alkalis but doing these he remarks ‘But it seemed to me to be a point of special interest to inquire whether we could imitate the change of colour of arterial into that of venous blood, on the supposition that it arises from reduction'.

He then proceeds to describe his method of sample preparation that was to allow the sheep or ox blood obtained from a butcher to coagulate, cut the clot into small pieces and following rinsing, extract in water. He then shows his knowledge of chemistry in devising a series of solutions capable of reducing blood in the presence of alkalis. Such solutions contained ferrous sulphate (protosulphate of iron), tin chloride or ammonium sulphate. When these solutions were added to the blood samples ‘the colour is almost instantly changed to a much more purple red. The change of colour, which recalls the difference between arterial and venous blood, is striking enough, but the change in the absorption spectrum is far more decisive'. ‘If an additional quantity of the reagent is now added, the same effect is produced as at first, and the solution may thus be made to go through its changes any number of times'. By exposing the purple solution to air and providing that a small amount of reducing solution had been added, the colour change was reversible and the spectral lines observed reverted to those prior to reagent addition. He makes another key observation in that if the blood was in a shallow vessel, the colour change when exposed to air was rapid, but if the sample was in a tube, only blood in the top half of the tube reverted to the normal, red, colour whereas that in the bottom part remained purple. Therefore, Stokes' pioneering experiments showed for the first time that changes in the colour of blood were rapidly and repeatedly reversible and that exposure to air affects these changes.

There is a delightful section of the paper where Stokes makes his biological speculations. Thus: ‘I have purposely abstained from physiological speculations until I should have finished the chemico-optical part of the subject; but as the facts which have been adduced seem calculated to throw considerable light on the function of cruorine in the animal economy, I may perhaps be permitted to make a few remarks on this subject'. Stokes was unaware of Hoppe-Seyler's 1864 paper [18] in which the word haemoglobin had been used first to describe the colouring matter of blood. He had therefore used his own term, ‘cruorine', for this. The name derives from the Latin word ‘cruor' for blood, and Stokes acknowledges a Dr Sharpey for having suggested this term. He also noted that the word Haematin was being used specifically for the pigment generated by blood decomposition.

Some discussions are included as to whether the colour changes seen in his experiments in vitro correspond with those in vivo. He argues that many observations in the literature on the colour of venous blood involved the use of post-mortem material and that some changes in colour happen after death. He notes that Hoppe had proposed that the absorptive properties of blood could be used for forensic purposes, but argues that blood (cruorine) is relatively unstable so what should be used is the characteristic absorption bands of hematin which is far more stable.

He also discusses reports where changes in the optical properties and colour of blood occurred following the addition of water. Stokes speculates that many of these apparent changes in colour were due to changes in osmolarity of the solution that affected the shape of the red blood corpuscles. Thus, by exosmosis, corpuscles lose water, shrink and become more light refractive, whereas absorbing water by endosmosis, corpuscles swell and their refractive properties resemble that of the plasma in which they are suspended. This discussion shows Stokes' knowledge of biology as well as of fluidics. He concludes: ‘No doubt the form of the corpuscles is changed by the action of the reagents introduced; but to attribute the change of colour to this is, I apprehend, to mistake a concomitant for a cause, and to attribute, moreover, the change of colour to a cause inadequate to produce it’. His most important speculation concerns the transport of oxygen by blood. Thus: ‘It has been a disputed point whether the oxygen introduced into the blood in its passage through the lungs is simply dissolved or is chemically combined with some constituent of the blood….Now it has been shown in this paper that we have in cruorine a substance capable of undergoing reduction and oxidation, more especially oxidation, so that we have all that is necessary to account for the absorption and chemical combination of the inspired oxygen'. Hoppe-Seyler would shortly thereafter also speculate on the transport of oxygen by blood [19].

6. Haemoglobin

Now we know that haemoglobin, one of nature's most remarkable molecules, is not oxidized or reduced upon exchange of oxygen molecules. One molecule of haemoglobin, made up of four subunits, can carry four molecules of oxygen and the more oxygen it carries, the greater its strength of binding oxygen, a phenomenon called the Bohr effect [20]. It was only over a century after Stokes' original observations that Max Perutz, who had worked obsessively for decades on the structure of haemoglobin, determined the detailed molecular changes that occur in haemoglobin when oxygen binds [21]. However, Stokes' remarkable biological knowledge and pioneering observations have nevertheless made a significant contribution to scientific knowledge. Stokes was a remarkable communicator of science including biology as exemplified by his series of Burnett lectures on light he delivered in Aberdeen in 1885 [22]. Stokes' work on haemoglobin influenced others to use optical spectroscopy to investigate biomolecules. Thus, Charles McMunn [23], another Irish scientist from County Sligo and who had been taught in Trinity College Dublin by William Stokes, George's cousin, identified respiratory pigments in both animals and plants. These pigments were later called cytochromes. McMunn's published work at the time was heavily criticized by Hoppe-Seyler and consequently, he ceased his research. However, this early work was later taken up by David Keilin [24] who used optical spectroscopy to fully characterize the cytochromes, thereby linking his work with that of George Stokes [25].

In Horace Judson's magnificent book concerning the history of biology in the twentieth century [26], Stokes is acknowledged as one of the founding fathers of what became known as biophysics, a topic that dominated the first half of the twentieth century. Thus, although Stokes only used visible light for his experiments on biological materials, following their discovery by Röntgen at the end of the nineteenth century [27], X-rays replaced visible light. Because the very short wavelength of X-rays approximates that of inter-atomic distances, the internal structure of inorganic crystals, and when they became available, crystalized biological molecules, is revealed by the scattering of X-rays that pass through them, a phenomenon called X-ray diffraction [28]. In the first half of the twentieth century, X-ray diffraction in the hands of the Braggs [29], Astbury [30] and others became a major experimental tool for the elucidation of the structure of proteins, including haemoglobin [21] and in the hands of Rosalind Franklin [31], that of DNA [32,33].

7. Use of fluorescence in biology

Today, fluorescence is widely used in biology [3] and space does not permit a full discussion of this topic. However, in the twenty-first century, two Nobel Prizes in chemistry have been awarded for work involving fluorescence. The first, in 2008 to Osamu Shimomura, Martin Chalfie and Roger Y. Tsien, was for the development and application of use of green fluorescent protein (GFP) and its derivatives. These molecules have had a major impact on biology, being used for gene reporter assays, including transgenesis, fluorescent microscopy and protein colocalization assays. The second, in 2014 to Eric Betzig, Stefan W. Hell and William E. Moerner, was for the development of super-resolved fluorescence microscopy. Super-resolved fluorescence microscopy involves a combination of stimulated emission depletion microscopy developed by Stefan Hell and single-molecule microscopy developed by Eric Betzig and William Moerner allowing individual molecules to be visualized inside living cells. As an extension of adapting microscopy to the nanometre scale, or ‘nanoscopy', Shankar Balasubramanian and David Klenerman developed a two-colour coincidence single-molecule fluorescence method capable of detecting single biomolecules [34], thereby revolutionizing fields such as DNA sequencing, biology, archaeology and palaeontology. In the context of Nobel Prizes, one wonders how many Stokes would have obtained if they had been awarded at the peak of his scientific output.

One technology that depends both on the speed of fluorescence and the diversity of available fluorescent molecules is so-called flow cytometry. Here, mixtures of different cells or particles in suspension, labelled on the surface and/or internally by multiple fluorescent reagents (or fluorophores) pass at speeds of several metres per second in a jet of pressurized fluid through a sequence of focussed laser beams and their fluorescence emission analysed by multiple photomultiplier tubes each detecting particular emission spectra. Following hydrodynamic focussing, single cells or particles pass sequentially through the focal points of different lasers in times of microseconds and each laser excites different fluorophores having different Stokes shifts. With combinations of dichroic mirrors, long and short pass light filters, detectors aligned with the different incident lasers can distinguish individual fluorophores and hence individual cell types or particles labelled with combinations of fluorophores at speeds of 10⁴ to 10⁵ per second. With the ability to distinguish particular cell types and by sequestering individual cells in single electrically charged liquid droplets, they can be sorted from a heterogeneous mixture, thereby providing a powerful tool for biological research and this is a technology fundamentally based on fluorescence. For example, such technology is essential for understanding the inter-relationships between different cell types in the blood cell lineage [35,36]. Although conventional flow cytometry only measures the total and not the distribution of fluorescence either on the outside or inside of the particle [35]. The advent of cellular imaging by flow cytometry [37], whereby the distribution of fluorescence on the analysed cell can be visualized, has revolutionized this field. One can only imagine how Stokes would be in his element discussing the issues of fluidics, including hydrodynamic focussing, optics, physics and biology of this technology.

8. Conclusion

George Gabriel Stokes was the youngest of eight children of the Church of Ireland rector of the parish of Skreen (An Scrin) in County Sligo. His father, also Gabriel Stokes, came from a distinguished family of mathematicians, physicians and engineers and the mathematical skills of his son George were spotted at an early age. A full biographical account of this man from An Scrin can now be found in a book entitled ‘George Gabriel Stokes: Life, Science and Faith' [38]. The village of Skreen is now bypassed by the N59 road from Galway around the west of Ireland to Sligo-part of the ‘Wild Atlantic Way' [39]. However, for those interested in the history of science and Irish science in particular, a visit to the village to see the modest plaque (figure 1) commemorating George Gabriel Stokes in front of the site of the former rectory is well worth the detour.

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