Confirming Robert Brown's Observations of Brownian Movement, Proceedings of the Royal Microscopical Society, 31 (4): 316-321, 1996.

Confirming Robert Brown's Observations of

Brownian Movement

by Brian J Ford

Introduction

Robert Brown (1773-1858) was a distinguished microscopist and botanist. He discovered the naked ovule of the gymnospermae, a most exacting piece of microscopical investigation, and carefully documented the phenomenon now known as Brownian Movement. The idea that he did not observe this physical phenomenon has often surfaced in the past. It has been said that the optical capacity of his simple microscopes was too limiting, and many modern accounts erroneously state that Brown observed the movement of pollen grains. That is not correct: most are several orders of magnitude too large to exhibit the phenomenon. As a careful reading of Brown's original paper shows, he was observing the movement of minute particulates within pollen grains, a very different matter. It has been shown (Ford, 1985, 1991a) that the earliest simple microscopes can allow us to visualise structures as fine as 0.7mm, and Robert Brown could certainly have studied the cell nucleus with his own microscopes of the early 1800s.

These early microscopes comprised a single magnifying component, typically a hand-polished biconvex lens made from soda glass. It was with these instruments that the ground-work of modern biology was laid, including the discovery of the germinative cells of plants and animals and the realm of microorganisms.

Origins of Microscopy

There are several erroneous teachings associated with the dawn of microscopy. One is that the first microscopes were simple instruments, with compound microscopes following later. Another is the idea that the concept of refraction is itself a relatively modern development. This view is two millennia out of date, for Claudius Ptolemy in his Optics, sermo 5,

wrote of the principles of refraction in the second century B.C:

"In the passage [of light] from the rarer to the denser, the flexure is towards the perpendicular, and in the passage from the denser to the rarer [medium], the flexure is away from the perpendicular."

Later writings by Roger Bacon (1214?-1294) imply an understanding of magnification (Bridges, 1897); he wrote that 'refracted vision' could be used to make the greatest things appear small, 'and also the contrary'. Subsequent writers prior to the era of microscopy, including the Franciscus Maurolycus (1494-1575) and Fracastoro of Verona (1483-1553) understood the nature of magnification and laid the ground-work for the conceptual developments of later centuries. The revolutionary observations of Leeuwenhoek and, later, Robert Brown, were following a lengthy process of enlightenment (Ford, 1973). The recent objections to Brown's claims hang, in essence, on a simple conclusion: in the words of this latest submission, Brown was examining particles 'after the pollen grains burst' (Deutsch, 1996). This view is absurd; Brown's many observations disprove any such simplistic interpretation.

What did Brown study?

When Brown began his epoch-making observations in 1927, he was studying the north American angiosperm species Clarkia pulchella, [the genus being spelled Clarckia by Brown in his account]. He looked with particular care at the structure of the pollen-grains. These he took, not from already opened anthers, but from fully-formed pollen sacs that were yet to open and which he dissected at the bench. He suspended some of the pollen grains in water and examined them closely, only to see them 'filled with particles' that were 'very evidently in motion'. There is no question of Brown confusing his observations with other movements caused, perhaps, by evaporation currents. He made sure that the movement 'arose neither from currents in the fluid, nor from its gradual evaporation, but belonged to the particle itself'. He carried out careful experiments to disprove these alternative explanations, and it Brown was usually able to anticipate the later objections of those who would doubt his capacity to have observed what he claimed.

It would have been very tempting for Robert Brown to assume, as had other workers before him, that he was observing the very essence of life. Within the germinal cells of living organisms he could perceive movement without end. To a modern eye, well versed in the drama of scientific discovery as the twentieth century comes to its close, this is the immediate explanation. Many of the lay people who have seen the phenomenon conclude that they are watching life itself at work.

To Brown's great credit, he was not so easily persuaded. Robert Brown had read the accounts of many of the earlier workers who had seen this phenomenon, and noted that they tended to associate it with organic matter (on the assumption that is was linked with the mechanisms of life). He writes that it had been assumed they were:

'. . . elementary molecules of organic bodies, first so considered by Buffon and Needham, then by Wrisberg with greater precision, soon after and still more particularly by Muller, and, very recently, by Dr. Milne Edwards, who has revived the doctrine and supported it with much interesting detail.'

Having seen the phenomenon in a host of living plant specimens he was led to investigate whether it persisted in plants that were dead. As he was passing the proofs of his paper for the press, he wrote that he had seen the same phenomenon within pollen grains preserved for about eleven months in an alcoholic solution:

'. . . particularly of Viola tricolor, Zizania aquatica and Zea mays.'

Brown moved on to consider a host of clearly non-living specimens, including rocks 'of all ages' which yielded the particles 'in abundance'. In short, he concluded, any solid mineral would reveal the phenomenon subject to its being reduced to a sufficiently fine powdery form. He showed an admirable objectivity in taking up a topic well known to previous microscopists, yet in setting out a revolutionary explanation for its physical (as opposed to its biological or organic) nature. To this day, interest in the topic continues (Bown, 1992, |Deutsch, 1996).

Brown's Microscopy

Brown was an accomplished microscopist and an extraordinarily gifted observer of microscopic phenomena. Thus it was that Robert Brown identified the naked ovule in the gymnospermae. This is a difficult observation to make with a modern instrument even with the benefit of hindsight. To Brown this task was immeasurably greater. Typically, he tucked the report of his observation away in a more lengthy publication. To a paper by Captain P. P. King Brown added the following words:

'It would entirely remove the doubts that may exist respecting the point of impregnation, if cases could be produced where the ovarium was either altogether wanting, or so imperfectly formed, that the ovulum itself became directly exposed to the action of the pollen . . . such, I believe, is the real explanation of the structure of the Cycadeae, the Coniferae, of Ephedra, and even of Gnetum.' (King, 1827).

In later years, it was to the observation of the incessant agitation of minute suspended particles that Brown's name became inextricably linked. The effect, which was to become known as Brownian Movement, was first noticed by him in 1827 (Brown, 1827). His description of the phenomenon reveals that he was planning to continue his work on the mechanisms of fertilization in flowering plants. Having worked on the ovum, it was natural to direct attention to the structure of pollen and its interrelationship with the pistil.

Brownian Movement, or Brownian Motion?

All users of high-powered microscopes, simple or compound, are likely to observe Brownian movement. What we observe are tiny suspended particles in a fluid medium in unceasing movement back and forth. They are "jiggling on the spot" - not showing movement in a specific direction, but ceaselessly jerking about. Allusions to the phenomenon were found in many earlier accounts, but it was not until 1827 that Robert Brown wrote his precise and insightful account. Brown's usually wrote of this phenomenon as a 'movement', and this is the term recognised in the standard texts. In recent years the alternative usage 'Brownian motion' has gained more general acceptance in the world of physics. Strictly speaking, it is not correct.

Robert Brown

Robert Brown's father James was a Scottish Episcopalian minister with a reputation for independence. The young Robert was born at Montrose, Scotland, on December 21, 1773 and he clearly inherited this intellectual strength. He was educated at the Marischal College, Aberdeen, and later enrolled to study medicine at the University of Edinburgh. He joined the Fifeshire Regiment of Fencibles as Ensign and Surgeon's Mate at the age of twenty-one in 1795. When he was posted with his regiment to Ireland, he devoted much of his time to academic study. He noted that he was studying German grammar before breakfast, and then he worked on botanical documents until lunch-time. From 1.00pm to 3.00 pm he would see his patients as a doctor. Then, if his social commitments allowed, he continued his scientific work until midnight.

The successful young officer was sent to London to help find new recruits for the regiment. Robert Brown soon met the eminent botanist Sir Joseph Banks, being introduced to him as "a Scotchman, fit to pursue an object with constance and [a] cold mind". Banks was planning a voyage of discovery to the new territories now known as Australia, and his choice for a botanist to accompany him was the young Brown. The Captain was the young Matthew Flinders, and Brown was to be paid a salary of £420, a substantial sum. When they sailed on board the Investigator on July 18, 1801, both Robert Brown and Matthew Flinders were twenty-seven. Also on board was an artist and draughtsman of immense talent: the forty-one year old Ferdinand Bauer (brother of the equally renowned Francis).

They arrived on the territories of New Holland (as Australia was then called by the British) on December 8, 1801, landing at King George Sound, on the south-western corner of Australia. Brown collected more than 500 new species of plants within the first three weeks. Later he stayed for three months at Port Jackson, and ten months more on the island of Tasmania. The expedition returned to England in October 1805 with vast collections of drawings and notes and many zoological specimens, and with nearly 4,000 different species of plants. Robert Brown was offered a government grant to work on the material, and spent much of the next five years to describing 2,200 species, over 1,700 of which were previously unrecorded. Brown himself nominated 140 new genera.

From 1806-1822 Robert Brown served the Linnean Society of London as 'clerk, librarian and housekeeper' and when Sir Joseph Banks died on June 19, 1820, Brown took on Banks's home and collections in Soho Square. The stipulation of the bequest was that the collections would be in the care of Brown during his lifetime, and would pass to the new British Museum on Brown's death. In the event, matters moved quickly, for Robert Brown was able to negotiate the transfer of the specimens in 1827, on condition that they become a permanent part of the British Museum and that he remained curator for life. This gesture was an important event in the establishment of the great London museum collections which have since become so important in the world of science.

The Royal Society elected Brown to Fellowship in 1810, and he became a Fellow of the Linnean Society twelve years later. He was elected to the presidency of the Linnean from 1849 to 1853. He died in London on June 10, 1858, a week before Darwin received Wallace's paper on the theory of 'survival of the fittest', and the date of Brown's death ultimately led to the availability of a free date at which Darwin might have his epoch-making paper on the theory of evolution read to the Society.

Confirmation of Brown's Observations

One recent objection to the validity of Brown's observations on Brownian movement was published as a typed summary in a bulletin published by the American Physical Society (Deutsch, 1991). This publication is not subject to peer review, and has the avowed editorial policy of publishing short proposals and ideas without editorial selection on grounds of quality. This hasty comment was picked up and circulated elsewhere, and has recently re-emerged in the Proceedings (Deutsch, 1996). The notion of 'pseudo-Brownian motion' has been raised with the questionable justification that Brown may have observed the effects of freely suspended particles released from pollen grains.

There are two sources of refutation of this fanciful proposition. One is that Brown's own papers make it clear that he was, firstly, observing particulates in enclosed environments (pollen grains, for example, and vesicles within mineral specimens) and, secondly, was well aware of the consequences of turbulence or convection currents within a fluid medium. In this sense, Brown's writings and research methodologies were considerably superior to those of his latter-day critics.

The second refutation lies in the recreation of Brown's original experiments. His microscope, preserved at the Linnean Society of London, has often been dismissed as of poor optical quality (Anon, 1932). Yet, when properly used, it can provide images of gratifyingly good resolution. A similar microscope preserved at Kew Gardens (Ford, 1985) gives comparable results. Was it possible that some alternative effect, some form of 'pseudo-Brownian motion', was responsible for his reports? A recreation of his original experiments was undertaken to resolve the matter. Brownian movement is familiar to all microscopists, and the very different movement caused by evaporation currents induced by thermodynamic turbulence are well known to all experienced research workers.

True Brownian movement has unmistakable characteristics. Its kinetic force is directly related to particle size, and the vector of the force that gives rise to the movement is not in any way consistent, nor does it result in motion in a specific direction. To the eye of the microscopist it is, instantly and unmistakably, Brownian movement. The microscope now in the collections of the Linnean Society of London was, by resolution of the President and Council, taken from the collections in order to carry out the experiments at my own laboratory. The attempts were based on a central premise: to reveal the phenomena observed by Robert Brown in a form as close as possible to the view he obtained in 1827.

Much interest was aroused in the project, though the aim was not always fully understood. The response to an outline of the experiment published in London was to assume that image-intensification was the aim (Wheatley, 1992). It must be emphasised that this was not the case. The recreation of a pioneering experiment can only be recorded if it offers today's observer sight of what the original researcher could have seen. A modern glass slide may be preferred to a mica slip; a pearl bulb may be more convenient than an oil-lamp; a platinum loop may be more easily available than a Victorian dissecting needle; and any of these substitutions may be made if expedient.

What cannot be done is anything that provides a change in the appearance of the specimen as perceived by the observer. In the case of the Brown experiments, the single #1 lens was mounted on a modified objective tube of a Leitz microscope. The video camera, recording directly onto 8mm tape, was mounted ready to record the resultant images. No microscope eyepiece or field lens was used in the light path. The Clarkia pollen was obtained from anthers of C. pulchella in the herbarium collections of the Botanical Garden at Cambridge University, and pollen specimens from other species within the Oenotheraceae were also utilized. Exactly as Brown recorded, the experiments were carried out in the month of June and the pollen grains were mounted in water after removal from pre-dehiscent anthers. A 10mm graticule was recorded at the end of the demonstration in order to provide an on-screen calibration.

The phenomenon of Brownian movement was well resolved by the original microscope lens (Ford, 1991b, 1992b). Within the pollen grains, ceaseless movement could be observed. There is clearly no question of extraneous hydrodynamic phenomena in such a closed system, and evaporation-induced turbulence could equally be excluded. But this qualification is unnecessary to any seasoned microscopist: Brownian movement is instantly recognisable for what it is. The size of particles within the pollen was hard to estimate, though a working diameter of approximately 2mm could be offered. Correlated observations on colloidal systems in Indian ink and cow's milk show that particulate matter below this size can be clearly visualized by the Brown lens.

The video demonstration was exhibited for the first time to the delegates to a major meeting in the United States, and it has since been seen in London, Newcastle, Cambridge and elsewhere. The response was enthusiastic: Robert Brown's claims were clearly no exaggeration. Dr Walter McCrone, uniquely experienced at the observation of such phenomena, has concluded that the results make 'a beautiful video'. After 165 years, Brown's his pioneering observations and the clarity with which he assessed their implications were made available to modern microscopists. Frame-by-frame stills from the videomicrographs clearly display the phenomenon and were published in Nature (Ford, 1992b).

Brownian Movement and Albert Einstein

The analysis of Brownian movement by Albert Einstein in 1905 led to the formulation of the Boltzmann Constant, and shortly afterwards J. B. Perrin began his publications in Paris. They were later summarized in an extended paper which was later published in English translation as a book in its own right (Perrin, 1909). Not only did his account range across the many other workers who published since the time of Brown, but he also demonstrated the effect by projection microscopy. A copy of this book was given top the Author by Deutsch; intriguingly, the volume - which has no distinguishing marks to indicate reprinting - is a modern facsimile and not an original.

So the subject remains clouded in curiosity. To this day the implications of Brownian movement are debated; the mathematics of the phenomenon continues to occupy a significant tranche of contemporary publications in physics. A search through Current Contents for 'Brownian Motion' will often provide several citations each month in the modern literature.

In conclusion, Robert Brown took all precautions necessary to safeguard his observations from external perturbation. Many workers have doubted the abilities of the pioneers of microscopy to make the observations they recorded. Some have failed to repeat observations using the original equipment. These are manifestations of some modern observers' limited technique and narrow understanding, rather than a valid critique on the accuracy of the pioneer microscopists. Brown's self-critical stance was admirable, and others could do well to learn from it.

The issue we are faced with here is simple. Could Brown's microscope reveal Brownian movement? The answer is an unequivocal affirmative, and in due course we will place the video evidence on the world-wide web for the scrutiny of other microscopists. Brown recognised Brownian movement when he saw it. Unfortunately, some more recent microscopists are not as able to understand what they see. Their private obsessions must not stand in the way of a full appreciation of the considerable achievements of our predecessors.

ACKNOWLEDGEMENTS

I am grateful to the President and Council of the Linnean Society of London for permission to restore to use the Bancks microscope of Robert Brown (Ford, 1982) so that these historic experiments could be recreated, and to the Trustees of the Appleyard Fund and the Leverhulme Trust, for grants in aid of the research. Professor Max Walters of Cambridge provided the author with advice and assistance on the genus Clarkia and Dr Peter Yeo at the Cambridge University botanical Garden was kind enough to allow access to the herbarium material on C. pulchella and to members of the Oenotheraceae cultivated in the collections.

REFERENCES

Anon (1932) Centenary of Robert Brown's Discovery of the Nucleus. 'The Linnean Society account describes the instrument as 'surprisingly simple, being little more than a dissecting-microscope.' Journal of Botany, January.

Bown, W. (1992) Brownian Motion sparks renewed debate, New Scientist, 133: 25, 15 February.

Bridges, J.H., editor, (1897-1900) Bacon, Roger, Opticks, Oxford & London.

Brown, R. (1827) A Brief Account of Microscopical Observations, etc., London (not published), vide p 8.

Deutsch, D. H. (1991) Did Robert Brown observe Brownian Motion: probably not. Bulletin of the American Physical Society, 36 (4): 1374, April 1991. Reported in Scientific American, 265: 20.

———— (1996) Brownian Motion, now you see it, now you don't, Proceedings of the Royal Microscopical Society, 31 (3): 222-227.

Ford, Brian J. (1973) The Revealing Lens: Mankind and the Microscope, London: Harrap, September.

———— (1982) The Restoration of Brown's first Botanical Microscope, Microscopy, 34: 406-418, December.

———— (1985) Single Lens - Story of the Simple Microscope, London: Heinemann, New York: Harper & Row.

———— (1991a) The Leeuwenhoek Legacy, 185pp, Bristol: Biopress and London: Farrand Press, July. [Distributed in the United States by Lubrecht & Cramer].

———— (1991b) Robert Brown, Brownian Movement, and teethmarks on the hatbrim [leading paper], The Microscope, 39 (3 & 4): 161-171.

———— (1992a) The Controversy of Robert Brown and Brownian Movement, Biologist, 39 (3): 82-83.

———— (1992b) Brown's Observations Confirmed, Nature, 359: 265. ibid., Botanical Appendix: 534 et seq.

King, P. P. (1827) Character and Description of Kingia, [in] Narrative of a Survey of the Intertropical and Western Coasts of Australia, II: 536-563, London.

Perrin, J. B. (1909) [in] Annales de Chimie et de Physique, 8me series, September. Translated by F. Soddy F.R.S. (1910) Brownian Movement and Molecular Reality, London: Taylor and Francis.

Wheatley, D. (1992) Brown motionless, Biologist, 39 (4): 124.

Captions for illustrations

1) Robert Brown's microscope

This instrument is preserved in the collections at the Royal Botanic Gardens, Kew. The engraved silver plate records its ownership by Brown. Note an advanced feature of this simple microscope: a concentric fine-adjustment focussing control mounted on the pillar immediately above the substage mirror. I am grateful to Mr Gren Ll. Lucas for drawing my attention to this instrument, and to Dr David Cutler at the Jodrell laboratories for the use of facilities in connection with research on this instrument.

2) The Linnean Society microscope

These simple microscopes were manufactured by the father and son firm of Bancks in central London during the 1820's. This example, shown in its fitted case, was used by Robert Brown during his Presidency of the Linnean Society. After being in private hands for many years, the microscope was returned to the Society in time for the centenary (held in 1928) of the naming of the nucleus by Robert Brown. Note the screw-in brass cups (right) in which the single lenses are mounted.

3) The components of Brown's Microscope

The Linnean Society's microscope had been through many hands when the author restored it to working order. It had been wrongly re-assembled it in the recent past, and some of the components were strained. Prior to re-assembly, the microscope was photographed and accurately measured. Note the five single lenses Brown used for his observations. The circular stage and double-sided mirror were characteristic of these 'botanical' microscopes.

4) The Bancks microscope with fine adjustment

For an instrument made in the early nineteenth century, the Bancks microscopes show remarkable technical sophistication. In addition to the fine-adjustment control, this version (used by Charles Darwin) features a sub-stage condenser lens. At the top of the microscope body is the lens arm, which can be tracked across the area of the stage by means of a rack-and-pinion facility.

5) The construction of a sophisticated simple microscope

In this isometric drawing of Brown's microscope in the Kew collections, the constructional details are clarified. Three milled controls adjust the lens arm, coarse focussing on the main pillar, with the fine adjustment beneath. In this model, the entire pillar can be reclined through a quadrant fulcrum at the base. Though this microscope was not fitted with a substage condenser lens, a concave mirror was fitted.

6) The author working with Robert Brown's microscope

A sense of scale is conveyed by this photograph of the Linnean Society microscope in use. In reconstructing Brown's original observation, candle-light, lamp-light and daylight were utilised. Sunlight was shown to produce a vivid dark-ground image in which Brownian movement could be resolved. During us, the microscope is screwed into the lid of the mahogany box in which its components are normally stored.

7) Early doubts over the specifications of a Robert Brown microscope

After the Linnean Society were presented with Brown's microscope, a cursory description was published. It appeared in the Journal of Botany (1932) and dismissed it as 'little more than a dissecting microscope'. Such instruments are described in words like these in museum collections around the world. The facility for using them to maximum benefit has largely been lost during the last century. In practice, however, they provide results that belie their uncomplicated construction.

8) Cell nuclei and cytoplasmic inclusions under Robert Brown's microscope

An epidermal peel of Allium was lain on a coverslip without mountant for this micrograph. The dark-ground image was obtained by utilising oblique illumination from the concave side of the sub-stage mirror. The smallest constituents are of bacterial dimensions, and are clearly in active Brownian movement. The nuclei are conspicuous in this view, yet during the 1930s it was believed unlikely that so diminutive a structure could have been resolved with a simple microscope.

9) Enlargements from the videomicrograph of Brownian movement

Consecutive frames from milk fat globules in active movement show the random lateral displacement of these minute particles. The video recording, described by Walter McCrone HonFRMS as a 'beautiful' demonstration, clearly shows conventional Brownian movement of the kind all experienced microscopists

10 (transparency): The microscope of Robert Brown in its case

When not in use, the botanical microscope was disassembled and packed into a fitted case. Lined with green velvet, and fashioned from mahogany, the box bears a recessed boss in the centre of the lid. The base of the microscope was screwed home into this fitment, thus making the box into its base.

11 (transparency) Robert Brown's microscope in use

The microscope could be reclined in use for the comfort of the microscopist. Note the ivory sliders. Each bears about four countersunk apertures. Disks of mica are secured in each by means of circlips, a permanent preparation being held by pressure between the two. Sections of bone, wood, or entire specimens such as feathers were mounted in this fashion. Fresh botanical material was placed direct on the circular glass stage and observed from above with little other than fresh water as a mountant.

12 (transparency) The business end of a Bancks microscope

The circular stage of brass and its inset glass disc can be clearly seen in this close-up study of a Bancks microscope in use. A swing-out secondary stage was frequently provided. This allowed a specimen on a watch-glass to be easily changed, and the arrangement also helps to stabilise a specimen mounted on a slider. The projecting fulcrum served also as the support for stage forceps, used to inspect floral or arthropod specimens in the round.

13 (transparency) Epidermal cells of Epidinium

Robert Brown made his first observations of the cell nucleus in orchid epidermis, and this experiment recreates the original observation. A peel of epidermal tissue is mounted in a film of water on a glass slide and examined through a Brown's microscope. Not only are the stomatal guard cells clearly visible, but the cytoplasmic structures can be seen. Brownian movement can be discerned amongst the smallest of these particles.

14 (transparency) Cytoplasmic details revealed by Brown's microscope

Here we view parenchymatous tissues of Epidinium teased apart on the stage. The fine cytoplasmic inclusions are of bacterial dimensions (approximately 2mm in diameter). They are clearly seen to be in a state of Brownian movement. It should be noted that the constraints associated with a single lens - chromatic and spherical aberration - are not the severe limiting factor which it is popular to believe. The experienced eye will see chromatism in this image, but the clarity compares favourably with many routine micrographs in modern publications.

15 (transparency) Staminal hairs of Tradescantia under dark-ground

The staminal cluster of Tradescantia virginiana is packed with a tuft of fine hairs. Each is a monofilament of rectangular cells within which cytoplasmic bridles support a conspicuous nucleus. Under dark-ground illumination the low-power lenses of Brown's microscope vividly reveal the structure of these hairs, and the nuclei can be distinguished. The microscope was well able to make out the phenomenon of cytoplasmic streaming, which Brown was first to describe.

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