Colour has attracted the interest and attention of many of the most gifted intellects of all time. Ideas of early thinkers were not -and could not have been- grasped on a scientific level without knowledge of a kind that lay far in the future. One character that is being considered is the colourful surfaces of living tissues, which could hardly have been visualized without a corresponding reference to the microscale parallel. Millions of years before man made manipulated synthetic structures, biological systems were using nanoscale architecture to produce striking optical effects. Here we show the microsculpture of the adaxial surface of flower petals from the asphodel, the Stork&#x27;s-bill and the common poppy by using optical, scanning electron and atomic force microscopy. Microsculpture has been studied in leaves and pollen grains of higher plants. To the best of our knowledge imaging and nanoscale morphometry of petals has not been reported hitherto. Our findings on flower petals&#x27; microsculpture may be linked with aspects on colour revealed from ancient literature

Apostolis Argiropoulos

Danae Koukos

Demetrios Anglos

Demetris Gikas

Emmanuel Spanakis

Nikos Alexandredes

Sophia Rhizopoulou

English

Nature Precedings

 1
Title: Visual perception of colourful petals reminds us of classical fragments 
 
 
Sophia Rhizopoulou1, Apostolis Argiropoulos1, Emmanuel Spanakis2, Demetris 
Gikas1, Nikos Alexandredes1, Danae Koukos1& Demetrios Anglos2 
 
1 National and Kapodistrian University of Athens, Faculty of Biology, Department of 
Botany, Panepistimioupolis, Athens 157 84, Greece 
2 Foundation of Research and Technology, Institute of Electronic Structure & Laser, 
Heraklion 711 10 Crete, Greece 
 
Text 
Colour has attracted the interest and attention of many of the most gifted 
intellects of all time. Ideas of early thinkers were not -and could not have been- 
grasped on a scientific level without knowledge of a kind that lay far in the 
future. One character that is being considered is the colourful surfaces of living 
tissues, which could hardly have been visualized without a corresponding 
reference to the microscale parallel. Millions of years before man made 
manipulated synthetic structures, biological systems were using nanoscale 
architecture to produce striking optical effects1. Here we show the 
microsculpture of the adaxial surface of flower petals from the asphodel, the 
Stork’s-bill and the common poppy by using optical, scanning electron and 
atomic force microscopy. Microsculpture has been studied in leaves2, 3, 4, 5 and 
pollen grains6 of higher plants. To the best of our knowledge imaging and 
nanoscale morphometry of petals has not been reported hitherto. Our findings 
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on flower petals’ microsculpture may be linked with aspects on colour revealed 
from ancient literature. 
Empedocles, Democritus, Plato, Aristotle, Theophrastus, Descartes, Hooke, 
von Fraunhofer, Newton, Leonardo da Vinci (his notes reveal knowledge of 
Theophrastus text on colours), Goethe (his books reveal knowledge of Leonardo’s 
ideas about colours), Hegel, Schopenhauer, Young, Maxwell, Helmholtz, Hering and 
Schrödinger all have been intrigued by colour and have contributed to our knowledge 
of it. Also, colour had always been an interesting subject for philosophers, although 
their ideas could be neither confirmed nor disproved for nearly two thousand years. 
Democritus (460–370 B.C.) regarded colours as the visually perceived result of 
various shapes, orderings, and positions of atoms and he conceived of colours as 
quantities of energy (light) that reveals translucence as an incorporeal property 
ranging from bright to dark. Plato (427–347 B.C.) wrote that colours are generated 
through the interaction of certain defined kinds of elemental particles and visual 
organs7, 8. According to Aristotle (384–322 B.C.) colour is a visible corporeal quality9. 
The Pythagoreans named the corporeal surface colour; colour is either in the limits (of 
bodies) or is the limit itself9. Theophrastus (370–288 B.C.) was the first to come up 
with a description of colour10, 11 According to Zeno (332–261 B.C.) colours constitute 
the first determinant of the form of matter11, this extraordinary statement stands 
furthermore isolated among the statements about colour and has received very little 
attention12, 13. Newton (1642–1726) states that wavelength composition of a light 
beam serves to define its colour, i.e. waves of length 400 to 450 nm are violet, 450 to 
480 blue, 480 to 560 green, 560 to 590 nm yellow, 590 to 620 nm orange and 620 to 
800 mm red. So pervasive is this doctrine in contemporary life that the existence of 
other explanations of colour is considered a matter for history of science11, 14, 15. In the 
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ongoing development of colour theory over centuries, Johann Wolfgang von Goethe 
(1749-1832) rejuvenated classical, philosophical ideas on colour16. 
In the plant kingdom, pigments of flowers can be exploited as sources for 
natural pigments and structural patterns. It is likely that natural systems offer 
technologically unrealised photonic structures and design protocols2, while modern 
industry is conceived and nurtured largely by the demands for colour17. 
Flowering plants known from antiquity18 (presented by Theophrastus, named 
by C. Linnaeus and collected by J. Sibthorp18) rely on a remarkable visual strategy in 
order to attract pollinators. It is likely that colourful flowers have co-evolved with the 
way pollinators see them19, 20, while floral colour has been used by pollinators as a 
predictor of nutritional rewards21, 22, 23  and warmth24. 
We present microscopic images of the adaxial surfaces of off-white petals (Fig. 
1a) from Asphodelus ramosus L. (asphodel), of crimson petals (Fig. 1b) from 
Erodium malacoides (L.) L’ Hir. (Stork’s-bill) and red petals (Fig. 1c) from Papaver 
rhoeas L. (common poppy), consisting of two-three individual layers of cells, 
approximately 2–30 μm thick25, 26.Cuticle folds of the surface of petals were observed 
by using scanning electron (SEM) and atomic force microscopy (AFM). To perform 
our work we relied on a high resolution tool in order to detect the first topographical 
information of petal surfaces. 
Images of the adaxial surface of petals from the asphodel flower were obtained 
by using optical microscopy (Fig. 1a1), showing mature stomata. In SEM image, 
nearly parallel cuticle folds were observed (Fig. 1a2). AFM imaging, over a 4 μm2 
scan area reveals a periodicity of cuticle folds (Fig. 1a3) and a detailed surface relief 
(Fig. 1a4). According to Theophrastus10 what is smooth is white and all smooth 
surfaces are brilliant9; but brilliant substances must also have open passages and be 
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translucent10. Democritus calls white smooth and black rough and he refers to the 
shape of the atomic figures13. 
Epidermal cells of crimson Erodium flower petals display thickenings of the 
cell wall at their base in a radial distribution (Fig. 1b1) and the rounded cells have a 
conical-papillate shape (Fig. 1b2) that resembles images obtained by AFM (Fig. 1b3) 
and the surface relief is shown in Figure 1b4. According to Theophrastus crimson 
comes from white, red and black10; for thus it makes an appearance delightful to the 
senses, while its brilliance and lustre testify to the presence of white13, 15. Aristotle 
proposed purple as the strongest colour-energy after light itself, while warmth is 
associated with red27. In the nanoworld, a particle with a diameter of about 100 nm 
appears purple-pink, but the colour shifts to red for particles with diameters of around 
20 nm28. 
The adaxial surface of petals of Papaver (Fig. 1c) viewed by optical 
microscope possesses oblong, red, epidermal cells with anticlinal undular walls (Fig. 
1c1). Cuticle folds of petals form an irregular pattern, as is apparent in SEM (Fig. 1c2), 
in AFM imaging (Fig. 1c3) and from the surface relief (Fig. 1c4). According to 
Theophrastus red is composed of atoms whose form is the same as, but larger than, 
that of things which are warm10. 
Colour reflects the state of the object to which it belongs and it depends on a 
relationship between light and the corporeal quality of the matter. However, “true” 
and “pure” colours are never seen27, 29. It also seems likely that the perception of 
colour has been influenced by qualities and temperaments being associated with 
colours. In addition, colour can be seen on a surface, as a spirited thing and the word 
to describe it was often fittingly applied as an adjective meaning something related to 
the colour itself. On the other hand, colourful plant pigments concentrated in vacuoles 
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and plastids are dependent on a mixture of elements in the cells, and in the 
environment (e.g. pH, heat and moisture). Therefore, it might be an oversimplification 
that a wavelength of light determines a colour. 
 
Methods Summary 
Fully expanded, turgid petals were harvested from flowers of Asphodelus ramosus 
(Asphodelaceae), Erodium malacoides (Geraniaceae) and Papaver rhoeas 
(Papaveraceae) plants growing under field conditions (38°14.3´N, 23°47.8´E) during 
spring and then were carefully transferred to the laboratory. Fresh tissue samples were 
cut and the surface sections were directly examined with a Zeiss Axioplan II 
microscope (Zeiss, Oberkochen, Germany) equipped with a camera, using Kodac 
colour 400 film. Petal samples were carefully cut in 2–4 mm2 pieces and fixed in 3 % 
glutaraldehyde in phosphate buffer at pH 7 at room temperature for 2 h. The tissues 
were then postfixed in 1 % OsO4 in the same buffer at 4 °C for 4 hours and 
dehydrated in a graded acetone series. The dehydrated tissue samples were critical 
point dried, mounted with a double adhesive tape on stubs, sputter coated with gold30 
and observed with a scanning electron microscope (JEOL 6300 SEM, Japan); SEM 
pictures were digitally recorded. Floral tissues, in their natural state, are very sensitive 
to the mechanical pressures of atomic force microscopy (AFM); thus, a mild method 
of fixation was performed on petals by using glutaraldehyde, paraformaldehyde and 
osmium tetroxide. Segments (4 mm2) were cut from the adaxial petal surface and 
were immersed in a solution of 2 % glutaraldehyde plus 2% paraformaldehyde, in 0.1 
M sodium cacodylate buffer (SCB), at pH 7.2, for 2.5 h, at room temperature. The 
plant material was washed three times by immersion in SCB for 5 min, and postfixed 
in 2 % OsO4 for 5 h at 4°C. It was then washed three times in SCB for 30 min (each 
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time) at room temperature. The tissues, immersed in the buffer solution, remained at 
4°C until they were to be studied. In order to quantify surface profiles of petals from 
topographic images, the stained tissues were screened in tap mapping atomic force 
microscope (TM-AFM, Multimode SPM, Veeco, USA) and various parameters were 
analysed and processed by using the software package Nanoscope III (Veeco, USA). 
 
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Figure Legend 
Figure 1│ Morphometry of flower petal surface (open square indicates the area of 
the petal adaxial surface obtained by using microscopes). a. Asphodelus ramosus’ off-
white surface obtained by optical microscope (a1), SEM (a2) and TM-AFM (a3), with 
profile view of the line section (a4). b. Erodium malacoides’ crimson surface obtained 
by optical microscope (b1), SEM (b2) and TM-AFM (b3) image, with profile view of 
the line section (b4). c. Papaver rhoeas’ red surface obtained by optical microscope 
(c1), SEM (c2) and TM-AFM (c3), with profile view of the line section (c4).  
 
Acknowledgements. This work was carried out as part of the Pythagoras (70/3/8036) 
programme that is co-funded by the European Social Fund and National Resources 
(EPEAEK II). 
 
Author contributions. S.R., A.A. and D.A. designed the research. A.A., E.S. and 
N.A. performed the experiments. S.R., A.A., E.S., D.G., D.K. and D.A. analysed the 
data. S.R., A.A., D.K. and D.A. contribute to the preparation of the manuscript. S.R. 
and D.K. wrote the paper. 
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A study on functional and structural traits of the nocturnal flowers of Capparis spinosa

Atomic force microscopy of pollen grains, cellulose microfibrils, and protoplasts.

Bees associate warmth with floral colour Nature 442,

Colour mixtures according to Democritus and Plato.

Colour vision as an adaptation to frugivory in primates.

Coloured nectar: distribution, ecology and evolution of an enigmatic floral trait.

Doxographi Graeci (Abud Walter de Gruyter et Socios,

Flowers through insect eyes.

Greek color theory and the four elements, A Cosmological Interpretation

leaves: a study using X-ray photoelectron spectroscopy, time-of-flight secondary-ion mass spectrometry, atomic-force microscopy and scanningelectron microscopy.

On colours in Minor Works (translator

On sense and sensible objects, in Parva Naturalia (translator Hett,

On the senses, in: Theophrastus and the Greek physiological psychology before Aristotle (translator

Photonic structures in biology.

Pigment distribution, light reflection and cell structure in petals.

Surface properties of barley straw Colloids and Surfaces B:

Symbolic plants of the Olympic Games.

The colourful past (Abegg-Stiftung,

The Hellenistic Philosophers: Translations of the Principal Sources with Philosophical Commentary

Theophrastus and recent scholarship.

True colors. Nature nanotechnology 2, 521 (2007). N a t u r e P r e c e d i n g s : h d l : / n p r e .

Why the sky is blue: discovering the color of life

Visual perception of colourful petals reminds us of classical fragments

http://precedings.nature.com/documents/1523/version/1

Visual perception of colourful petals reminds us of classical fragments

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