Pigment Grains Research Articles

Non-destructive pigment analysis of artefacts by Raman microscopy

When integrated with a microscope, Raman spectroscopy provides a uniquely specific and sensitive means of identifying pigment grains of sub-micrometre dimensions in a broad range of artefacts. This article reviews the methodology of Raman microscopy and its application to the study of mediaeval manuscripts.

The changed appearance of oil paintings due to increased transparency

Using the Kubelka and Munk formula (and i?- the absence of adequate experimentally determIned data, hypothetical values), the effect of ~ge-induced increase in the transparency of oil paint IS shown to produce distortions in the rendering of forms when the thickness of the paint and dark grounds or underpainting are the basis of the artist's technique. A commonly observed result of the increase in the transparency of oil paint with age is the effect it has on paintings with a dark ground or with a localized dark underlayer, the modelling being produced by controlling the paint thickness and the extent to which the dark is obscured. While the values in the thickly painted parts hardly change with time, the mid-tones produced by thinner applications of paint become darker and merge into the shadows. In extreme cases the modelling of forms, which is expected to be grad- ual, becomes sharper; smooth rounded forms tend towards two-tone renderings with sharp transitions from light to dark. The increased transparency is usually ex- plained in art technology literature in terms of an increase in the refractive index of the oil medium. Reflection at the interface between oil and pigment grains is lessened when the index for the oil (no) increases, becoming closer to that of the pigment (!II). The reflectance R is quoted as:

Nuclear microscopy of inhomogeneous thick samples

The long range of MeV protons in matter means that PIXE analysis of inhomogenous thick samples is subject to uncertainty if the elemental composition changes with depth. Samples which are particularly affected by this include rock and mineral samples and material of archaeological interest, such as pigments. In this study, elemental mapping of lead-tin yellow pigment grains using electron probe microanalysis (EPMA) is compared with PIXE using microfocused 2 and 3 MeV protons. It is found that, although well-defined grains can be seen using EPMA, which is sensitive only to a thin surface layer, the structure is lost using PIXE, because of Pb or Sn X-rays induced from deep within the sample. Simultaneous mapping using backscattered particles (BS) allows the grains to be seen clearly, and by taking different energy slices from the BS energy spectra the grains can be mapped at different depths below the surface. It is concluded that simultaneous BS analysis is essential when carrying out PIXE analysis of thick inhomogeneous samples to confirm that the sample is uniform at the point where the spectrum is taken. This is illustrated using a point analysis of a thin lead-tin grain overlying a region of organic material.

Functional aspects and eye organization of black and white morphs of the New Zealand beach beetle Chaerodes trachyscelides (Tenebrionidae)

The tenebrionid beetle Chaerodes trachyscelides occurs in two distinct colour varieties in New Zealand: dark beetles are found on black sandy beaches, white beetles on white sandy beaches. No differences were encountered between black and white beetles with regard to eye size, facet dimension, ommatidial number and gross anatomical organization. The 38 μm-thick cornea is biconvex with outer and inner radii of curvature being 35 and 12 μm, respectively, and the eye being of the acone, apposition type. There are 8 retinula cells per ommatidium (2 central, 6 peripheral). Each retinula cell is 88 μm long, of which 40 μm are occupied by the rhabdom. Eyes of both black and white beetles contain large amounts of electron-dense screening pigment grains in primary and secondary pigment cells as well as in the retinula cells, where they are distributed evenly from distal tip to basement membrane. Differences were found with regard to intracellular organelles and the organization of the rhabdom microvilli. Eyes of black beetles displayed greater signs of light- or darkness-induced microvillar disruptions, whereas eyes of white morphs contained subcellular structures and organelles indicative of increased metabolic activity. Both kinds of sand, but in particular the black one, strongly attenuate impinging light right across the entire spectrum within the uppermost few millimetres. Electroretinogramme recordings revealed the great absolute sensitivity of the beetles' eyes to white light when compared with that of a similarly tested fly and also showed a broad sensitivity peak in the bluegreen-green part of the spectrum. Secondary peaks were not obvious and flicker fusion frequency did not exceed 20 Hz. It is concluded that the dark-sand environment not only has led to the dark-coloured variety of beetle, but has also influenced the degree to which retinula cells tolerate light despite the fact that gross structural organization and eye morphology had remained seemingly unaffected.

<b>A STUDY OF UNUSUAL INTRACELLULAR ORGANELLES AND ULTRASTRUCTURAL ORGANISATION OF THE EYE OF </b><i><b>GAMMARUS OCEANICUS</b></i><b> (SEGERSTRALE 1947) FIXED IN THE MIDNIGHT SUN OF THE SPITSBERGEN (SVALBARD) </b><b>SUMMER </b>

The structural organization of the eye of Gammarus oceanicus conforms to that of other gammaridean species. Individual facets and a compartmentalization of the eye cuticle do not occur, though internally ommatidial groups are clearly separated from one another. The cones are bipartite; the rhabdoms are centrally fused and reach a maximum length and width of 30 μm and 10 μm, respectively. Four large retinula cells and one small retinula cell per ommatidium are developed with nuclei located below the fenestrated membrane. All eyes, though fixed at 0045, displayed the light-adapted condition with screening pigment granules surrounding the entire rhabdom and insulating the latter from the reflecting vesicles of auxiliary cells. With regard to quality and quantity of cell organelles, the retinula cells proved extraordinarily richly endowed: there were photic vesicles of 0.04 μm diameter along the edges of the rhabdom, multivesicular bodies with and without limiting membranes, two types of electron-dense spherical organelles (i.e. screening pigment grains and carotenoid-containing structures), mitochondria, microtubules, and unusual multilamellate bodies. The latter, surrounded by a membrane and associated with a grainy ground substance, are thought to be somehow related to the continuous light of the Arctic summer that G. oceanicus is subjected to. It is hypothesized that the unusual lamellate bodies are either involved in the task of meeting extra energy requirements during times of illumination or represent one stage in the conversion of shed microvillar material to residual bodies.

Single electrode studies on the retina of the butterflyPapilio

1. The butterfly retina exhibits strong interactions between photoreceptor responses recorded intracellularly. In general, the receptor which is locally giving the largest response suppresses the responses of neighbouring receptors that are less strongly stimulated. The effect enhances the differences between the primary photo-receptors and reduces responses to stimuli that excite all receptors together. 2. The interaction is explained in terms of a high extracellular resistance, so that receptor currents pass through other receptors in the opposite direction to those of their own responses. The result is that receptors are actively turned off by colours away from their own peak wavelength. 3. This effect applies most strongly to colour and to polarization plane when the stimulus is a point source on axis, and is therefore strong between the receptors of the same ommatidium. 4. The result is that spectral sensitivity peaks and angular sensitivity peaks are narrower, and polarisation sensitivity is greater, than expected from single retinula cells in isolation. The sensitivities measured electrophysiologically cannot be easily related to the physical properties of the visual pigments. Polarisation sensitivity (PS) can reach 50. 5. There are four types of primary photoreceptor, with peak near 380 nm, 450 nm, 550 nm and 610 nm. Cell marking usually reveals these as single retinula cells. Near the peak spectral sensitivity the responses are up to 60 mV positive-going, but away from the peak they can be negative-going. 6. Anatomically the retina ofPapilio has four distal, four proximal retinula cells, and a ninth basal cell. Narrow pigment cells and tracheoles squeeze through the substantial basement membrane along with each bundle of nine axons. 7. Two of the distal retinula cells contain red pigment grains near the rhabdom. The distal retinula cells are UV or blue sensitive. Green sensitive cells are proximal and can be coupled in opposite pairs. Red sensitive cells are proximal. 8. The UV sensitive cell with peak near 380 nm is the most sensitive of the cell types when measured by the position of theV/logI curve on the intensity axis at the spectral peak of each type. The red-sensitive cells are also sensitive. By its inhibitory effect, interaction between receptors reduces the sensitivity measurement on this scale. 9. Angular sensitivities measured with positive-going responses near the spectral peaks are narrow (Δρ-2°); when measured with negative-going responses they are wider (Δρ=3° to 5°). 10. One type of unit has only negative-going responses to −60 mV, with Δp=2° to 5°, spectral peak near 550 nm and sometimes also 380 nm or 450 nm. This type has not been marked and is regarded as a restricted channel for return current. ItsV/logI curve extends over an intensity range of 106. 11. The variety of the units suggests that their responses are not due to a simple regular network with all units connected indiscriminately to all others at all times through their terminals. There are selective channels for current flow and some retinula cells appear to be little influenced by others. 12. Theory shows that when there is a direct electrical coupling between a pair of retinula cells (not passing through the extracellular space) it is possible to balance out the negative interactions caused by current flow through their terminals. Far from degenerating the signals, direct electrical coupling can cancel the negative interaction, and this may be its normal function.

The effects of light and temperature on the structural organization of the eye of the Antarctic amphipod Orchomene plebs (Crustacea)

The Antarctic amphipod Orchomene plebs lives in an environment which is characterized by stable temperatures of — 2 to 0 °C, and low ambient light intensities. The microanatomy of the eye of dark adapted and light-adapted animals, kept at 0 °C, was studied by light and electron micro­scopy and compared with that of individuals kept at + 10 °C for 7 h. The eye of a 19 mm (total body length) specimen consists of approxi­mately 260 ommatidia, each measuring 40-50 μm across. The cornea is smooth and external facets are not developed. The crystalline cones, which are the intracellularly secreted products of two cone cells per ommatidium, possess cores that stain more intensely than their peripheral regions. The ultrastructure of the core reveals a regular lattice of 30 nm particles, which are interpreted as glycogen granules. The rhabdom, whose microvilli measure 85-100 nm in diameter, is spindle-shaped and of the centrally fused type. The ratio of rhabdom length: width is 3.2 in light- adapted and 2.8 in dark-adapted animals. There are five retinula cells per omma, one of them being consistently smaller than the rest. Spherical (0.4 μm) granules of screening pigment, present only in the five retinula cells, exhibit migrations that are not controlled by an internal rhythm but are the direct response to fluctuations in the ambient light intensity. In the light-adapted state the pigment grains form a sleeve around the entire rhabdom, but in the dark-adapted eye they cluster around the proximal ends of the cones. The nuclei of the retinula cells are found below the basement membrane, but those of the cells occupying the spaces between ommatidial groups are located above it. These ‘inter­stitial’ cells lack pigment granules, but contain many vesicular organelles, which in the dark-adapted state only resemble miniature sea-urchins of 0.4 μm diameter. Heat-stressed dark-adapted animals exhibit grotesquely deformed rhabdoms with microvilli whose membranes are severely disrupted. The position of the screening pigment is similar to that of light adapted material, but rhabdom dimensions as well as the contents of the inter­stitial cells are indicative of dark-adapted eyes. When heat-stressed ani­mals after a stay of 7 h at + 10 °C are returned to water of 0 °C and examined 7 h later, their photoreceptors (based on five eyes each) show a remarkable recovery: the visual membranes have reorganized, microvillus patterns have reformed and the rhabdom structure has regenerated. The screening pigments, too, have begun to migrate into the characteristic position of the dark-adapted condition. The experiments show that light and temperature cause separate but intricately related effects that must not be studied in isolation from each other.

The eyes of mesopelagic crustaceans

The compound eyes of the mesopelagic eupausiid Thysanopoda tricuspidata were investigated by light-, scanning-, and transmission electron microscopy. The eyes are spherical and have a diameter that corresponds to 1/6 of the carapace length. The hexagonal facets have strongly curved outer surfaces. Although there are four crystalline cone cells, only two participate in the formation of the cone, which is 90-120 micrometer long and appears to have a radial gradient of refractive index. The clear zone, separating dioptric structures and retinula, is only 90-120 micrometer wide. In it lie the very large oval nuclei of the seven retinula cells. Directly in front of the 70 micrometer long and 15 micrometer thick rhabdom a lens-like structure of 12 micrometer diameter is developed. This structure, known in only a very few arthropods, seems to be present in all species of Euphausiacea studied to date. It is believed that the rhabdom lens improves near-field vision and absolute light sensitivity. Rod-shaped pigment grains and mitochondria of the tubular type are found in the plasma of retinula cells. The position of the proximal screening pigment as well as the microvillar organization in the rhabdom are indicative of light-adapted material. The orthogonal alignment of rhabdovilli suggests polarization sensitivity. Behind each rabdom there is a cup-shaped homogenous structure of unknown, but possibly optical function. Finally, the structure and the function of the euphysiid eye are reviewed and the functional implications of individual components are discussed.

The ommatidium of the lacewing Chrysopa (Neuroptera).

The eye is a clear zone eye with extensive movement of retinula cells on adaptation to light. The ommatidium has three types of rhabdomere, at different levels, so that the eye necessarily abstracts at least three kinds of information simultaneously from the incoming rays. In the lightadapted state light can enter each ommatidium only via a crystalline tract that is surrounded by dense pigment grains. A small distal rhabdomere (cell 7) always lies at the end of this tract. In the dark-adapted eye the retinula cell nuclei and distal rhabdomere move to the cone tip and the crystalline tract is drawn into the cone. There is then a region of the retinula cell column, between cone tip and proximal rhabdoms, across which there is no structure that could act as a light guide. A key question, therefore, is how the light is focused across this clear zone in the darkadapted state. As shown by the wide angular distribution of eyeshine when a parallel beam is incident on the dark-adapted eye, rays are poorly focused upon the columns of the large rhabdoms. The wide visual fields of receptors 1-6 in the dark-adapted eye, inferred from the observation of eyeshine, are seen as a way of narrowing the bandwidth of spatial frequencies, so that only the largest objects in the visual field contribute to motion-detection. This would improve the signal-to-noise ratio, not in the receptors themselves, but in the neural mechanism, by simplifying the incoming signal.

Unit studies on the retina of dragonflies

The dorsal part of the adult eye contains mainly uniform blue-sensitive receptors with peak near 380 nm in Anax and near 410 nm in Libellula. This is not a simple rhodopsin type of curve. 2. The larval eye and the ventral part of the adult eye contain receptors with a wide range of spectral sensitivity curves, with peaks from 420–520 nm in Anax and 450–550 nm in Libellula. Many of these can be derived from a typical rhodopsin curve when allowance is made for self-absorption in very long receptors. 3. For most adult receptors, rotation of the plane of polarization by 90° from the optimum is equivalent to a decrease of only 60–70 % in intensity. 4. The acceptance angle curve is approximately a Gaussian normal curve with width of 1.0–1.8° at linear half height. A few units have complex fields which may be artefacts. 5. Retinula unit responses in larval compound eyes resemble those of the adult but the larval units are slower in response and usually more sensitive to rotation of the plane of polarization. 6. There are eight retinula cells arranged in two tiers in each ommatidium. 7. In the dark, pigment grains move away from the tip of the cone and the palisade around the rhabdom increases. These effects could increase the sensitivity by a change in the anatomy. 8. In an eye of this type the ERG is an unsatisfactory tool, the angles of acceptance must be measured very carefully, and experiments with a single electrode have only a limited interpretation.

The Action of Atebrin on Bird Malaria

1. Atebrin given during the incubation period delays the appearance of parasites in the blood of birds infected by direct blood inoculation with Plasmodium relictum.2. If given to birds infected by the bites of mosquitoes it does not retard the appearance of parasites in the blood, when given either before, or after, the infective bites, but it has some action in diminishing the severity of the attack and the degree of splenic enlargement.3. Parasites appear in the blood of birds, infected by sporozoites, actually during the course of atebrin treatment if this is prolonged beyond the normal incubation period.4. When asexual parasites appear in the blood of mosquito-infected birds after atebrin treatment they are abnormal in appearance and devoid of pigment grains; gametocytes are rarely seen in the blood of such birds, but if present they have normal pigment granules.5. Treatment with atebrin produces peculiar bodies in the blood cells of canaries.