High-Performance Polarization Microscopy Reveals Structural Remodeling in Rat Calcaneal Tendons Cultivated In Vitro.

Collagenous tissues exhibit anisotropic optical properties such as birefringence and linear dichroism (LD) as a result of their structurally oriented supraorganization from the nanometer level to the collagen bundle scale. Changes in macromolecular order and in aggregational states can be evaluated in tendon collagen bundles using polarization microscopy. Because there are no reports on the status of the macromolecular organization in tendon explants, the objective of this work was to evaluate the birefringence and LD characteristics of collagen bundles in rat calcaneal tendons cultivated in vitro on substrates that differ in their mechanical stiffness (plastic vs. glass) while accompanying the expected occurrence of cell migration from these structures. Tendon explants from adult male Wistar rats were cultivated for 8 and 12 days on borosilicate glass coverslips (n = 3) and on nonpyrogenic polystyrene plastic dishes (n = 4) and were compared with tendons not cultivated in vitro (n = 3). Birefringence was investigated in unstained tendon sections using high-performance polarization microscopy and image analysis. LD was studied under polarized light in tendon sections stained with the dichroic dyes Ponceau SS and toluidine blue at pH 4.0 to evaluate the orientation of proteins and acid glycosaminoglycans (GAG) macromolecules, respectively. Structural remodeling characterized by the reduction in the macromolecular orientation, aggregation and alignment of collagen bundles, based on decreased average gray values concerned with birefringence intensity, LD and morphological changes, was detected especially in the tendon explants cultivated on the plastic substrate. These changes may have facilitated cell migration from the lateral regions of the explants to the substrates, an event that was observed earlier and more intensely upon tissue cultivation on the plastic substrate. The axial alignment of the migrating cells relative to the explant, which occurred with increased cultivation times, may be due to the mechanosensitive nature of the tenocytes. Collagen fibers possibly played a role as a signal source to cells, a hypothesis that requires further investigation, including studies on the dynamics of cell membrane receptors and cytoskeletal organization, and collagen shearing electrical properties.

Toluidine blue staining for cell and tissue biology applications.

Toluidine blue (TB) staining either alone or in association with other methodologies has the potential to answer a variety of biological questions regarding the human, animal and plant tissues or cells. In this brief review, we not only report the primary use of TB to detect the anionic substrates and availability of their binding sites, but also unveil the resulting applications of TB staining in biological research. Among these applications, the uses of TB staining to identify the changes in chromatin DNA-protein complexes, nucleolus location, and extracellular matrix proteoglycan complexes associated with different physiological and pathological events are described. The usefulness of TB staining to monitor the effects elicited by environmental insults on chromatin and intercalation of drugs into the DNA is also included.

Polarization Microscopy and Infrared Microspectroscopy of Integument Coverings of Diapausing Larvae in Two Distantly Related Nonsocial Bees.

The larvae of the two distantly related nonsocial bees Ericrocis lata (Apidae) and Hesperapis (Carinapis) rhodocerata (Melittidae), which develop mostly under arid desert areas of North America, and that differ in that they either spin (E. lata) or do not spin (H. rhodocerata) protective cocoons before entering diapause, produce transparent films that cover the larval integument. To understand the nature of these films, their responses to topochemical tests and their characteristics when examined with fluorescence and high-performance polarization microscopy and microspectroscopy were studied. A positive staining by Sudan black B, birefringence of negative sign, and a Fourier transform-infrared (FT-IR) spectrum typical of lipids were detected for the integument covering of both species. The FT-IR signature, particularly, suggests a wax chemical composition for these lipid coverings, resembling the waxes that are used as construction materials in the honey cells produced by social bees. Considering the arid environmental conditions under which these larvae develop, we hypothesize that their covering films may have evolved as protection against water depletion. This hypothesis seems especially appropriate for H. rhodocerata larvae, which are capable of undergoing a long diapause period in the absence of a protective cocoon.

Chiral supramolecular order revealed during the formation of calf thymus and phage DNA crystals.

The control of DNA packaging has been reported to be dependent on an ordered liquid-crystalline state. However, the textural characteristics that are typical of crystals and that resemble mesophases have not been reported for highly polymerized or even shorter types of DNA filaments under in vitro conditions that favor crystallization. Because DNA crystals are expected to exhibit particular textural optical anisotropies, pure and highly polymerized calf thymus DNA and simpler λ phage DNA were crystallized from solution drops and were analyzed using high-performance polarization microscopy with and without differential interference contrast (DIC) optics. Both types of DNA formed crystals that exhibited chiral supramolecular textures resembling the twist-grain boundary (TGB) columnar mesophases described for liquid crystals and exhibited intrinsic negative birefringence. To the best of our knowledge, this is the first observation using polarization/interference optics of pure DNA crystals that have TGB columnar mesophase-like textural characteristics. A comparison of the crystals formed from the highly polymerized calf thymus DNA and those formed from the shorter phage DNA strands revealed textural differences. Compared to the phage DNA crystals, the crystals of highly polymerized thymus DNA exhibited a more intertwisted columnar distribution and a fibrous texture between their columnar structures. In addition, a form birefringence phenomenon was detected only in the thymus DNA crystals. These characteristics are presumed to reflect the higher level of supramolecular order, self-assembly and chirality in highly polymerized calf thymus DNA crystals relative to that of crystals formed from the simpler, shorter, λ phage DNA. The higher-order supramolecular organization revealed here for in vitro DNA preparations raises the possibility that this structure could also occur, possibly to a smaller degree, during DNA self-aggregation under specific in vivo conditions. Whether the DNA crystal properties presently described play a role in the establishment of higher-order levels of hierarchical chromatin structure and consequently in chromatin physiology, should be further investigated.

The Feulgen reaction: A brief review and new perspectives.

The Feulgen reaction has been proposed by Robert Feulgen and Heinrich Rossenbeck for the identification of DNA nearly a hundred years ago. Since then, many other applications of this cytochemical/topochemical procedure at qualitative and quantitative level have been proposed in relation to DNA and its role in chromatin in human, animal and plant cells. In this article, we briefly review some fundamental aspects of the Feulgen reaction and current applications of such a method in studies of altered chromatin texture, including its association with or preceding changes in transcriptional activities and effect on epigenetic marks. Further perspectives on the use of the Feulgen reaction will depend of the proposal of innovative biological questions in which its reveals appropriate.

DNA Methylation Changes in Valproic Acid-Treated HeLa Cells as Assessed by Image Analysis, Immunofluorescence and Vibrational Microspectroscopy.

Valproic acid (VPA), a well-known histone deacetylase inhibitor, has been reported to affect the DNA methylation status in addition to inducing histone hyperacetylation in several cell types. In HeLa cells, VPA promotes histone acetylation and chromatin remodeling. However, DNA demethylation was not checked in this cell model for standing effects longer than those provided by histone acetylation, which is a rapid and transient phenomenon. Demonstration of VPA-induced DNA demethylation in HeLa cells would contribute to understanding the effect of VPA on an aggressive tumor cell line. In the present work, DNA demethylation in VPA-treated HeLa cells was assessed by image analysis of chromatin texture, the abundance of 5-methylcytosine (5mC) immunofluorescence signals and Fourier transform-infrared (FT-IR) microspectroscopy centered on spectral regions related to the vibration of-CH3 groups. Image analysis indicated that increased chromatin unpacking promoted by a 4-h-treatment with 1.0 mM VPA persisted for 24 h in the absence of the drug, suggesting the occurrence of DNA demethylation that was confirmed by decreased 5mC immunofluorescence signals. FT-IR spectra of DNA samples from 1 mM or 20 mM VPA-treated cells subjected to a peak fitting analysis of the spectral window for-CH3 stretching vibrations showed decreased vibrations and energy of these groups as a function of the decreased abundance of 5mC induced by increased VPA concentrations. Only the 20 mM-VPA treatment caused an increase in the ratio of -CH3 bending vibrations evaluated at 1375 cm-1 in relation to in-plane vibrations of overall cytosines evaluated at 1492 cm-1. CH3 stretching vibrations showed to be more sensitive than-CH3 bending vibrations, as detected with FT-IR microspectroscopy, for studies aiming to associate vibrational spectroscopy and changes in DNA 5mC abundance.

Topochemistry, optical anisotropy and FT-IR microspectroscopy of the cocoon of Lithurgus chrysurus (Hymenoptera, Megachilidae).

A previous study has not revealed the participation of a mucous component in the cocoon wall of the solitary bee, Lithurgus chrysurus, differing from the cocoon structure reported for many other bee species. However, uncertainty remains, because only the median and rear zones of this cocoon type have thus far been analyzed. Here, we studied the front zone of this cocoon, searching its components and their organization, to fill this knowledge gap. Topochemical assays, polarization microscopy and Fourier transform-infrared (FT-IR) microspectroscopy were used to study cross sections from L. chrysurus cocoon. Three main layers differing in structural organization were found to compose the cocoon wall. Silk fibroins were assumed to constitute the filamentous threads of the inner and outer layers and the laminar structure of the intermediate layer. Deduced from its topochemical properties and FT-IR spectral signature, a foamy material containing mucin glycoproteins and carboxylated acid glycosaminoglycans was found in the intermediate layer. FT-IR analysis using a Savitzky-Golay 2nd-derivative and absence of linear dichroism and birefringence phenomena suggest that a random-coil secondary structure predominates in the foam component. Co-existence of α-helical and ß-sheet conformations is also hypothesized for the fibroin component of this cocoon. It is thus concluded that in addition to fibroin elements, a mucous component, likely contributed by a Malpighian tubule secretion, integrates the composition of the front zone of the cocoon wall of L. chrysurus. In addition, the FT-IR analysis of the inner layer silk of this cocoon suggests significant differences in comparison to the silk fibroins of the silkworm, and some minor spectral differences in comparison to published data on the honeybee silk, with respect to protein secondary structure.

FT-IR Microspectroscopy of Rat Ear Cartilage.

Rat ear cartilage was studied using Fourier transform-infrared (FT-IR) microspectroscopy to expand the current knowledge which has been established for relatively more complex cartilage types. Comparison of the FT-IR spectra of the ear cartilage extracellular matrix (ECM) with published data on articular cartilage, collagen II and 4-chondroitin-sulfate standards, as well as of collagen type I-containing dermal collagen bundles (CBs) with collagen type II, was performed. Ear cartilage ECM glycosaminoglycans (GAGs) were revealed histochemically and as a reduction in ECM FT-IR spectral band heights (1140-820 cm-1) after testicular hyaluronidase digestion. Although ear cartilage is less complex than articular cartilage, it contains ECM components with a macromolecular orientation as revealed using polarization microscopy. Collagen type II and GAGs, which play a structural role in the stereo-arrangement of the ear cartilage, contribute to its FT-IR spectrum. Similar to articular cartilage, ear cartilage showed that proteoglycans add a contribution to the collagen amide I spectral region, a finding that does not recommend this region for collagen type II quantification purposes. In contrast to articular cartilage, the symmetric stretching vibration of -SO3- groups at 1064 cm-1 appeared under-represented in the FT-IR spectral profile of ear cartilage. Because the band corresponding to the asymmetric stretching vibration of -SO3- groups (1236-1225 cm-1) overlapped with that of amide III bands, it is not recommended for evaluation of the -SO3- contribution to the FT-IR spectrum of the ear cartilage ECM. Instead, a peak (or shoulder) at 1027-1016 cm-1 could be better considered for this intent. Amide I/amide II ratios as calculated here and data from the literature suggest that protein complexes of the ear cartilage ECM are arranged with a lower helical conformation compared to pure collagen II. The present results could motivate further studies on this tissue under pathological or experimental states involving ear cartilage.

Optical anisotropy reveals molecular order in a mouse enthesis.

Entheses are specialized biological structures that functionally anchor tendons to bones. The complexity, mechanical characteristics and properties of the entheses, particularly those related to exercise, mechanical load and pathologies, have been extensively analyzed; however, the macromolecular organization of the enthesis fibers, as assessed by polarization microscopy, has not yet been investigated. Morphological and optical anisotropy characteristics, such as birefringence, linear dichroism (LD) and differential interference contrast (DIC-PLM) properties, are thus analyzed in this study of a healthy adult mouse calcaneal tendon-bone enthesis. The molecular and supramolecular order of collagen and GAGs was determined for the collagen bundles of this enthesis. Based on a birefringence plot pattern as well as on metachromasy and linear dichroism after toluidine blue staining at pH 4.0, a similarity between the calcaneal tendon-bone enthesis and cartilage during ossification may be assumed. This similarity is assumed to favor the adequacy of this enthesis to support a compressive load. Considering that the collagen-proteoglycan complexes and the enthesis fibers themselves have a chiral nature, these structures could be acting via reciprocal signaling with the cellular environment of the enthesis.

Fluorescence, aggregation properties and FT-IR microspectroscopy of elastin and collagen fibers.

Histological and histochemical observations support the hypothesis that collagen fibers can link to elastic fibers. However, the resulting organization of elastin and collagen type complexes and differences between these materials in terms of macromolecular orientation and frequencies of their chemical vibrational groups have not yet been solved. This study aimed to investigate the macromolecular organization of pure elastin, collagen type I and elastin-collagen complexes using polarized light DIC-microscopy. Additionally, differences and similarities between pure elastin and collagen bundles (CB) were investigated by Fourier transform-infrared (FT-IR) microspectroscopy. Although elastin exhibited a faint birefringence, the elastin-collagen complex aggregates formed in solution exhibited a deep birefringence and formation of an ordered-supramolecular complex typical of collagen chiral structure. The FT-IR study revealed elastin and CB peptide NH groups involved in different types of H-bonding. More energy is absorbed in the vibrational transitions corresponding to CH, CH2 and CH3 groups (probably associated with the hydrophobicity demonstrated by 8-anilino-1-naphtalene sulfonic acid sodium salt [ANS] fluorescence), and to νCN, δNH and ωCH2 groups of elastin compared to CB. It is assumed that the α-helix contribution to the pure elastin amide I profile is 46.8%, whereas that of the B-sheet is 20% and that unordered structures contribute to the remaining percentage. An FT-IR profile library reveals that the elastin signature within the 1360-1189cm(-1) spectral range resembles that of Conex-Toray aramid fibers.

Effect of Aloe vera application on the content and molecular arrangement of glycosaminoglycans during calcaneal tendon healing.

Although several treatments for tendon lesions have been proposed, successful tendon repair remains a great challenge for orthopedics, especially considering the high incidence of re-rupture of injured tendons. Our aim was to evaluate the pharmacological potential of Aloe vera on the content and arrangement of glycosaminoglycans (GAGs) during tendon healing, which was based on the effectiveness of A. vera on collagen organization previously observed by our group. In rats, a partial calcaneal tendon transection was performed with subsequent topical A. vera application at the injury site. The tendons were treated with A. vera ointment for 7 days and excised on the 7(th) , 14(th) , or 21(st) day post-surgery. Control rats received ointment without A. vera. A higher content of GAGs and a lower amount of dermatan sulfate were detected in the A. vera-treated group on the 14(th) day compared with the control. Also at 14 days post-surgery, a lower dichroic ratio in toluidine blue stained sections was observed in A. vera-treated tendons compared with the control. No differences were observed in the chondroitin-6-sulfate and TGF-ß1 levels between the groups, and higher amount of non-collagenous proteins was detected in the A. vera-treated group on the 21(st) day, compared with the control group. No differences were observed in the number of fibroblasts, inflammatory cells and blood vessels between the groups. The application of A. vera during tendon healing modified the arrangement of GAGs and increased the content of GAGs and non-collagenous proteins.

Changes in liver cell DNA methylation status in diabetic mice affect its FT-IR characteristics.

BACKGROUND: Lower levels of cytosine methylation have been found in the liver cell DNA from non-obese diabetic (NOD) mice under hyperglycemic conditions. Because the Fourier transform-infrared (FT-IR) profiles of dry DNA samples are differently affected by DNA base composition, single-stranded form and histone binding, it is expected that the methylation status in the DNA could also affect its FT-IR profile. METHODOLOGY/PRINCIPAL FINDINGS: The DNA FT-IR signatures obtained from the liver cell nuclei of hyperglycemic and normoglycemic NOD mice of the same age were compared. Dried DNA samples were examined in an IR microspectroscope equipped with an all-reflecting objective (ARO) and adequate software. CONCLUSIONS/SIGNIFICANCE: Changes in DNA cytosine methylation levels induced by hyperglycemia in mouse liver cells produced changes in the respective DNA FT-IR profiles, revealing modifications to the vibrational intensities and frequencies of several chemical markers, including νas -CH3 stretching vibrations in the 5-methylcytosine methyl group. A smaller band area reflecting lower energy absorbed in the DNA was found in the hyperglycemic mice and assumed to be related to the lower levels of -CH3 groups. Other spectral differences were found at 1700-1500 cm(-1) and in the fingerprint region, and a slight change in the DNA conformation at the lower DNA methylation levels was suggested for the hyperglycemic mice. The changes that affect cytosine methylation levels certainly affect the DNA-protein interactions and, consequently, gene expression in liver cells from the hyperglycemic NOD mice.

Polarization microscopy of extended chromatin fibers.

Analysis of the formation of extended chromatin fibers (ECFs) in response to the action of gravity following lysis by hypertonic and detergent solutions is a useful technical procedure relevant for studies of the positioning of particular DNA signals on chromatin filaments. Additionally, if toluidine blue molecules are allowed to bind electrostatically to available DNA phosphates on ECFs, the birefringence brightness generated in these filaments, as observed by polarization microscopy, facilitates the description of the frequency of ECF formation and extension of the chromatin filaments generated. Thus, different patterns of DNA-nuclear matrix protein associations related to varying transcriptional activities and chromatin organization in isolated cells can be assessed. A technique for producing ECFs in different isolated cell types under variable physiological and/or pathological conditions is detailed in this chapter.

Image analysis of chromatin remodelling.

Chromatin packaging plays a significant role in regulating gene transcription. Study of the higher-order packing states of chromatin by image analysis at the light microscope level, especially when validated by methods of molecular biology, immunochemistry, and/or immunocytochemistry, enabled the detection of changes involved in the processes associated with or preceding alterations in transcriptional activities. Here, we recommend and describe the use of relatively simple methods for staining and detecting chromatin remodelling by image analysis.

Highly purified collagen coating enhances tissue adherence and integration properties of monofilament polypropylene meshes.

INTRODUCTION AND HYPOTHESIS: Complications related to tissue integration of polypropylene implants used in the treatment of pelvic organ prolapse are relatively prevalent. Collagen, a biocompatible, less immunogenic material with modulating properties on the inflammatory process, may improve polypropylene integration. The objective was to study biomechanical and histological effects of monofilament polypropylene mesh coated with purified collagen gel. METHODS: Forty rats were implanted with two fragments of polypropylene mesh in their abdominal walls (one on each side of the linea alba). One of the fragments had a collagen gel coating (group I) while the other one did not (group II). The animals were euthanized at 7, 14, 90, and 180 days after implantation and their abdominal walls were excised for analysis. RESULTS: The biomechanical study showed that mesh adherence to neighboring tissue increased significantly in group II (p < 0.05). Acute (p < 0.001) and chronic (p = 0.004) inflammatory responses as well as granulation tissue formation (p = 0.001) were less intense in group II at 7 and 14 days. Granulomatous inflammation and foreign body reaction was less significant at 7 days in group II (p = 0.029 and p < 0.001). The birefringence analysis showed higher mean brightness density in the late phase of implantation in group II meshes (p = 0.000). CONCLUSION: Polypropylene mesh coated with purified collagen gel increases adherence to tissue, promotes a less intense and lasting inflammatory response and triggers a greater organization and packing arrangement of collagen fibers in the late phase of implantation.

Using the FT-IR linear dichroism method for molecular order determination of tendon collagen bundles and nylon 6.

Polarized Fourier transform-infrared (FT-IR) was used to compare the orientation of vibrational chemical groups of bovine tendon collagen bundles (CBs) to that of nylon 6, a simpler polyamide model, in terms of linear dichroism (LD). Subtraction of spectral profiles identified the most significant differences regarding the amide regions. At 1630cm(-1), the CBs displayed higher peak areas and absorbance when positioned perpendicularly (A⊥) to the plane of polarized light, in comparison with nylon 6. In contrast, at the 1526cm(-1) amide II spectral region the inverse occurred. In the amide III region (1232cm(-1)), the LD was positive and higher for CBs. Dichroic ratios (DR=A||/A⊥) calculated from the average of ten measured spectra for CBs and nylon 6 revealed that the values for CBs were <1.0 in the 3298-1655cm(-1) wavenumber range and >1.0 in the 1536-1234cm(-1) wavenumber range. From 1535 to 1120cm(-1), nylon 6 displayed DR values higher than those of CBs. The LD band integrated areas were higher in CBs than in nylon 6. The LD differences between CBs and nylon 6 are probably due to a more complex chemical composition and supramolecular oriented architecture in CBs in comparison to nylon 6.

Contribution of AT-, GC-, and methylated cytidine-rich DNA to chromatin composition in Malpighian tubule cell nuclei of Panstrongylus megistus (Hemiptera, Reduviidae).

The Malpighian tubule cell nuclei of male Panstrongylus megistus, a vector of Chagas disease, contain one chromocenter, which is composed solely of the Y chromosome. Considering that different chromosomes contribute to the composition of chromocenters in different triatomini species, the aim of this study was to determine the contribution of AT-, GC-, and methylated cytidine-rich DNA in the chromocenter as well as in euchromatin of Malpighian tubule cell nuclei of P. megistus in comparison with published data for Triatoma infestans. Staining with 4',6-diamidino-2-phenylindole/actinomycin D and chromomycin A(3)/distamycin, immunodetection of 5-methylcytidine and AgNOR test were used. The results revealed AT-rich/GC-poor DNA in the male chromocenter, but equally distributed AT and GC DNA sequences in male and female euchromatin, like in T. infestans. Accumulation of argyrophilic proteins encircling the chromocenter did not always correlate with that of GC-rich DNA. Methylated DNA identified by immunodetection was found sparsely distributed in the euchromatin of both sexes and at some points around the chromocenter edge, but it could not be considered responsible for chromatin condensation in the chromocenter, like in T. infestans. However, unlike in T. infestans, no correlation between the chromocenter AT-rich DNA and nucleolus organizing region (NOR) DNA was found in P. megistus.

Biochemical and anisotropical properties of tendons.

Tendons are formed by dense connective tissue composed of an abundant extracellular matrix (ECM) that is constituted mainly of collagen molecules, which are organized into fibrils, fibers, fiber bundles and fascicles helicoidally arranged along the largest axis of the tendon. The biomechanical properties of tendons are directly related to the organization of the collagen molecules that aggregate to become a super-twisted cord. In addition to collagen, the ECM of tendons is composed of non-fibrillar components, such as proteoglycans and non-collagenous glycoproteins. The capacity of tendons to resist mechanical stress is directly related to the structural organization of the ECM. Collagen is a biopolymer and presents optical anisotropies, such as birefringence and linear dichroism, that are important optical properties in the characterization of the supramolecular organization of the fibers. The objective of this study was to present a review of the composition and organization of the ECM of tendons and to highlight the importance of the anisotropic optical properties in the study of alterations in the ECM.

Analysis of the deep digital flexor tendon in rats submitted to stretching after immobilization.

Few studies have analyzed the effect of stretching after immobilization on the structural and biochemical properties of tendons. Here, the effect of stretching and immobilization on the proximal (p), intermediate (i), and distal (d) regions of the deep digital flexor tendon in rats was analyzed. The d region was subjected to compression and tension forces, the i region was subjected to compressive forces and the p region received tension forces. Rats were separated into five groups: GI--control for GII; GII--immobilized rats; GIII--control for GIV and GV groups; GIV--immobilized and stretched rats; and GV--immobilized rats which were allowed free cage activity. GII showed a higher molecular organization in the d and p regions as detected by measuring optical retardation, a lower concentration of hydroxyproline in the i region and a significant decrease in noncollagenous proteins found in the three regions of the tendon. Regarding the glycosaminoglycans, diminishing dermatan sulfate and the absence of chondroitin sulfate in the i region were observed in GII when compared to GI. However, in the same region of GIV, higher concentrations of chondroitin and dermatan sulfate were observed along with a strong metachromasy. An increase in hydroxyproline content in the i region and a higher molecular organization in the d and p regions were observed in GIV. Apparently, the active isoforms of metalloproteinase-2 also increased after stretching in all regions. These results suggest that stretching after immobilization contributed to the increase in molecular organization and to the synthesis of extracellular matrix components.

Butterfly scale form birefringence related to photonics.

Wings of the butterflies Morpho aega and Eryphanis reevesi were investigated in the present study by fluorescence, polarization and infra-red (IR) spectroscopic microscopy with the aim of identifying the oriented organization of their components and morphological details of their substructures. These wings were found to exhibit a strong iridescent glow depending on the angle of the incident light; their isolated scales exhibited blue fluorescence. Parallel columns or ridges extend from the pad and sockets to the dented apical scale's region, and they are perpendicular to the ribs that connect the columnar ridges. The scales reveal linear dichroism (LD) visually, when attached on the wing matrix or isolated on slides. The LD was inferred to be textural and positive and was also demonstrated with IR microscopy. The scale columns and ribs are birefringent structures. Images obtained before and after birefringence compensation allowed a detailed study of the scale morphology. Form and intrinsic birefringence findings here estimated and discussed in the context of nonlinear optical properties, bring to the level of morphology the state of molecular order and periodicity of the wing structure. FT-IR absorption peaks were found at wavenumbers which correspond to symmetric and asymmetric (-N-H) stretching, symmetric (-C-H) stretching, amide I (-CO) stretching, amide II(-N-H), and ß-linking. Based on LD results obtained with polarized IR the molecular vibrations of the wing scales of M. aega and E. reevesi are assumed to be oriented with respect to the long axis of these structures.