Mariana De Niz

Gavin R Meehan

Nicolas M B Brancucci

Matthias Marti

Brice Rotureau

Luisa M Figueiredo

Friedrich Frischknecht

Intravital imaging of host-parasite interactions in skin and adipose tissues

Intravital microscopy (IVM) allows the visualization of how pathogens interact with host cells and tissues in living animals in real time. This method has enabled key advances in our understanding of host-parasite interactions under physiological conditions. A combination of genetics, microscopy techniques and image analysis have recently facilitated the understanding of biological phenomena in living animals at cellular and sub-cellular resolution. In this review, we summarize findings achieved by intravital microscopy of the skin and adipose tissues upon infection with various parasites and we present a view into possible future applications of this method.

Introduction

Over the course of almost a century, the implantation of imaging windows on animals for microscopic observation has been widespread, and within this time frame it has evolved significantly. Now coined "intravital microscopy" (IVM) the technique of using optical windows to visualize phenomena at cellular or sub-cellular resolution has come a long way since its first use in 1824, when it was applied to visualize the rolling of leukocytes on the vascular endothelium of living frogs [START_REF] Dutrochet | Recherches anatomiques et physiologique sur la structure intime des animaux et des végétaux et sur leur motilité[END_REF][START_REF] Wagner | Erlauterungstafeln zur Physiologie und Entwicklungsgeschichte[END_REF]. These studies, and an additional series of first discoveries were key for understanding endothelial physiology. Various animal models (including worms, fish, insects, amphibians, reptiles, and mammals) have been imaged since, by IVM. In parallel, a series of technological breakthroughs over the past few decades in the field of physics, optics, and genetics have transformed IVM from an exotic tool to a commonly used platform to dissect processes of health and disease in living animals. Advances in image analysis have also transformed the use of IVM from mostly qualitative to yield quantitative results (reviewed by [START_REF] Coombes | Dynamic imaging of host-pathogen interactions in vivo[END_REF]).

In parasitology, even though animal models exist for a considerable range of parasites, many questions remain understudied in vivo. In this review, we present findings that have been achieved by IVM focusing on vector-borne parasites. Furthermore, we provide insights into the optimized methodology, surgical procedures, imaging, and anaesthetic techniques that have made these findings possible. We aim to convey the relevance of IVM to the parasitology community, given the significant findings it has already allowed. Equally importantly, it has allowed us to reassess ideas and questions proposed by in vitro studies, which were not possible to observe in vivo, or not possible to quantify. With current technology, we have been able to re-visit these questions, and in many cases find unexpected features relevant to pathology, or cell biology. The use of animals and live imaging in particular, has been extremely valuable for the parasitology field in the absence of relevant in vitro or in silico models that can faithfully reproduce host-parasite interactions of clinical relevance to human health.

Biological relevance of the skin and adipose tissue for parasitology

Parasitic diseases are transmitted in various ways, including through ingestion of contaminated water, food, or soil; sexual contact; transcutaneous penetration, or bites of infected vectors. Vector-borne parasites introduced into the skin during a bite include Plasmodium spp., Wuchereria and Brugia spp. (filarial worms) transmitted by mosquitoes; Trypanosoma brucei spp. transmitted by tsetse flies; Onchocerca volvulus transmitted by Simulium (black) flies; and Leishmania spp. transmitted by sandflies. Other parasites entering the host skin by routes other than vector bites include Trypanosoma cruzi, transmitted in triatome faeces or urine, Schistosoma spp. where the swimming cercariae can actively penetrate the skin, and Dracunculus spp. (Guinea worms) which is ingested orally, but subsequently invades sub-cutaneous tissues, from where it slowly egresses upon contact with water. However, the skin is much more than a route of entry into the host, as most parasites spend at least part of their existence there and often initiate a first host response.

The skin can also serve as an anatomical reservoir of parasites and is a recurring theme in arthropod-borne human diseases, probably because skin invasion for enhanced transmission to the next host is likely a powerful evolutionary force.

The skin is the largest organ of the human body, and it represents the first line of immunological defence against many infections, with extensive crosstalk between epithelial, stromal, and immune cells to ensure homeostasis. Most parasites have developed mechanisms to evade detection and successfully establish an infection either in the skin itself, or elsewhere in the host. Anatomically, the skin can be divided into three distinct compartments: the epidermis, which is an avascular layer mostly composed of keratinocytes and Langerhans cells; the dermis, which is highly perfused by blood and draining lymphatic vessels (Figure 1) and the sub-cutaneous adipose tissue. The structure of the skin provides an interface between the vascular and lymphatic circulations, as well as the interstitial space. The latter is a fluid-filled anatomical compartment defined by a complex lattice of collagen bundles, found within and between tissues including the dermis [START_REF] Benias | Structure and distribution of an unrecognized interstitium in human tissues[END_REF]. Until recently, the physiological importance and extent of the interstitium had been largely understudied, yet this compartment is very likely to be of relevance for host-pathogen interactions defining phenomena such as extravasation and sequestration of different parasites.

Direct in vivo and in situ imaging of the skin has facilitated the understanding and identification of host and pathogen factors relevant for effective parasite transmission upon vector biting (or parasite traversal itself, as is the case of eg. Schistosoma spp.). They have also allowed the study of general aspects of parasite development including proliferation, migration, and interactions with the host immune system.

A tissue that together with the skin has gradually gained momentum and interest for its relevance as a parasite reservoir is the adipose tissue (reviewed by [START_REF] Tanowitz | Adipose tissue -a safe haven for parasites?[END_REF]), although its exact function (s) have not yet been established. In various fields of research, the adipose tissue went from being regarded as an inert site of energy storage or 'fat', to being considered now as a complex, multi-component site of paramount relevance to metabolism and systemic immunity (reviewed by [START_REF] Tilg | Adipocytokines: mediators linking adipose tissue, inflammation and immunity[END_REF]). This importance has permeated into parasitology and it is possible that the adipose tissue modulates/impacts on parasite biology. It might serve as a source of nutrients, allow modulation of immunity and pathology, and/or support transmission (in the case of subcutaneous adipose tissue). Coincident with the change in conception of the relevance of the adipose tissues, observations of some parasites at this anatomical site were already reported decades ago but very few functional, dynamic, or IVM studies were pursued, or reported until very recently. The potential of IVM to this tissue is further discussed in this review.

Optical windows and direct imaging: Intravital microscopy of the skin and adipose tissue IVM of the mouse skin includes non-invasive methods such as imaging of the ear-pinna, foot-pad (Figure 2A and2B) or tail; semi-invasive methods such as the dorsal skinfold chamber (Figure 2C) or invasive methods such as the skin flap (Figure 2D). Additionally, a relatively less explored location which has been successfully imaged by IVM is the skin flank (Figure 2E). The advantages and limitations of all methods are summarized in table 1. Skin at distinct anatomical sites exhibits important differences in, for example, thickness, composition, vascularization and cytokine profiles. In recent years, several groups have investigated the relevance of cellular heterogeneity across skin sites with tools including IVM [START_REF] Driskell | Distinct fibroblast lineages determine dermal architecture in skin development and repair[END_REF][START_REF] Jain | Shedding light on cutaneous innate immune responses: the intravital microscopy approach[END_REF][START_REF] Tong | The Skin Immune Atlas: Three-Dimensional Analysis of Cutaneous Leukocyte Subsets by Multiphoton Microscopy[END_REF]. This led the generation of a 3D immune cell atlas of mouse skin [START_REF] Tong | The Skin Immune Atlas: Three-Dimensional Analysis of Cutaneous Leukocyte Subsets by Multiphoton Microscopy[END_REF], demonstrating different densities of adaptive and immune cell populations at various depths of the epidermis and dermis; functional cellular niches in specific anatomical locations differing in the tail, ear, flank and feet; and the relevance of structures such as the vasculature, and fibroblast-rich networks of collagen and elastin, for variations in resident immune cell populations.

Equally, cutaneous immune responses mediated by Langerhans cells (which act as potent antigen presenting cells), have been shown to vary dramatically across skin sites, impacting T cell responses, and immunization outcomes [START_REF] Wang | Langerin expressing cells promote skin immune responses under defined conditions[END_REF]. Importantly, these and other findings of skin cell heterogeneity have helped reconcile disparate results across studies in other fields, which used different skin locations for observations. Although few studies in parasitology have directly considered these differences, they are very relevant in the context of infection, and should therefore influence the choice for imaging by IVM.

Each of the imaging sites for skin IVM mentioned above has specific advantages and limitations. Ear and footpad imaging (Figures 2A and2B) allow observation over long periods of time. It is relatively easy to perform due to the non-invasive nature of the procedure, and that neither site is heavily affected by cardio-respiratory motion. In both cases, careful hair removal is necessary to minimize autofluorescence, and to prevent absorption of laser light by hair. Both sites allow non-invasive, easily accessible longitudinal imaging of complex processes including stem cell dynamics via imaging hair follicle development, and the contributions of stem cell populations to tissue regeneration [START_REF] Pineda | Intravital imaging of hair follicle regeneration in the mouse[END_REF]. Protocols developed for longitudinal imaging in the ear pinna are a strong alternative to more complex windows relevant for other skin locations, such as dorsal or ventral skin in the mid-body.

Conversely, a skin flap requires surgically generating a flap to expose the inner and outer surfaces of the dorsal or ventral skin. This procedure is more invasive than ear or footpad imaging, but it provides a larger imaging surface and a different anatomical location, which might be relevant depending on the biological question addressed. If performed without the purpose of implementing an optical chamber, this procedure allows imaging once, for relatively long periods of time, but cannot extend over the course of days (Figure 2D). The more sophisticated surgical procedure for skin imaging is the dorsal skinfold chamber (Figure 2C), which has evolved significantly since its first use in 1924 [START_REF] Algire | An adaptation of the transparent chamber technique to the mouse[END_REF][START_REF] Arfors | A titanium rabbit ear chamber: Assembly, insertion and results[END_REF][START_REF] Sandison | A new method for the microscopic study of living growing tissues by the introduction of a transparent chamber in the rabbit's ear[END_REF][START_REF] Amon | Heme oxygenase and nitric oxide synthase mediate cooling-associated protection against TNF-alpha-induced microcirculatory dysfunction and apoptotic cell death[END_REF][START_REF] Knappe | Effect of systemic hypothermia on local soft tissue trauma-induced microcirculatory and cellular dysfunction in mice[END_REF][START_REF] Lehr | Dorsal skinfold chamber technique for intravital microscopy in nude mice[END_REF]. Modern optical windows are made of titanium [START_REF] Menger | Viewing the microcirculation through the window: some twenty years experience with the hamster dorsal skinfold chamber[END_REF]. Alternatively, they can be made of non-metallic polymer materials that allow their use with radiation and magnetic resonance imaging (MRI) [START_REF] Erten | MR and fluorescence imaging of doxorubicin loaded nanoparticles using a novel in vivo model[END_REF][START_REF] Gaustad | Tumor vascularity assessed by magnetic resonance imaging and intravital microscopy imaging[END_REF], and are lighter in weight thus causing less discomfort to the mice. The method for generating an optical window for skin IVM consists of placing two symmetrical frames to "sandwich" an extended double layer of skin. The skin is covered with a removable glass coverslip, which can be incorporated into one of the frames, allowing visualization with an objective. This setup allows for repetitive analysis over a period of 2-4 weeks [START_REF] Lehr | Dorsal skinfold chamber technique for intravital microscopy in nude mice[END_REF], and it requires a 2-3-day recovery period following implantation. Finally, skin flank IVM is amongst the least explored methods in the context of host-pathogen interactions, yet one worth considering in future applications in parasitology given the neural, lymphatic, vascular, and cellular composition at this site. At a dorso-lateral anatomical location (Figure 2E), the flank has been a preferred site in the context of immunology studies, engraftment, and viral dynamics, including that of herpes simplex virus 1 (HSV-1) [START_REF] Ariotti | Tissue-resident memory CD8+ T cells continuously patrol skin epithelia to quickly recognize local antigen[END_REF]. The surgical procedure is described by [START_REF] Moalli | The Rho regulator Myosin IXb enables nonlymphoid tissue seeding of protective CD8+ T cells[END_REF], and involves creating incisions at the skin flank to separate it from the peritoneum, and generating an imaging platform by inserting a stainless steel disk under the dermis.

Different types of windows can be used to perform IVM in adipose tissue depots, depending on the tissue being accessed (Figure 2F). However only one window type was so far used in parasitology (Plasmodium) [START_REF] De Niz | The machinery underlying malaria parasite virulence is conserved between rodent and human malaria parasites[END_REF]. This work used a terminal abdominal window, whereby the surgery consisted on a small incision in the dorso-lumbar area and the careful externalization of the perigonadal adipose tissue. This was followed by the careful attachment of the surrounding skin, with a removable glass coverslip that can be directly placed on the microscope stage. Alternative to the terminal window used in this study (allowing imaging for up to 8 hours), an analogue of the dorsal skinfold chamber described above exists for the abdominal wall, which can be applied to most abdominal organs including adipose tissue depots. Although surgically much more challenging, the abdominal chamber provides the advantage of long-term, repetitive imaging in the range of 2-4 weeks.

New parasite biology revealed by IVM Plasmodium spp.

Plasmodium development in the mammalian host, eventually causative of malaria, is initiated when a female Anopheles mosquito injects motile Plasmodium sporozoites into the skin of a host upon probing for a blood meal [START_REF] Amino | Quantitative imaging of Plasmodium transmission from mosquito to mammal[END_REF][START_REF] Matsuoka | A rodent malaria, Plasmodium berghei, is experimentally transmitted to mice by merely probing of infective mosquito, Anopheles stephensi[END_REF][START_REF] Medica | Quantitative dynamics of Plasmodium yoelii sporozoite transmission by infected Anopheline mosquitoes[END_REF][START_REF] Sidjanski | Delayed migration of Plasmodium sporozoites from the mosquito bite site to the blood[END_REF][START_REF] Vanderberg | Intravital microscopy demonstrating antibody-mediated immobilisation of Plasmodium berghei sporozoites injected into skin by mosquitoes[END_REF][START_REF] Yamauchi | Plasmodium sporozoites trickle out of the injection site[END_REF]. Successful continuation of the parasite's life cycle depends on the sporozoites leaving the skin, and traveling to the liver where an enormous expansion of the parasite mass will occur via asexual replication.

The idea that mosquitoes inject Plasmodium sporozoites into the dermis of the host, rather than directly into blood circulation, was first proposed in the 1930s. However, the in vivo investigation of the migration of sporozoites after transmission and the discovery of the dermal phase of infection only became possible in the past decade.

Excision of the bite site showed that mosquito-injected Plasmodium sporozoites remained in the skin for at least 5 to 15 minutes, before entering the blood stream. It was hypothesized at the time, that sporozoite migration to the blood vessels following inoculation was delayed either because of anti-sporozoite antibodies or a cutaneous hypersensitivity reaction to the mosquito bite [START_REF] Sidjanski | Delayed migration of Plasmodium sporozoites from the mosquito bite site to the blood[END_REF]. While decades earlier it was already demonstrated that antisporozoite antibodies could immobilize sporozoites in vitro [START_REF] Stewart | Plasmodium berghei sporozoite invasion is blocked in vitro by sporozoite-immobilizing antibodies[END_REF][START_REF] Vanderberg | Studies on the Motility of Plasmodium Sporozoites[END_REF], revisiting this question with IVM on mice immunized with irradiated sporozoites and subsequently infected with WT parasites revealed a striking cessation in motility within the skin.

IVM showed in vivo a) probing of the skin by the mosquito proboscis; b) mosquito ingestion of blood either by direct puncture of a blood vessel, or pool feeding from hematomas formed upon vessel rupture by the mosquito proboscis; c) the various types of sporozoite deposits during a mosquito feed, which could be linear or fountain-like; d) demonstration of sporozoite migration following a brief delay after their deposition in the dermis and e) entry into both blood and lymph vessels [START_REF] Amino | Quantitative imaging of Plasmodium transmission from mosquito to mammal[END_REF][START_REF] Vanderberg | Intravital microscopy demonstrating antibody-mediated immobilisation of Plasmodium berghei sporozoites injected into skin by mosquitoes[END_REF].

Although the first IVM studies were performed in the ear pinna, various groups later imaged mosquito bites in both the ear pinna and the ventral and dorsal midline skin [START_REF] Jin | Direct microscopic quantification of dynamics of Plasmodium berghei sporozoite transmission from mosquitoes to mice[END_REF][START_REF] Kebaier | Kinetics of mosquito-injected Plasmodium sporozoites in mice: Fewer sporozoites are injected into sporozoite-immunized mice[END_REF][START_REF] Yamauchi | Plasmodium sporozoites trickle out of the injection site[END_REF]. Interestingly, this consistently resulted in fewer inoculated sporozoites in the ear, but the kinetics of migration were conserved between sites. IVM also revealed that up to 15-20% of inoculated sporozoites are transported via lymphatic vessels to the lymph nodes [START_REF] Amino | Quantitative imaging of Plasmodium transmission from mosquito to mammal[END_REF][START_REF] Yamauchi | Plasmodium sporozoites trickle out of the injection site[END_REF]; and that a small fraction of parasites that remain in the skin can begin developing within this organ [START_REF] Coppi | The malaria circumsporozoite protein has two functional domains, each with distinct roles as sporozoites journey from mosquito to mammalian host[END_REF][START_REF] Gueirard | Development of the malaria parasite in the skin of the mammalian host[END_REF]. The remaining majority of sporozoites are either eliminated, or successfully travel to the liver endothelium via blood vessels.

Finally, IVM has allowed studying and quantifying the speeds of sporozoite migration at different bite sites of the dermis, the dynamics of sporozoite contact with host blood vessels, and the subsequent migration of sporozoites from the skin into the bloodstream [START_REF] Amino | Quantitative imaging of Plasmodium transmission from mosquito to mammal[END_REF][START_REF] Hellmann | Environmental constraints guide migration of malaria parasites during transmission[END_REF][START_REF] Hopp | Longitudinal analysis of Plasmodium sporozoite motility in the dermis reveals component of blood vessel recognition[END_REF] (Figure 1A-B). These studies revealed that the sporozoite migration path is determined by the environment [START_REF] Hellmann | Environmental constraints guide migration of malaria parasites during transmission[END_REF]; that sporozoites migrate through cells using a secreted perforin-like protein [START_REF] Amino | Host cell traversal is important for progression of the malaria parasite through the dermis to the liver[END_REF], and slow down when migrating in close proximity to blood capillaries [START_REF] Hopp | Longitudinal analysis of Plasmodium sporozoite motility in the dermis reveals component of blood vessel recognition[END_REF].

These results also informed in vitro studies at the intersection of physics, and biology [START_REF] Hellmann | Environmental constraints guide migration of malaria parasites during transmission[END_REF][START_REF] Muthinja | Microstructured blood vessel surrogates reveal structural tropism of motile malaria parasites[END_REF], that use patterned environments to address questions that could not be answered by IVM. For example, the different migration path in the skin of ear or tail could be assigned to the different architecture of the environment (reviewed in [START_REF] Muthinja | Tailored environments to study motile cells and pathogens[END_REF]). Small differences in sporozoite migration in the skin were observed by IVM in mice lacking a specific integrin, possibly suggesting that this molecule interacts with a sporozoite surface protein [START_REF] Dundas | Alpha-v-containing integrins are host receptors for the Plasmodium falciparum sporozoite surface protein, TRAP[END_REF]. Finally, a recent elegant study revealed that the sporozoite surface protein CSP protects the parasites from the action of its own perforins, which are necessary for migration through cells in the skin [START_REF] Aliprandini | Cytotoxic anti-circumsporozoite antibodies target malaria sporozoites in the host skin[END_REF].

One of the first in vivo clues of the relevance of adipose tissue to Plasmodium asexual blood stages, came from a study using bioluminescence to characterize P. berghei sequestration in rodents [START_REF] Fishelson | Murine malaria parasite sequestration: CD36 is the major receptor, but cerebral pathology is unlinked to sequestration[END_REF]. This phenomenon is the ligand-mediated arrest of Plasmodium schizonts and trophozoites in the host vasculature of humans, and is believed to be associated with many malaria complications. The human P. falciparum parasite sequesters by interacting with a variety of human receptors, such as CD36, ICAM, EPCR, and CSA. A study using human skin transplants on SCID mice identified the rolling behaviour of P. falciparuminfected RBCs in living mice [START_REF] Ho | Visualization of Plasmodium falciparum-endothelium interactions in human microvasculature: mimicry of leukocyte recruitment[END_REF]. While the P. berghei ligand for sequestration remains unidentified, recent IVM-based work showed that the overall machinery for parasite sequestration is conserved between human and rodent Plasmodium spp. [START_REF] De Niz | The machinery underlying malaria parasite virulence is conserved between rodent and human malaria parasites[END_REF]. This work showed that P. berghei parasites lacking two central proteins for the Plasmodium export machinery (MAHRP1a and SBP1), lose sequestration in the adipose tissue and lungs, decreasing virulence and improving survival.

IVM showed significantly different parasite numbers and dynamics at this site when comparing WT and mutant parasite lines. Specifically, non-sequestering parasite lines were found circulating within erythrocytes in the bloodstream, while sequestering lines were static in the vessel walls adjacent to activated macrophages. Importantly, both, sequestering and non-sequestering lines showed a strictly intravascular localization early in infection, consistent with sequestration rather than extravasation.

Trypanosoma brucei

Human African Trypanosomiasis (HAT), or sleeping sickness, is caused by two main subspecies of Trypanosoma brucei that are exclusively transmitted through the bite of Glossina tsetse flies.

Within the fly, T. brucei undergoes a complex developmental cycle culminating in the production of salivary metacyclic forms that can infect mammalian hosts [START_REF] Rotureau | Through the dark continent: African trypanosome development in the tsetse fly[END_REF]. Like Plasmodium and other vector-borne pathogens, the presence of T. brucei changes the composition of the saliva and modifies the feeding behaviour of the tsetse fly in a way that enhances the chances of parasite transmission to the host (van den [START_REF] Van Den Abbeele | Trypanosoma brucei modifies the tsetse salivary composition, altering the fly feeding behavior that favors parasite transmission[END_REF]. Tsetse flies are pool-feeders and lacerate the skin of their host rather than inserting a proboscis directly into the vasculature. After causing significant local damage and inflammation at the bite site that often result in a transient chancre, the insects feed on the resultant pool of capillary blood and lymph [START_REF] Bouchet | Solenophagy and telmophagy: biting mechanisms among various hematophagous insects[END_REF][START_REF] Goodwin | The pathology of African Trypanosomiasis[END_REF]. However, our knowledge on the early interface between the parasites and the host skin in vivo remained limited until recently.

Various studies have documented skin reactions to the parasite in different animal models by histology, fluorescence-based imaging, electron microscopy [START_REF] Akol | Early events following challenge of cattle with tsetse infected with Trypanosoma congolense: development of the local skin reaction[END_REF][START_REF] Dwinger | Development of Trypanosoma congolense, T. vivax and T. brucei in the skin reaction induced in goats by infected Glossina morsitans centralis: a light and electron microscopical study[END_REF][START_REF] Goodwin | Pathological effects of Trypanosoma brucei on small blood vessels in rabbit ear-chambers[END_REF][START_REF] Goodwin | The pathology of African Trypanosomiasis[END_REF][START_REF] Ikede | Pathological changes in cattle infected with Trypanosoma brucei[END_REF][START_REF] Gray | An extravascular site of development of Trypanosoma (Nannomonas) congolense[END_REF][START_REF] Mwangi | Trypanosoma congolense infection in sheep: Ultrastructural changes in the skin prior to development of local skin reactions[END_REF][START_REF] Mwangi | Cellular phenotypes in Trypanosoma congolense infected sheep: The local skin reaction[END_REF][START_REF] Sbarbati | Subcutaneous adipose tissue classification[END_REF][START_REF] Thuita | Trypanosoma brucei rhodesiense Transmitted by a Single Tsetse Fly Bite in Vervet Monkeys as a Model of Human African Trypanosomiasis[END_REF], thermographic imaging, or molecular and flow cytometric methods [START_REF] Caljon | The dermis as a delivery site of Trypanosoma brucei for tsetse flies[END_REF]. Nevertheless, IVM recently brought new perspectives in our understanding of the crucial role of the skin in trypanosome biology. By multiple imaging approaches, including intravital imaging in the ear pinna, African trypanosomes were confirmed to first develop in the skin at the bite site before they reach the blood and lymph vasculatures. Also, the resulting neutrophil recruitment was further proven to enhance early onset of infection (Figure 1C-D). At the same time, a multiplex kinetic intravital imaging approach [START_REF] Calvo-Alvarez | A new chimeric triple reporter fusion protein as a tool for in vitro and in vivo multimodal imaging to monitor the development of African trypanosomes and Leishmania parasites[END_REF] revealed that the entire dermis is an important anatomic reservoir of trypanosomes where a significant number of parasites was constantly available for transmission to tsetse vectors [START_REF] Capewell | The skin is a significant but overlooked anatomical reservoir for vector-borne African trypanosomes[END_REF]. This imaging approach consisted of combining whole animal bioluminescence imaging, spinning-disk confocal microscopy on the ear pinna, and two-photon microscopy on the abdominal flank, in the same animals. In parallel, a functionally adapted population of parasites was shown to occupy adipose tissues in a mouse infection [START_REF] Trindade | Trypanosoma brucei parasites occupy and functionally adapt to the adipose tissue in mice[END_REF] and it is possible that the adipocyte-rich hypo-dermal layer of the skin may attract, host and/or maintain at least part of the skin-dwelling trypanosome population by providing them an immunological niche and/or a stable lipid-rich nutritive environment [START_REF] Tanowitz | Adipose tissue -a safe haven for parasites?[END_REF].

Although the existence of a significant extravascular population of T. brucei parasite is not novel [START_REF] Goodwin | The pathology of African Trypanosomiasis[END_REF], their enrichment in the skin of HAT patients and asymptomatic latent carriers ( [START_REF] Capewell | The skin is a significant but overlooked anatomical reservoir for vector-borne African trypanosomes[END_REF], Camara et al., unpublished) questions our understanding of trypanosome transmission and HAT epidemiology. This is especially important when considering the possibility of a significant silent reservoir of trypanosomes in the skin of latent carriers readily accessible to tsetse vectors [START_REF] Büscher | Do cryptic reservoirs threaten Gambiense-sleeping sickness elimination?[END_REF]. The presence of parasites in extravascular sites (skin, adipose tissue, brain) for extended periods raises several fundamental questions on parasite biology. Indeed, parasites in the blood and adipose tissue appear functionally specialized as they can catabolize fatty acids only in the fat [START_REF] Trindade | Trypanosoma brucei parasites occupy and functionally adapt to the adipose tissue in mice[END_REF]. This in turn raises several questions regarding how parasites proliferate, how differentiation is triggered preferentially in transmissioncompatible tissues, how parasites migrate, and whether and how they sequester.

Leishmania spp.

Leishmania parasites are transmitted by the bite of infected Phlebotomus and Lutzomyia sandflies. The sandfly bite leads to wounding of the microvasculature, creating a haemorrhagic pool from which the sandfly feeds (Belkaid et al., 2000;Belkaid et al., 2000;[START_REF] Kamhawi | Protection against cutaneous Leishmaniasis resulting from bites of uninfected sand flies[END_REF][START_REF] Teixeira | Saliva from Lutzomyia longipalpis induces CC chemokine ligand 2/Monocyte chemoattractant protein-1 expression and macrophage recruitment[END_REF] (Figure 1E-F). Upon infection, three types of leishmaniasis can occur depending on the infectious species: cutaneous, mucocutaneous, or visceral. Cutaneous leishmaniasis causes nodules or ulcers in the skin, often resulting in deforming scars.

Mucocutaneous leishmaniasis may develop from cutaneous leishmaniasis, forming sores in the mouth, nose or larynx. Visceral leishmaniasis affects organs including the liver, spleen and bone marrow, and if severe, can be fatal.

Regarding visualization of Leishmania transmission in the skin, IVM has been a key tool to elucidate mechanisms of entry and invasion which were ground-breaking in the field ( [START_REF] Peters | In vivo imaging reveals an essential role for neutrophils in leishmaniasis transmitted by sand flies[END_REF][START_REF] Ng | Migratory dermal dendritic cells act as rapid sensors of protozoan parasites[END_REF], reviewed by [START_REF] Beattie | Leishmania-host interactions: What has imaging taught us?[END_REF][START_REF] Ritter | Are neutrophils important host cells for Leishmania parasites?[END_REF]). While it was known that the definitive host of Leishmania parasites are macrophages, neutrophils were shown to be the first transient host cells in the skin [START_REF] Peters | In vivo imaging reveals an essential role for neutrophils in leishmaniasis transmitted by sand flies[END_REF]. A combination of transgenic parasites (L.m.-RFP) and transgenic mice (LysM-GFP, a reporter for monocytes, macrophages and neutrophils), together with 2-photon IVM of the ear pinna, showed that neutrophils slowly infiltrate into the sandfly bite site, and continue to be recruited for an extended period of time.

IVM showed that Leishmania parasites within neutrophils were viable, and that following apoptosis of the neutrophils, parasites were released within the vicinity of surrounding macrophages (the definitive host cell), thereby contributing to the progression of the disease [START_REF] Peters | In vivo imaging reveals an essential role for neutrophils in leishmaniasis transmitted by sand flies[END_REF] (Figure 1E-F). Importantly, upon neutrophil depletion, a reduction in viable parasites was detected, corroborating a previously suggested role for neutrophils in Leishmania pathogenesis [START_REF] Van Zandbergen | Cutting Edge: Neutrophil granulocyte serves as a vector for Leishmania entry into macrophages[END_REF].

Another IVM-based study demonstrated the unique role of dermal dendritic cells (DDCs) in Leishmania sensing [START_REF] Ng | Migratory dermal dendritic cells act as rapid sensors of protozoan parasites[END_REF]. Two-photon IVM of the ear pinna revealed that upon Leishmania infection, the highly motile DDCs ceased their motility. This was followed by a) the extension of long and highly dynamic pseudopods or dendrites capable of tracking and engulfing parasites, b) retraction of the pseudopods towards the cell body, and c) incorporation of parasites into cytosolic vacuoles. This rapid cessation of motility and rapid tracking and engulfment was specific for parasites, and not observed towards inert particles. Both IVM-based studies shed light into the role of innate immune cells at the skin interface following a sandfly bite, providing important advances in the understanding of this disease, which are impossible to detect in vitro.

Lastly, a more recent study used two-photon IVM to image for the first time, the entire time course of L. major infection in the ear skin, following the bite of an infected sand fly. This study carefully describes various events relevant to host pathogen interactions, pathology, and parasite dynamics previously unknown. This method takes advantage of various features of IVM to perform this long-term imaging in a non-invasive manner, including deep penetration, and little photodamage [START_REF] Carneiro | Use of two-photon microscopy to study Leishmania major infection of the skin[END_REF].

The parasites so far discussed are transmitted by the direct bite of infected vectors.

Additional parasites also transmitted by vector bites include the filarial worms Onchocerca volvuluscausative agent of onchocerciasis, Wuchereria bancrofti and Brugia malayi -causative of filariasis, and the Thelieria parva and Babesia parasites, causative of theileriosis and babesiosis. However, to date no studies have used IVM to investigate these parasites. Other parasites transmitted via the skin, but not via infected bites, include Trypanosoma cruzi and Schistosoma. IVM-based findings on both parasites are discussed below.

Trypanosoma cruzi

Triatomines are the hematophagous insect vectors of Trypanosoma cruzi -the causative agent of Chagas disease. Triatomines are vessel feeders, and after piercing the host skin will probe via rapid whip-like intradermal movements of the maxillae until a vessel is found. Like mosquitoes and tsetse flies, triatomines possess various molecules in the saliva, which reduce hemostasis and have anti-inflammatory properties [START_REF] Ribeiro | Purification and characterization of prolixin S (nitrophorin 2), the salivary anticoagulant of the blood-sucking bug Rhodnius prolixus[END_REF][START_REF] Pereira | Anticoagulant activity of Triatoma infestans and Panstrongylus megistus saliva (hemiptera/triatominae)[END_REF]Ribeiro et al., 1995 (1); [START_REF] Ribeiro | Role of arthropod saliva in blood feeding: Sialome and Post-Sialome Perspectives[END_REF]. Like for mosquitoes and tsetse flies, host-to-vector transmission occurs when the triatomine feeds on an infected individual. However, unlike other pathogens, T. cruzi is unique in that a person only becomes infected when the triatomine defecates on the host skin during or after a feed (Figure 1G). The parasites are in the faeces and enter the body through mucous membranes (i.e. eyes or mouth) or itch-induced scratching that leads to breaks in the skin.

Despite the importance of the skin as an entry site of the parasite, T. cruzi interactions at the skin interface in vivo remain poorly understood. Only two studies have used IVM in the context of T. cruzi, and both focused on the patterns of triatomine feeding and salivation at the mouse skin, rather than vector-to-host parasite transmission [START_REF] Soares | Intravital microscopy and image analysis of Rhodnius prolixus (Hemiptera: Reduviidae) hematophagy: The challenge of blood intake from mouse skin[END_REF][START_REF] Soares | Salivation pattern of Rhodnius prolixus (Reduviidae; Triatominae) in mouse skin[END_REF]. The first study used the ear pinna of mice, together with triatomine saliva labelled with acridine orange. Using epifluorescence microscopy, it was found that salivation occurs throughout all feeding phases (probing and engorgement) of the triatomine Rhodnius prolixus, and measured the frequency of saliva emissions [START_REF] Soares | Salivation pattern of Rhodnius prolixus (Reduviidae; Triatominae) in mouse skin[END_REF].

The second study investigated the interface between the feeding process of the triatome, and the host's response at the vascular endothelium (Figure 1H). This work used epifluorescence microscopy, as well as stereomicroscopy and an electromyogram. The main findings included a) the induction of vascular permeability alterations following a triatomine bite via injection of dyes; b) the immediate platelet and leukocyte aggregation at the venular endothelium following a bite, and c) the integration of imaging and analysis methods in vector and host, which enabled monitoring vessel wall pulsations to register movements during blood pumping, as well as the evaluation of blood flow through the triatomine's head [START_REF] Soares | Intravital microscopy and image analysis of Rhodnius prolixus (Hemiptera: Reduviidae) hematophagy: The challenge of blood intake from mouse skin[END_REF]. Although there are no IVM studies yet that investigate parasite transmission at the skin, one ex vivo study addressed the kinetics of skin penetration of T. cruzi trypomastigotes in mice [START_REF] Schuster | Trypanosoma cruzi: Skin-penetration kinetics of vectorderived metacyclic trypomastigotes[END_REF]. A drop of vector faeces or urine containing trypomastigotes was placed on the bite site of a triatomine, and the skin was surgically removed after different periods of time. This showed that the minimal exposure period necessary for infection was as little as 5 minutes with longer periods of exposure time correlating with higher infection rates. This showed that T. cruzi can rapidly invade the host, and that some parasites can be carried away from the bite site immediately [START_REF] Schuster | Trypanosoma cruzi: Skin-penetration kinetics of vectorderived metacyclic trypomastigotes[END_REF] and sets the stage for similar studies as those conducted in T. brucei and Plasmodium to reveal the dynamics of infection.

Schistosoma

Various Schistosoma species are the causative agents of schistosomiasis. The sexual reproduction of the parasite occurs in humans as well as other hosts, while asexual reproduction occurs in freshwater snails (Biomphalaria spp.). Schistosoma are motile in all life stages, and this motility is relevant to their capacity to search for, and invade the host skin, and to circulate within the host following invasion. Schistosomes are phototrophic, which leads the cercariae to preferentially localize to the surface of shallow waters, where they can maximize their contact with humans [START_REF] Stirewalt | Schistosoma manonsi: Cercarial penetration of host epidermis at the ultrastructural level[END_REF]. Cercariae then respond to thermal gradients in order to find the skin of the host, and then to chemical cues that allow them to complete invasion [START_REF] Fishelson | Murine malaria parasite sequestration: CD36 is the major receptor, but cerebral pathology is unlinked to sequestration[END_REF][START_REF] Haas | Schistosoma mansoni cercariae: stimulation of acetabular gland secretion is adapted to the chemical composition of mammalian skin[END_REF][START_REF] Salter | Schistosome invasion of human skin and degradation of dermal elastin are mediated by a single serine protease[END_REF]. Most of the interactions of Schistosoma with the skin have been explored using a range of imaging, molecular and chemical methods (reviewed by [START_REF] Mckerrow | Invasion of skin by Schistosoma cercariae[END_REF]). One study used IVM to monitor Schistosoma interactions with the skin to understand excretory/secretory (ES) molecule release by the parasite during skin invasion [START_REF] Paveley | Fluorescent imaging of antigen released by a skin-invading helminth reveals differential uptake and activation profiles by antigen presenting cells[END_REF] (Figure 1I-J). Using carboxyfluorescin diacetate succinimidyl ester (CFDA-SE), a fluorescent amine reactive tracer, live cercariae were labelled to investigate their interactions with innate immune cells in the mouse ear pinna. Time-lapse confocal imaging showed that as cercariae move through the skin, CFDA-SE-labelled material is released via the oral sucker. This material is thought to be a mixture of digestive proteases that aid in the migration of the parasite.

Ex vivo work investigating the dynamics of ES molecule uptake by macrophages and dendritic cells showed that, depending on the relative abundance of each cell type, and differential rates of antigen processing by these cells, this might be key to the success of adaptive immune priming in response to Schistosoma infection [START_REF] Paveley | Fluorescent imaging of antigen released by a skin-invading helminth reveals differential uptake and activation profiles by antigen presenting cells[END_REF].

Future directions

Considering its ease, IVM of parasites in the skin has focused mainly on the use of the ear pinnae of mice. While this has advantages in terms of accessibility and non-invasiveness, the few studies in Plasmodium and T. brucei using other skin sites to image parasite dynamics have shown differences between locations. This is particularly important as the skin is physiologically different between anatomical sites and parasites may adapt to the respective environment. The use of skinfold chambers, which allow visualization within a much larger surface area, for a much longer period of time will be a useful tool to study new tissue locations. Interestingly, most IVM studies have focused on vector-to-host transmission, with only one having imaged host-to-vector transmission after a bite. The importance of the skin and adipose tissue as reservoirs and for disease pathology remains largely obscure and requires more attention in the future.

Technological advances including the increasing range of available fluorescent reporter mice, and transgenic parasites expressing different fluorescent proteins alongside tagged genes potentially involved in transmission will lead to further advances in our understanding of parasite biology. These technologies, in combination with the use of single-cell transcriptome analysis and immune profiling, may reveal new tissue-specific interfaces between parasites and their hosts that have not been observed previously.

An exciting area of IVM that is becoming more popular for parasite research is the use of humanised models. Whilst conventional animal models are invaluable in providing insights into parasite behaviour, they cannot fully replicate human disease due to differences in parasite strains, cell morphology and humoral immune responses. As a result, there has been a drive in recent years to examine human parasites directly. One way to achieve this is through the use of humanized models. By incorporating human tissues and stem cells into immunocompromised animals, these models successfully mimic human physiology allowing human parasites to be studied in an in vivo environment. They have been particularly popular for studying P. falciparum infection as they allow certain aspects of malaria to be studied in a way not possible using murine Plasmodium spp (reviewed by [START_REF] Minkah | Humanized mouse models for the study of human malaria parasite biology, pathogenesis, and immunity[END_REF]).

One of these aspects is the sequestration of the P. falciparum asexual stages in the microvasculature. Although this also occurs in certain murine malaria infections, the binding ligand of P. falciparum is unique, limiting our abilities to study this behaviour closely. This has been addressed in a number of studies by utilising mice implanted with human skin grafts. Through IVM they have observed the rolling and adherence behaviour of parasitized red blood cells in both post-capillary venules and arterioles and demonstrated that this behaviour could be reversed using antibodies that blocked the parasites binding ligands [START_REF] Ho | Visualization of Plasmodium falciparum-endothelium interactions in human microvasculature: mimicry of leukocyte recruitment[END_REF][START_REF] Yipp | Differential roles of CD36, ICAM-1, and p-selectin in Plasmodium falciparum cytoadherence in vivo[END_REF]. Similar models using human subcutaneous fat, a more prominent site of sequestration that closely mimics brain endothelium [START_REF] Moxon | Loss of endothelial protein C receptors links coagulation and inflammation to parasite sequestration in cerebral malaria in African children[END_REF], have yet to be fully realised but are in development (Meehan et al, unpublished). Aside from these limited studies using P. falciparum, little IVM research has been carried out on parasites using humanised animals but as this technology develops further, it will likely be utilised by an increasing number of researchers in the future. invasive method (C) includes the dorsal skinfold chamber, which requires the surgical implantation of two titanium or polymer frames that can hold a ring with a glass cover-slip through which imaging is performed. An invasive method is the generation of a skin flap (D), whereby a skin flap is generated, exposing a large imaging area. This procedure is invasive, and terminal. A less commonly used method for IVM imaging is the skin flank (E) which requires the generation of an incision at a dorso-lateral location, and either direct imaging, or mounting on a stainless steel disc for stable image acquisition. For adipose tissue imaging, various types of window exist to visualize various depots (marked by X). To image the peri-gonadal adipose tissue a terminal lower abdominal window (F) was generated.

Figure 1 .Figure 2 .

 12 Figure 1. Anatomically, the skin is divided into 3 compartments: the epidermis (an avascular layer mostly composed of keratinocytes and Langerhans cells), the dermis (highly perfused by blood

Table 1 . Summary of windows for skin and AT imaging

 1

	Technique	Key points	Complexity	Advantages	Limitations
	Foot pad	Intact skin. Hair	Low	• Simple to perform	• Auto-fluorescence if
		removal necessary to avoid auto-fluorescence.		• Not invasive • Longitudinal imaging through	hair present • Limited vascularization and
				various days.	imaging area.
				• Saves animals
				(consistent with
				3Rs)
	Ear pinna	Intact skin. Hair	Low	• Simple to perform	• Auto-fluorescence if
		removal necessary to avoid auto-fluorescence.		• Not invasive • Longitudinal imaging through	hair present • Limited vascularization and
				various days.	imaging area.
				• Saves animals	• Rich in cartilage but
				(consistent with	poor in sub-
				3Rs)	cutaneous adipose
					tissue.
	Tail	Intact skin. Hair	Low	• Simple to perform	• Auto-fluorescence if
		removal necessary to avoid auto-fluorescence.		• Not invasive • Longitudinal imaging through	hair present • Poor in sub-cutaneous adipose
				various days.	tissue.
				• Saves animals
				(consistent with
				3Rs)
	Dorsal	Semi-invasive.	High	• Longitudinal	• Could present post-
	skinfold	Requires the surgical		imaging through	surgical complications
	chamber	implantation of a	Important to cause as	various days.	including infection
		chamber, exposing a small skin area. The chamber remains in place and allows longitudinal imaging with recovery of the mouse.	little damage as possible: the animal should survive surgery, implantation and multiple rounds of imaging.	skin sites. • Reduces use of animals (3Rs) • Different vascular environment as that presented in more accessible	and inflammation. • Chronic chamber may introduce artefacts • Extreme care should be taken to prevent pain.
	Skin flap	Exposure of a large	Medium to high	• Large imaging area	• It is terminal
		skin area by generating a skin flap that can be imaged through a simple window (e.g. a coverslip). It is	Important to cause as during imaging bleeding or artefacts possible to avoid little damage as	• Different vascular skin sites. more accessible that presented in environment as	Possibility of ischemia.
		invasive and terminal.
	Skin flank	Relatively easy access.	Medium to high	• Large imaging area	• In some modalities, it
		Exposure of a relatively large skin area.	during imaging bleeding or artefacts possible to avoid little damage as Important to cause as	more accessible those presented in environments as lymphatic nerve, and • Different vascular,	is terminal.
				skin sites.
	Abdominal	Exposure of a large	Medium to high	• Large imaging area	• It is terminal
	window for	AT area that can be
	AT access

Acknowledgements

We thank Prof. Volker Heussler (IZB, University of Bern, Switzerland), for his input and training on the techniques here reviewed, and to Prof. James M. Brewer (Wellcome Centre for Molecular Parasitology), for input on discussions for future applications of IVM. MDN was funded by Long Term EMBO Postdoctoral fellowship ALTF 1048-2016. GRM was funded by Wellcome Trust grant 095507. LMF was funded by FCT (IF/01050/2014) and ERC (FatTryp, ref.771714). The authors declare no conflicts of interest.

(red) and draining lymph vessels (green)). (A) Plasmodium parasites are transmitted by infected Anopheles mosquitoes. Inoculated Plasmodium sporozoites can either remain in the skin; be transported to lymph nodes via lymph; or to the liver via blood vessels. Sporozoites show different dynamics at different skin sites or when in proximity to blood capillaries. (B) Maximum projection of P. berghei sporozoite tracks (green) in the proximity of blood vessels (CD31, magenta). Scale bar = 25µm. [START_REF] Hopp | Longitudinal analysis of Plasmodium sporozoite motility in the dermis reveals component of blood vessel recognition[END_REF]. (C) Trypanosoma brucei parasites are transmitted by the bite of infected tsetse flies (Glossina spp). They first develop in the skin at the bite site before they reach the blood and lymph vasculatures, and the entire dermis is an important parasite reservoir. (D) Extravascular trypanosomes (red) imaged in the skin vessels of Flk1-GFP mice (green) using spinning disc confocal microscopy Scale bar: 10 µm. [START_REF] Capewell | The skin is a significant but overlooked anatomical reservoir for vector-borne African trypanosomes[END_REF]. (E) Leishmania spp. are transmitted by the bite of infected Phlebotomine or Lutzomyia sandflies. Neutrophils are recruited to the bite site, and are crucial for the dissemination of parasites. (F) Two-photon IVM still from a LysM-GFP mouse (neutrophils, green) 2h after infection with Leishmania major (red). Scale bar = 20µm. [START_REF] Peters | In vivo imaging reveals an essential role for neutrophils in leishmaniasis transmitted by sand flies[END_REF]. (G) Trypanosoma cruzi is transmitted via the faeces of the triatomine Rhodnius prolixus.

(H) Vascular permeability as shown by FITC-Dextran leakage from the blood vessel upon insect probing. Scale bar = 100µm. [START_REF] Soares | Intravital microscopy and image analysis of Rhodnius prolixus (Hemiptera: Reduviidae) hematophagy: The challenge of blood intake from mouse skin[END_REF]. (I) Schistosoma spp. undergo asexual reproduction in freshwater snails (Biomphalaria spp.). Schistosoma spp. cercariae are motile and invade the host skin.

(J) Schistosoma excretory/secretory molecule release (using a fluorescent tracer (green)) during parasite skin invasion. Scale bar = 100µm. [START_REF] Paveley | Fluorescent imaging of antigen released by a skin-invading helminth reveals differential uptake and activation profiles by antigen presenting cells[END_REF].


Eric Haramburu
email: eric.haramburu@gmp.iut-tlse3.fr-francis.collombet@gmp.iut-tlse3.fr

* -Francis Collombet

Bernard Ferret
email: bernard.ferret@gmp.iut-tlse3.fr

-Jean-Stéphane Vignes
email: jean-stephane.vignes@microturbo.snecma.fr

Pierre Devos
email: pierre.devos@industrie.gouv.fr

Christophe Levaillant
email: levailla@enstimac.fr-schmidt@enstimac.fr

Fabrice Schmidt

Short-fibre-reinforced thermoplastic for semi structural parts: process-properties

Keywords: Short-fibre-reinforced composites, Fibre orientation, Manufacturing process, computational structure mechanics

This work concerns the high pressure injection of semi structural polymer -short fibre reinforced-parts. A research group, with three aeronautic firms of Toulouse and two academic laboratories of the French Midi-Pyrenees Region, intends to deal with these whole problems from the manufacturing process to the structural analysis of the injected parts. More particularly, this work features the interfacing of the industrial computation tools of fibres orientation and mechanical response of structures. Starting from the orientation data, the computation and the localization of the distributions of homogeneous elastic properties are performed for three industrial parts respectively a body of aircraft pressure valve (Liebherr Aerospace), a first stage stator (Microturbo) and a fan wheel (Technofan).

Introduction

The presented work lies within the scope of a research group which relates to the high pressure injection of semi structural technical polymer -short fibres reinforcedparts. A brainstorming carried out jointly by equipment suppliers of the aeronautical sector of the Midi-Pyrenees Region made an increasing need emerge related to the replacement of metallic materials by polymeric composites. Competition, in the sector of aeronautics, justifies the interest in thermoplastic short fibre reinforced composites in order to reduce the current production cost of certain parts with high mechanical characteristics and a precise geometry.

In a more detailed way, there is an interest in lightening the on board systems, in increasing the production series and the productivity, to fit to for function integration. On the other hand, a certain number of obstacles can be noted such as a strong heterogeneity of materials, a dependence with the manufacturing process both the final characteristics and geometrical forms as well as an inexperience in the control of the process and the application of the non destructive control techniques. Added to the requirements of aeronautics, the project is not reduced to a simple substitution of metallic materials by advanced technical materials but must be linked to a new design of the part.

The industrial aspect is aimed to develop, to carry out and to validate composite parts (Figure 1), a body of aircraft pressure valve, a fan wheel and a first stage stator for a gas turbine based on the industrial competences of the Midi-Pyrenees. The objective of the research task consists in characterizing the coupling between the design and the manufacture of semi-structural composite parts injected with short fibres, in order to carry out a stress analysis representative of heterogeneity of the injected item. It is well-known that the reality of the composite material only exists in the achieved part and strongly depends on the manufacturing process. However there is no optimization method of the process, no design tool at the disposal of the industrial engineering and design departments allowing to account for this reality. It represents an impediment of the economic interest of the composite solution regarding the traditional metal solutions.

For the injected short fibre composites, the knowledge of the local orientation of the fibres or the presence of welded joints implied by the filling mode of the moulds or the voids is the main feature. Indeed to design the mould, the plastic moulder has to know the distributions of the fibres orientation in order to control the shrinkages while cooling. In the same way, before the design of the mould, the mechanical engineer has also to get the fibres orientation to realise a mechanical design faithful to the heterogeneity of the material. Thus, the orientation mechanisms during the filling are the common denominator between the plastic moulder and the mechanical engineer's works.

The research group thus intends to deal with these whole problems in the experimental and numerical aspects, from the manufacturing process to the structural analysis and the final control of the defects within the injected parts. At the sides of the industrial firms, the academic partners are the research team PRO²COM of the LGMT and the CROMeP of the Engineering School of the Mines in Albi-Carmaux (EMAC). Three PhD. works are linked to there. This paper more particularly features the assessment about research team PRO²COM of the LGMT (PhD.1 in progress by E. Haramburu, jointly supervised with MICROTURBO company).

A main part of this work to do within the research strategy (Figure 2) is the interfacing of the industrial computational tools of fibres orientation and mechanical response of structures. The mathematical representation of the fibres orientation follows Advani's method (Advani and Tucker,87). It is integrated within the calculation methods of the mechanical properties, based on homogenization techniques.

Figure 2. Research strategy

As far as the application is concerned, this interface allows to gather the results of a numerical simulation of injection with fibre orientation data (components of the Advani's tensor), one finite element after the other and in the thickness of the piece. An estimation of the homogenized mechanical properties is carried out locally thanks to the Mori and Tanaka's method. The interface then builds the numerical pattern in relation to a code by associating the calculated local properties with the finite elements and a chosen mesh (from a same CAD pattern for the two analysis types). The results corresponding to the various stages carried out by the interface program are at the disposal of the design department engineer in order to check its relevance. The quantitative validation of this approach known as "wholly numerical", in particular thanks to some tests on industrial structures, is currently done.

Elastic properties estimation method

The heterogeneous nature of the injected short fibre composites represents an application field well adapted to the micromechanical modelling from homogenization techniques. Indeed, these methods consist in determining the elastic properties of an equivalent homogeneous material from the properties of the various components. Moreover, they can be extended to the nonlinear behaviour and damage problems (Collombet et al.,97), (Dunn and Ledbetter,97), (Wang and Weng,92).

The homogenisation is carried out on average of an elementary representative volume (ERV) of the material with the condition of choosing an ellipsoidal geometrical representation of heterogeneities. The ERV is the volume of the material containing all the heterogeneities (microscopic scale) influencing the mechanical response (macroscopic scale). In a classical way, a first stage called "the representation stage" requires to choose the various heterogeneities types and their geometrical dimensions associated with their ellipsoidal representation. This choice often knows a numerical fitting with the experiment on elementary specimens and controlled conditions of injection.

The stage of "localization" consists in defining average constitutive laws between the micro and macro-scale (Hill,63). Thanks to strain-and stressconcentration tensors A and B as ratios between the average heterogeneity strain (or stress) and the corresponding average in the composite, stiffness and compliance tensors C and S of composite are given by:

( ) ( ) ∑ ∑ = = ⋅ - + = ⋅ - + = N 1 i i 0 i i 0 N 1 i i 0 i i 0 B S S f S S or A C C f C C [1]
where superscript i indicates quantities associated with the N heterogeneities of the ERV, and superscript 0 denotes a matrix quantity. Symbol f represents the volume fraction for matrix or heterogeneity phases.

Equation [1] gives dual generic expressions for stiffness and compliance tensors in terms of strain-and stress-concentration tensor A and B. Then, the different micromechanical approaches in the literature provide different ways to approximate A or B.

Eshelby's modelling

Eshelby (Eshelby,57 and 61) calculates the disturbance of the strain field in the ERV because of the presence of a heterogeneity of given elastic properties (principle of equivalent inclusion). Eshelby obtains the strain-concentration tensor (in the principal local directions of heterogeneity i) such as:

( ) [ ] 1 0 i 0 i i E C C S E I A - - ⋅ ⋅ + =
where symbol I represents the fourth-order unit tensor and E i denotes the Eshelby's tensor in accordance to the shape of the ellipsoidal heterogeneity by its aspect ratio r=l/d (with l and d, respectively the length and the diameter) and of the elastic properties of the isotropic matrix by the Poisson's ratio (Mura,82).

Moreover, if we consider an orientation of the heterogeneity i in the global directions of the ERV, one obtains (Pettermann et al.,97):

( ) ( ) [
] ( )

1 i i 1 0 i 0 i i i i E , T C C S E I , T A - -         φ θ ⋅ - ⋅ ⋅ + ⋅ φ θ =
where ( )

i i , T φ θ
is the transformation tensor for fourth-order tensors in terms of Euler angles θ i and φ i which performs the rotation from the local system of heterogeneity i R LOC to the global ERV system R ERV (Figure 3).

Figure 3. Local directions R LOC in the heterogeneity i in the global directions R ERV

However, Eshelby's modelling represents the elementary situation of an isolated heterogeneity. It does not take into account the interactions between the ERV phases. From the model given by Eshelby, Mori and Tanaka have determined a disturbance on average in the macroscopic fields due to the each heterogeneity presence in the ERV.

Mori & Tanaka's method

The aim of Mori & Tanaka's method (Mori and Tanaka,73) is to take into account the influence of the local interactions between the phases on the stress and strain fields of the ERV. According to Benveniste formulation (Benveniste,87), the obtained strain-concentration tensor is as follows:

1 N 1 j j E j 0 i E i MT A f I f A A - =         + = ∑ [2]
From [1] and [2] the ERV equivalent stiffness is given by:

( ) ∑ ∑ = - =         + ⋅ ⋅ - + = N 1 i 1 N 1 j j E j 0 i E 0 i i 0 ERV MT A f I f A C C f C C [3]

Coupling of the Advani's tensor and Mori and Tanaka's method

A mathematical representation of fibres orientation distribution (FOD) in injected composite material with a symmetric rank 2 tensor is proposed by Advani (Advani and Tucker,87). As much as this quantity is commonly used to provide fibre orientation results from simulation or measurements, Advani's tensor does not allow a direct estimation of the overall set of elastic properties.

The use of the Advani's tensor in an estimation of the elastic properties of an equivalent homogeneous material has to be performed in accordance to the geometrical meaning of the components a ij . In particular, the components a 11 , a 22 and a 33 show the probability of presence of a fibres population in the primary global basis associated with the given Advani's tensor. The off axis components (a 12 , a 13 and a 23 ) are essential to the three-dimensional geometrical representation of FOD but it is difficult to underline the physical reality of their contributions.

Thus, the eigendirections of the Advani's tensor corresponding to ERV vectors basis represent always a fair situation in order to use FOD into Mori & Tanaka's method. By meaning of the corresponding eigenvalues α i as probability of presence of three heterogeneities, the fibres volume fraction f in the ERV is spread in the eigendirections with f . i α . The relation [3] becomes:

( ) ∑ ∑ = - =         α + ⋅ ⋅ - α + = 3 1 i 1 3 1 j j E j 0 i E 0 i i 0 ERV MT A f I f A C C f C C [4]
In the above relation, we note that Eshelby's strain-concentration tensors i E A depends on the transformation tensor in terms of Euler angles which performs the rotation from local system of each heterogeneity in each eigendirection to principal basis of Advani's tensor.

The elastic properties thus calculated are expressed in the principal directions of the Advani's tensor. Finally, the shift from the principal directions to the primary directions of Advani's tensor is performed thanks to the transformation tensor built with the normalised eigenvectors.

To sum up, the probability of presence of fibres in a given direction is represented by the diagonal Advani's tensor. Its principal directions define the great axis of the three heterogeneities for the corresponding fibres groups. They represent the directions of the local axis of the heterogeneities whose respective fibre contents are a weighting of the total fibre content by the eigenvalues of the Advani's tensor. Mori and Tanaka's estimation of the stiffness matrix of the equivalent homogeneous material is carried out in these axes. The last stage consists in coming back to the primary basis.

Interfacing the simulation tools

Calculating the elastic properties from the orientation tensors is used to interface the tools of the injection simulation through the orientation prediction and the computer codes of structure.

Interfacing strategy

For the design of a part, the engineer has to fulfil the feasibility and mechanical behaviour requirements. FOD is the bridge between results of the injection condition simulation and a stress analysis, starting from determining the homogeneous mechanical properties. This tool has to be usable by the design department. For that the designer must have means of graphically representing the intermediate results for a non stop analysis from the process to the structure design.

MTD (Mori-Tanaka-DOS) is the generic name of the program allowing the processing of orientation measures coming from the analysis of MEB images or from a simulation thanks to Moldflow® software for example.

In this way, the series of operations leading to a stress analysis starts with a CAD model of the part (Figure 4). The part is then meshed and imported in Moldflow® injection simulation software used in this study. According to the adjustments of the specified injection parameters (pressure, temperature, gate(s) localization and so on), the calculation of the orientation prediction is made. As output data, the orientation results are given in the form of one or several Advani's tensors per finite element (several Advani's tensors for the given finite element represent the FOD through the thickness).

Starting from the orientation data, the calculation and the localization of the distributions of homogeneous elastic properties are performed along the injected parts. A first possibility is to calculate mechanical characteristics to the finite elements defined for a Moldflow® simulation. The cell used for the injection model, can be use for a stress analysis. Apart from the Moldflow® meshing, the designer can juxtapose a mesh devoted to the stress analysis (with other types of finite elements, thinness, and so on). It should be emphasized that this assignment of properties is done by means of a proximity criterion. This criterion gets a sense only if the two meshes (Moldflow® and stress analysis) have been created from the same CAD model of the studied part and with some obvious conditions of common sense.

MTD interface patterns

Interface MTD is a program written in FORTRAN 77 allowing a wide use under MS-DOS and UNIX with various blocks of routines representing more than 10000 lines of code. The user interface allows the acquisition of input data via a command file with a MTD specific syntax. The designer has some "user" information currently operating at his disposal such as the data card reports or warnings and errors during MTD execution. A graphic interface called XMTD can be the user's assistant for the edition of the command files, the executions of calculations and the visualization of the intermediate results provided by MTD.

Numerical results

Several illustrations of the various stages of the calculation sequence are presented for the three industrial parts.

The orientation prediction

The fibres orientation data are obtained at the end of filling simulation of the volume of the part (performed by Moldflow®) with the injection parameters defined thanks to the moulder. The study is limited to the mere filling phase. The runners, the global cycle of injection including the packing and cooling times have not been modelled. These reductions are justified because only the FOD are required. In this case, we suppose that only the filling phase has an effect on the FOD (Figure 5).

Computation and assignment of elastic properties

The orientation tensors are the input data of calculation, in each finite element (ERV), of the elastic properties for their use in a stress analysis via the MTD interface. The visualization of the properties is not possible with the commercial computer codes such as for example the codes used by the project partners (Samcef®, Ansys®, I-deas® and Nastran®). Visualization options can be activated by MTD interface for real noting the distributions of some Young's moduli of the composite parts (Figure 6). Figure 6 shows for example with grey levels the distribution of the Young's modulus in the direction of the paddle height of the Microturbo stator blade. The aspect ratio r equal to 15 has been obtained after a numerical fitting by MTD thanks to simple standard traction specimens (Figure 6). This result recovers an experimental characterisation of the length distribution of fibres carried out by C. Levaillant's research team and Liebherr Aerospace. In more detailed way, the injection and filling conditions impose a single direction of the fibres along the great axis of the specimen. The experimental value of the Young's modulus in this direction is of 9.8 GPa. Figure 6 shows a strong sensitivity of the Young's modulus versus the aspect ratio. Indeed, a corresponding value for r = 10 is about 9 GPa. In this situation, it is obvious to get the accurate aspect ratio value thanks to homogenization.

Stress analysis

The stiffness matrices calculated on each finite element of the Moldflow® mesh can give birth to the following alternative. They can be used for the export of elastic moduli on the same elements and finally for the edition of a data file for a computer code containing the coordinates of the nodes, connections of the finite elements and the elastic moduli of the composite (what implies in our example to re-use the triangles of the Moldflow® model). The stiffness values can be assigned on geometrical points thus making possible to associate elastic moduli with the finite elements of another mesh type which would come to be superimposed instead of that of the Moldflow® model. In this case, MTD writes a data file for a computer code containing only the characteristics of the materials assigned to each finite element of the mesh chosen for the stress analysis.

Finally, the output data of MTD interface allow to start a stress analysis for any model. The nodes, the finite elements and the properties of materials being defined, it remains then to provide the modelling elements relating to the boundary conditions for example to end mechanical calculations (Figure 8). With the confidential industrial specifications of the modelling stages, a numerical simulation of the stator blade with quadratic tetrahedral finite elements is performed, body valve and fan wheel as well.

Conclusion

The main problem of the short fibres composite structures is the lack of information concerning the heterogeneity in the composite parts. The heterogeneous nature depends on both the process and the design phase. It is an impediment of the development of this solution in industry for a low cost. The cost reduction of composite parts depends on the development of an advanced numerical tool capable to loop from the manufacturing conditions to the mechanical response of a part. The MTD interface is right now used by the industrial partners with their own codes of structure. This interface allows the designer to take into account the influence of the injection process on the distribution of the mechanical properties. The experimental validation is in progress thanks to industrial partners on several scales, starting from the specimens to the industrial parts in service conditions.

Figure 1 .

 1 Figure 1. Industrial goals (in Ultem 2300)

Figure 4 .

 4 Figure 4. Interfacing strategy and operation sequence

Figure 5 .

 5 Figure 5. Filling of industrial parts at 0.5 second (Moldflow® software)

Figure 6 .

 6 Figure 6. Distribution of the mechanical characteristics (Young's moduli in Pa)

Figure 7 .

 7 Figure 7. MTD distribution of the Young's modulus (in Pa) values after injection in the main axis of the specimen

Figure 8 .

 8 Figure 8. Von Mises stress map for the three parts under their mechanical operation by Samcef software after the backing of the Moldflow® orientation predictions

This work features interactions with two other PhD. in progress (M. Wesselmann and G. Saint-Martin) supervised by CROMeP and LIEBHERR and TECHNOFAN firms.

8 th Euro-Japanese Symposium. Volume X -No. X/2001

8 th Euro-Japanese Symposium. Volume X -No. X/2001

Acknowledgements

The authors would like to thank Claude Rossignol and Matthias Wesselmann (Liebherr Aerospace), Olivier Darnis and Gilles Saint-Martin (Technofan) for their helpful technical

collaboration. This work was done with the financial support of the Midi-Pyrenees Regional Council and the French Agency of Technical Research (ANRT).