Shao-Min Zou1

Patrick Erbacher

Jean-Serge Remy

Jean-Paul Behr
email: behr@aspirine.u-strasbg.fr

Systemic linear polyethylenimine (L-PEI)mediated gene delivery in the mouse. +

Background: Several nonviral vectors including L-PEI confer a pronounced lung tropism to plasmid DNA when injected in the mouse tail vein in a non-ionic solution.

Methods and Results:

We have optimized this route by injecting 50 µg DNA with excess L-PEI (N/P=10) in a large volume of 5% glucose (0.4 ml). In these conditions, 1-5% percent of lung cells were transfected (corresponding to 2 ng luciferase/mg protein), the other organs remaining essentially refractory to transfection (1-10 pg luciferase/mg protein). Beta-galactosidase histochemistry confirmed alveolar cells, including pneumocytes, to be the main target, thus leading to the puzzling observation that the lung microvasculature must be permeable to cationic L-PEI/DNA particles of ca. 60 nm. A smaller injected volume, premixing of the complexes with autologous mouse serum, as well as removal of excess free L-PEI, all severely decreased transgene expression in the lung. Arterial or portal vein delivery did not increase transgene expression in other organs.

Conclusions: These observations suggest that effective lung transfection primarily depends on the injection conditions: the large nonionic glucose bolus prevents aggregation as well as mixing of the cationic complexes and excess free L-PEI with blood. This may favour vascular leakage in the region where the vasculature is dense and fragile, i.e. around the lung alveoli. Cationic particles thus can reach the epithelium from the basolateral side where their receptors (heparan sulfate proteoglycans) are abundant.

INTRODUCTION

Non-viral vectors continue to arouse interest because of their safety and low immunogenicity profiles. Major drawbacks still remain, such as low efficacy and short duration of transgene expression. However, in several recent reports describing cationic lipid formulations that were optimised for systemic delivery [START_REF] Osaka | Pharmacokinetics, tissue distribution, and expression efficiency of plasmid [P-33] DNA following intravenous administration of DNA/cationic lipid complexes in mice: Use of a novel radionuclide approach[END_REF][START_REF] Li | In vivo gene transfer via intravenous administration of cationic lipid-protamine-DNA (LPD) complexes[END_REF][START_REF] Hara | In vivo gene delivery to the liver using reconstituted chylomicron remnants as a novel nonviral vector[END_REF] by exploring either new structures [START_REF] Stephan | A new cationic liposome DNA complex enhances the efficiency of arterial gene transfer in vivo[END_REF][START_REF] Mclean | Organ-specific endothelial cell uptake of cationic liposome-DNA complexes in mice[END_REF], formulation conditions [START_REF] Thierry | Systemic gene therapy: Biodistribution and longterm expression of a transgene in mice[END_REF][START_REF] Hofland | In vivo gene transfer by intravenous administration of stable cationic lipid DNA complex[END_REF][START_REF] Templeton | Improved DNA: Liposome complexes for increased systemic delivery and gene expression[END_REF][START_REF] Liu | Factors influencing the efficiency of cationic liposomemediated intravenous gene delivery[END_REF] or additives [START_REF] Hong | Stabilization of cationic liposome-plasmid DNA complexes by polyamines and poly(ethylene glycol)-phospholipid conjugates for efficient in vivo gene delivery[END_REF][START_REF] Li | Characterization of cationic lipid-protamine-DNA (LPD) complexes for intravenous gene delivery[END_REF][START_REF] Barron | Effects of complement depletion on the pharmacokinetics and gene delivery mediated by cationic lipid-DNA complexes[END_REF], promising expression levels were reached. Polylysine, a member of the other class of synthetic vectors, has been extensively tested for systemic gene delivery as well [START_REF] Wagner | Delivery of drugs, proteins and genes into cells using transferrin as a ligand for receptor-mediated endocytosis[END_REF][START_REF] Frese | Targeting of genes to the liver with glycoprotein carriers[END_REF], especially for targeting the liver [START_REF] Wu | Targeting genes -Delivery and persistent expression of a foreign gene driven by mammalian regulatory elements invivo[END_REF]. The low efficacy of intracellular release of polylysine/DNA complexes [START_REF] Chowdhury | Fate of DNA targeted to the liver by asialoglycoprotein receptor-mediated endocytosis invivo -Prolonged persistence in cytoplasmic vesicles after partial hepatectomy[END_REF] has impeded further development. The discovery that some cationic polymers, such as polyethylenimine (PEI) [START_REF] Boussif | A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethylenimine[END_REF] and polyamidoamine dendrimers [START_REF] Tang | In vitro gene delivery by degraded polyamidoamine dendrimers[END_REF][START_REF] Kukowska-Latallo | Efficient transfer of genetic material into mammalian cells using Starburst polyamidoamine dendrimers[END_REF], facilitate endosomal release, may lead to a revival of this class of vectors. Indeed, recent work shows that PEI is a good vehicle for polynucleotide delivery in vivo following lung instillation [START_REF] Ferrari | ExGen 500 is an efficient vector for gene delivery to lung epithelial cells in vitro and in vivo[END_REF], kidney perfusion [START_REF] Boletta | Nonviral gene delivery to the rat kidney with polyethylenimine[END_REF], intracerebral injection [START_REF] Boussif | A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethylenimine[END_REF][START_REF] Abdallah | A powerful nonviral vector for in vivo gene transfer into the adult mammalian brain: Polyethylenimine[END_REF][START_REF] Goula | Size, diffusibility and transfection performance of linear PEI-DNA complexes in the mouse central nervous system[END_REF] or systemic delivery to the lung [START_REF] Goula | Polyethylenimine-based intravenous delivery of transgenes to mouse lung[END_REF] or to the liver [START_REF] Kren | In vivo site-directed mutagenesis of the factor IX gene by chimeric RNA/DNA oligonucleotides[END_REF][START_REF] Chemin | Liver-directed gene transfer: a linear polyethlenimine derivative mediates highly efficient DNA delivery to primary hepatocytes in vitro and in vivo[END_REF]. Among the various molecular weights (700 -8.10 5 Da) and topological isomers (branched or linear skeleton) available for PEI, the linear 22 kDa compound (L-PEI) has shown the most favourable properties. Indeed, L-PEI-mediated transfection remains effective in serum [START_REF] Boussif | Optimized galenics improve in vitro gene transfer with cationic molecules up to 1000-fold[END_REF][START_REF] Boussif | Transfert de gènes par des polymères cationiques[END_REF] and, when formulated with DNA in low salt, leads to small (ca. 60 nm) particles that are less prone to size-restricted diffusion in vivo [START_REF] Goula | Size, diffusibility and transfection performance of linear PEI-DNA complexes in the mouse central nervous system[END_REF].

In this report, L-PEI/plasmid complexes were injected into the venous and arterial blood stream. Analysis of the spatial and temporal expression profiles showed the lung to be the only organ significantly (though transiently) transfected. Histochemistry revealed the pattern of cells expressing the transgene. Finally, several additional experiments allow us to propose a hypothesis for the lung expression tropism that is generally observed following tail vein DNA delivery with cationic vectors.

MATERIALS AND METHODS

Intravenous injection of the complexes into the tail vein and luciferase expression

pCMVLuc and L-PEI (Exgen 500, Euromedex, Souffelweyersheim, France; N/P=10) were diluted separately in 200 µl of 5% glucose. After 10 min, solutions were mixed, homogenised immediately and left for 10 min. Female Balb/C mice (5 weeks old, CERJ, Le Genest-St-Isle, France) were placed in a restrainer and 400 µl of PEI/DNA complexes/mouse (n>4) were injected in the tail vein within 30 sec, using a 1/2 inch 26-gauge needle and a 1 ml syringe. Animals were anaesthetised after 24 h by breathing ether and decapitated to drain the blood off. Heart, lung, spleen, kidneys and 120-140 mg of the liver were collected in 1.5 ml Eppendorf tubes. The tissues were rapidly frozen in dry ice, then in liquid nitrogen. After thawing, samples were transferred into 10 ml plastic tubes containing 1 ml precooled lysis buffer (Promega, Cergy Pontoise, France) supplemented with a protease inhibitor cocktail (1 ml/20 g tissue, Sigma, Saint Quentin Fallavier, France). Tissues were homogenized with an Ultra-Turrax T25 (Janke & Kunkel, Staufen, Germany) at 20,000 rpm for 30 s and subjected to three (-80°C/37°C) freeze/thaw cycles. The homogenates were centrifuged at 14,000g for 4 min at 4°C and 20 µl supernatant aliquots were assayed for luciferase activity using a commercial kit (Promega) and a luminometer (Biolumat LB 9500, Berthold, Paris, France). Results were expressed as light units integrated over 10s ± s.d., per mg of cell protein using the BCA assay (Pierce, Paris, France).

Luciferase content was determined from a standard curve obtained with Photinus pyralis firefly luciferase (Boehringer Mannheim, Meylan, France) using the equation ng luciferase = (RLU + 1.25X10 6 ) / (5.03X10 7 ). β-Galactosidase histochemistry in the lung L-PEI/DNA complexes were prepared with 50 µg pCMVßgal (N/P=10) and injected in the tail vein as described above. Mice were sacrificed 24h later by decapitation. After windowing the right atrium, a plastic catheter was placed inside the ascending aorta via the left ventricle. Mice were first perfused at room temperature with 5 ml PBS containing 1% glutaraldehyde, 2% paraformaldehyde, 0.01% sodium deoxycholate and 0.02% Nonidet P-40 (PBS fixative solution), and then for 3 h at 4°C with the same solution. A last perfusion was performed with PBS only for 1 h. Lungs were collected and incubated at 37°C for 24 h in a solution containing 1 mg/ml X-gal (stock solution in dimethylformamide at 40 mg/ml, Euromedex), 5 mM K 3 Fe(CN) 6 , 5 mM K 4 Fe(CN) 6 , 2 mM MgCl 2 in 10 mM PBS, pH 7.4. The lungs were washed three times with 3% DMSO in PBS and photographed before being embedded in paraffin. Continuous 6 µm-thick sections were cut off, mounted on glass slides, and counterstained with nuclear red.

Measurement of zeta potentials and of the amount of PEI in the complexes

DNA (40 µg pCMVLuc) and the desired amount of L-PEI were each diluted in 500 µl of a 5% glucose or a 150 mM NaCl solution. After 10 min, solutions were mixed, homogenized and left for 10 min at room temperature. The zeta potential of the complexes was measured electrophoretically [START_REF] Erbacher | Transfection and physical properties of various saccharide, poly(ethylene glycol), and antibody-derivatized polyethylenimines (PEI)[END_REF] using a Zetasizer 3000 (Malvern Instrument, Orsay, France). In order to measure the fraction of PEI present in the PEI/DNA complexes, PEI was labelled with fluorescein isothiocyanate (Sigma) in a 0.2 M borate buffer, pH 8.4. The conjugate was purified by gel filtration on a PD10 column (Sephadex G-25 M, Pharmacia Biotech, Sweden) in water. The concentration of PEI amine functions (N) was determined with the ninhydrin assay. The level of fluorescein conjugation per amino group of PEI was 1% as measured by absorption spectroscopy at 495 nm. Complexes were prepared with 50 µg DNA and the desired amount of PEI-fluorescein in a total volume of 500 µl 5% glucose. The solution of PEI/DNA complexes was filtered by centrifugation (500 g, 20 min, 20°C) through a Centricon-100 membrane (100,000 Da cut-off, Amicon, Beverly, MA). The amount of free PEI-fluorescein in the filtrate was determined by absorbance at 495 nm, from which the fraction of PEI that was associated with DNA was calculated.

RESULTS

Calibration of reporter gene expression and selection of the cationic vector

In vivo, the most commonly used reporter genes are those of the firefly luciferase for assessing overall transfection levels, and of the bacterial ß-galactosidase for characterizing the percentage and type of transfected cells. Photometric detection of luciferase activity is so sensitive that it may lead to misleading conclusions. Histochemical tissue analysis following X-gal staining, while meaningful, is not convenient for percentages <0.1%. As a prerequisite to animal experiments, we first decided to set up a conversion chart between these reporter genes, using data obtained for transfection of various cell types in vitro with a cationic lipid (Transfectam) and a cationic polymer (PEI). Luciferase levels, that were expressed as relative light units (RLU)/mg total cell proteins [START_REF] Boussif | Optimized galenics improve in vitro gene transfer with cationic molecules up to 1000-fold[END_REF], which is apparatus-dependent, were also plotted as nanogram reporter protein per milligram cell protein, a dimensionless number (Figure 1).

Comparison of the percentage of ß-galactosidase-positive cells with overall transfection levels showed that when virtually all cells were transfected, they produced ca. 100 ng luciferase/mg cell protein, i.e. 0.01% of their protein mass as exogeneous protein. The interesting transfection levels were found within the range 0.1-100 ng luc/mg protein (10 7 -10 10 RLU/mg protein), where the percentage of ßgal-positive cells (0.5-100%) was roughly proportional to the overall luciferase level. The lower limit of this range is still >1,000-fold over background (Figure 1), which highlights the extreme sensitivity of photonic detection: a single transfected cell can be detected among 10 7 cells! As a take-home rule, 1 ng luc / mg protein thus corresponds to 1% transfected cells. This gives an idea of luciferase levels to be reached in animals.

With regard to the vector, several polyethylenimines are available [START_REF] Remy | Gene transfer with lipospermines and polyethylenimines[END_REF][START_REF] Kichler | Polyethylenimines: a family of potent polymers for nucleic acid delivery[END_REF], that differ from one another in molecular weight (15-1,000 kDa) and in topology (linear or branched). While most were of comparable efficacies in vitro, only the linear 22 kDa polymer (L-PEI) remained insensitive to up to 30% serum during transfection [START_REF] Boussif | Transfert de gènes par des polymères cationiques[END_REF]. In addition, preliminary in vivo screening of polymers showed branched PEI to be very toxic after tail vein injection: animals died within a few hours, presumably as a consequence of the numerous microemboli found in their lung tissues.

Figure 1. A conversion chart between overall transfection levels (as measured by luciferase activity) and the percentage of ß-galactosidase-positive cells.

Cells were transfected in 24-well plates using 2µg DNA (pCMVluc or pCMVßgal) complexed with a cationic lipid (Transfectam, N/P=6) or with a cationic polymer (PEI, N/P=9). Luciferase activity after 24 h was normalised per mg of cell protein as described in Materials and Methods. Alternatively, cells that were transfected with PEI/pCMVßgal were fixed and counted for blue staining following incubation with X-gal. Raw data are taken from ref [START_REF] Boussif | Optimized galenics improve in vitro gene transfer with cationic molecules up to 1000-fold[END_REF]. L-PEI was not lethal when formulated in isotonic NaCl, but led respectively to one thousand-and ten-fold lower expression than with isotonic glucose in the lung and in the heart; other organs were not affected. We thus used L-PEI/DNA complexes in 5% glucose [START_REF] Goula | Size, diffusibility and transfection performance of linear PEI-DNA complexes in the mouse central nervous system[END_REF][START_REF] Goula | Polyethylenimine-based intravenous delivery of transgenes to mouse lung[END_REF][START_REF] Ogris | The size of DNA/transferrin-PEI complexes is an important factor for gene expression in cultured cells[END_REF] for all experiments. In these salt-free conditions, complexes remain small because electrostatic repulsion is stronger than Van der Waals attraction [START_REF] Erbacher | Transfection and physical properties of various saccharide, poly(ethylene glycol), and antibody-derivatized polyethylenimines (PEI)[END_REF].

Body distribution of transgene expression following tail vein injection

Increasing amounts of pCMVluc/L-PEI complexes in 400 µl 5% glucose were injected within 30 seconds in the tail vein of BalbC mice. The amount of luciferase found in the lung, heart, spleen, liver and kidney after 24 hours increased 50-100 fold when the amount of DNA was increased from 20 µg to 50 µg (Figure 2). Higher amounts (100 µg) became toxic (only two animals survived out of four) and did not significantly increase transfection. Expression was ca. one hundred-fold higher in the lung than in the other organs. Mice injected with up to 50 µg DNA complexed with L-PEI showed no signs of toxicity. Injection of 100 µg DNA complexed with PEI led to 50% mortality within 24 hours. Autopsy showed frequent scattered red embolism centers in the lung and necrosis centers in the liver. To assess the influence of the amount of L-PEI on gene delivery, 50 µg pCMVluc were injected with increasing amounts of cationic polymer (Figure 3). Since cell entry relies on electrostatic interaction of the complexes with the cell surface [START_REF] Behr | Efficient gene transfert into mammalian primary endocrine cells with lipopolyamine-coated DNA[END_REF][START_REF] Labatmoleur | An electron microscopy study into the mechanism of gene transfer with lipopolyamines[END_REF], it is convenient to express the PEI/DNA ratio as amine over phosphate. Complexes that were electroneutral in physiological salt concentration (N/P=3, see zeta potentials below) did not transfect significantly better than naked DNA (N/P=0). Increasing N/P ratio (4 to 10) led to increasing levels of transfection in all organs. Very high ratio (N/P=20, not shown) were lethal. Interestingly, after the onset of transfection (N/P>3, Figure 3), the amount of luciferase found in the lung was always a hundred-fold higher than that found in the other organs. Such pronounced lung expression tropism following tail vein injection has been described by several authors, irrespective of the cationic lipid or polymer formulation used [START_REF] Li | In vivo gene transfer via intravenous administration of cationic lipid-protamine-DNA (LPD) complexes[END_REF][START_REF] Thierry | Systemic gene therapy: Biodistribution and longterm expression of a transgene in mice[END_REF][START_REF] Liu | Factors influencing the efficiency of cationic liposomemediated intravenous gene delivery[END_REF][START_REF] Hong | Stabilization of cationic liposome-plasmid DNA complexes by polyamines and poly(ethylene glycol)-phospholipid conjugates for efficient in vivo gene delivery[END_REF][START_REF] Goula | Polyethylenimine-based intravenous delivery of transgenes to mouse lung[END_REF][START_REF] Zhu | Systemic gene expression after intravenous DNA delivery into adult mice[END_REF]. According to the chart of Figure 1, transfection levels found in the other organs are very low (although well over background). Luciferase levels found in the lung (2 ng/mg protein), however, corresponded to the range of one percent transfected cells (see previous paragraph). Although transgene expression decreased by two orders of magnitude after 2 days (Goula et al. [START_REF] Goula | Polyethylenimine-based intravenous delivery of transgenes to mouse lung[END_REF] and data not shown), presumably because of cytotoxicity, it was therefore of interest to find out which were the transfected cell types.

Lung Heart Liver Spleen Kidney

Histochemical analysis of transgene expression in the lung

Tail vein injection was repeated with 50 µg pCMVßgal and L-PEI (N/P=10). Organs were perfused and stained in toto with X-gal. As expected from the luciferase experiments, only the lung tissue showed visible transgene expression. A control experiment (Figure 4a) performed with pCMVluc/L-PEI complexes confirmed the lung to be devoid of endogeneous ß-galactosidase staining after 24 h. In contrast, macroscopic examination of a lung lobe from a mouse transfected with pCMVßgal showed an intense blue staining that was evenly spread all-over the organ (Figure 4b). Examination of thin sections under the microscope (Figure 4c) showed 1-5% of lung cells to be transfected. This level is somewhat higher than that obtained by Goula et al. [START_REF] Goula | Polyethylenimine-based intravenous delivery of transgenes to mouse lung[END_REF] using 125 µg DNA and N/P=4. Transfection was restricted to the distal region of the airway tree, in the vicinity of the alveoli. Epithelial cells of the small conducting airways were not transfected (inset in Figure 4c). A closer (X400) examination of the alveolar tissue sections showed that transfected cells were essentially pneumocytes (Figure 4d) and some endothelial cells (inset in Figure 4d). This histologic profile led to the conclusion that the lung vascular endothelium was permeable to PEI/DNA particles as large as 60 nm.

Heart

Why is transgene expression so high in the lung?

Although nonlethal experimental conditions could be worked out, tail vein injection of 0.4 ml (ca. 20% of the mouse total blood volume) transfection mix over 30 s with a 26-gauge needle may simultaneously occlude the tail vein blood circulation during the injection procedure, push a glucose bolus throughout the general circulation back towards the lung and eventually generate hydrostatic pressure. Besides the known effects of pressure on vascular leakage and transfection [START_REF] Wolff | Direct gene transfer into mouse muscle in vivo[END_REF][START_REF] Budker | Naked DNA delivered intraportally expresses efficiently in hepatocytes[END_REF][START_REF] Zhang | Expression of naked plasmid DNA injected into the afferent and efferent vessels of rodent and dog livers[END_REF][START_REF] Liu | Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA[END_REF], this protocol could prevent most of the L-PEI/DNA complexes from binding to blood proteins responsible for decreased cell-surface binding. To test this hypothesis, the L-PEI/DNA complexes in 0.2 ml were incubated for 15 min with 0.2 ml autologous mouse serum prior to injection. Although ionic strength was increased, complexes remained small due to coating by serum proteins [START_REF] Ogris | The size of DNA/transferrin-PEI complexes is an important factor for gene expression in cultured cells[END_REF] hence animals histology showed no sign of microemboli (not shown). The lung expression tropism, however, was lost (Figure 5). In another experiment where complexes were injected in a fourfold smaller volume, expression in the lung was decreased eightfold (Figure 5). These findings thus emphasize the critical importance of the injection conditions in successful gene delivery to the lung. Complexes were injected in the tail vein (n = 3). Organs were collected 24h later and luciferase activity in homogenates was determined as described in Materials and Methods. Another crucial and eloquent factor is the L-PEI/DNA (N/P) ratio: the onset of lung transfection (N/P>3, Figure 3) correlates well with a particle surface charge (zeta potential, Figure 6) becoming positive when measured in 150 mM NaCl rather than in glucose. This is evidence that complexes experience a physiological salt environment after vascular escape. Furthermore, transfection of all organs is very much increased when N/P increases from 4 to 10 (a similar trend was found for cationic lipids [START_REF] Liu | Factors controlling the efficiency of cationic lipid-mediated transfection in vivo via intravenous administration[END_REF], although the surface charge (Figure 6) and size [START_REF] Goula | Size, diffusibility and transfection performance of linear PEI-DNA complexes in the mouse central nervous system[END_REF] of the complexes remain constant. This latter remark led us to measure the amount of PEI that was effectively bound to DNA. Indeed, in the asymptotic zeta potential regime, most of the extra PEI is not associated with the complexes (Figure 6). The beneficial effect of excess free PEI on transfection is, however, very pronounced (Figure 3). In an attempt to assess its exact role, we preinjected the mice with approximately the amount of free PEI found in solutions where N/P=10 (i.e. corresponding to N=6) and injected the cationic complexes made at N/P=4 some 15 min later: transfection results in all organs (not shown) were comparable to those found for N/P=4 (see Figure 3), showing that excess PEI, to be beneficial, must be present simultaneously with the complexes.

We next checked whether specific lung expression could be redirected to another organ. Injection of L-PEI/pCMVluc complexes into the portal vein decreased lung expression fifteen-fold (Figure 7); injection into the left ventricle of the heart decreased lung expression 80-fold. Remote injection thus decreased the lung tropism, in agreement with the aforementioned hypothesis. However portal vein injection did not increase luciferase expression in the liver, nor did intracardiac injection increase expression in organs with a direct arterial blood supply. L-PEI/DNA complexes were prepared in 400 µl of 5% glucose with 50 µg pCMVLuc (N/P=10). For tail vein injection, see Materials and Methods. For portal vein injection, mice were anaesthetised by intramuscular injection of ketamine hydrochloride (500 µg/20 g of body weight). The mesenteric vein was surgically exposed by a 1.5 cm abdominal midline incision and complexes diluted to 1 ml with 5% glucose were injected in the portal vein over at least one minute, using a 1/2 inch 30-gauge needle and a 1 ml syringe (n=3). For intracardiac injection, mice were anaesthetised as described above. A median sternal incision of the skin exposed the left thorax. A left ventricular puncture was performed between the 4th and 5th intercostal space on the left clavicular midline using a 1/2 inch 30-gauge needle and a 1 ml syringe; complexes were injected over 30s (n=3). Organs were collected and processed as described in Materials and Methods. In a nonionic glucose solution, the surface charge of the complexes reaches a limiting cationic value for N/P of 2-3. Accordingly, the amount of PEI in the complexes should then remain constant, the extra amount of PEI being free polymer in solution. This was roughly verified, e.g. for N/P=10, 30% of PEI is complexed, i.e. in the complexes, N/P has reached its limiting value of 3.

PEI in complexes (%)

CONCLUSION

Altogether, these results show that organs are generally not significantly transfected by systemic delivery of cationic complexes. Only their vascular endothelium is accessible to the particles, and primary endothelial cells are known to be difficult to transfect [START_REF] Boussif | Optimized galenics improve in vitro gene transfer with cationic molecules up to 1000-fold[END_REF], presumably because they lack the desired receptor (heparan sulfate proteoglycan HSPG [START_REF] Labatmoleur | An electron microscopy study into the mechanism of gene transfer with lipopolyamines[END_REF][START_REF] Mislick | Evidence for the role of proteoglycans in cation-mediated gene transfer[END_REF][START_REF] Mounkes | Proteoglycans mediate cationic liposome-DNA complex-based gene delivery in vitro and in vivo[END_REF]) on their apical membrane [START_REF] Gotte | Endocytosis of decorin by bovine aortic endothelial cells. off[END_REF]. The pharmacokinetics of cationic DNA complexes distribution has shown that cationic DNA complexes are essentially taken up by the lung and the liver [START_REF] Osaka | Pharmacokinetics, tissue distribution, and expression efficiency of plasmid [P-33] DNA following intravenous administration of DNA/cationic lipid complexes in mice: Use of a novel radionuclide approach[END_REF][START_REF] Mclean | Organ-specific endothelial cell uptake of cationic liposome-DNA complexes in mice[END_REF][START_REF] Mahato | In vivo disposition characteristics of plasmid DNA complexed with cationic liposomes[END_REF]. Liver uptake takes place in Kupffer cells [START_REF] Mahato | In vivo disposition characteristics of plasmid DNA complexed with cationic liposomes[END_REF], yet macrophages too are difficult to transfect [START_REF] Mclean | Organ-specific endothelial cell uptake of cationic liposome-DNA complexes in mice[END_REF]. The lung endothelium represents a large surface that may be mechanically and osmotically fragile since the alveolar endothelial/epithelial junction must be thin for fast gas exchange to occur. In the particular experimental setup described above, the small L-PEI/DNA complexes present in nonionic glucose solution may thus escape from a transiently leaky vasculature and reach the neighbouring polarized cells from the basolateral side where the anionic HSPG receptor is expressed [START_REF] Duan | Polarity influences the efficiency of recombinant adenoassociated virus infection in differentiated airway epithelia[END_REF], hence the exceptionally high level of transfection found in the lung.

Unfortunately, this surprising result requires conditions that may never be scaled up to large animals (it would correspond to a 1 liter glucose bolus injected within minutes in a human; but see also ref [START_REF] Zhang | Expression of naked plasmid DNA injected into the afferent and efferent vessels of rodent and dog livers[END_REF]). More reasonable goals for systemic delivery of particulate DNA vectors are targeting of mitotic integrin-expressing endothelial cells of tumors [START_REF] Erbacher | Gene transfer with synthetic virus-like particles via the integrinmediated endocytosis pathway[END_REF], or of asialoglycoprotein-expressing hepatocytes [START_REF] Zanta | In vitro gene delivery to hepatocytes with galactosylated polyethylenimine[END_REF][START_REF] Bettinger | Size reduction of galactosylated PEI/DNA complexes improves lectin-mediated gene transfer into hepatocytes[END_REF] that are accessible through the fenestrated liver endothelium. These targets require the complexes to remain active and small in the blood, an objective which is being actively pursued by several groups [START_REF] Hong | Stabilization of cationic liposome-plasmid DNA complexes by polyamines and poly(ethylene glycol)-phospholipid conjugates for efficient in vivo gene delivery[END_REF][START_REF] Barron | Effects of complement depletion on the pharmacokinetics and gene delivery mediated by cationic lipid-DNA complexes[END_REF][START_REF] Ogris | PEGylated DNA/transferrin-PEI complexes: reduced interaction with blood components, extended circulation in blood and potential for systemic delivery[END_REF][START_REF] Dash | Factors affecting blood clearance and in vivo distribution of polyelectrolyte complexes for gene delivery[END_REF].

Figure 2 .

 2 Figure 2 . Intravenous injection of L-PEI/DNA complexes in the mouse leads to detectable reporter gene expression in various organs.Expression was ca. one hundred-fold higher in the lung than in the other organs. Mice injected with up to 50 µg DNA complexed with L-PEI showed no signs of toxicity. Injection of 100 µg DNA complexed with PEI led to 50% mortality within 24 hours. Autopsy showed frequent scattered red embolism centers in the lung and necrosis centers in the liver.

Figure 3 .

 3 Figure 3. Gene delivery in vivo requires cationic complexes.L-PEI/DNA complexes were prepared with 50 µg pCMVluc and various amounts of L-PEI and injected into the tail vein of mice (4<n<6) according to Materials and Methods. In physiological salt conditions, complexes become positively charged for N/P>3 (see surface charge in figure6).

Figure 4 .

 4 Figure 4. Expression pattern of ß-galactosidase in the lung A) Control experiment: section of a Xgal-incubated lung after transfection with L-PEI/pCMVluc complexes (magnification X100). B) After ß-galactosidase gene delivery, the left lung lobe shows intense and evenly distributed blue staining. C) Section through the alveolar region shows 1-5% ßgal-positive cells (X100); inset: epithelial cells of the small conducting airways are not transfected. D) Higher magnification of transfected alveoli (X400) and of a capillary (inset X800).

Figure 5 .

 5 Figure 5. Gene delivery to the lung is decreased by tail vein injection of L-PEI/DNA complexes in a smaller volume or by mixing with serum. Plasmid (50 µg pCMVLuc) and L-PEI (N/P=10) were each diluted separately in 50, 100 or 200 µl 5% glucose. After 10 min, solutions were mixed, homogenized immediately and left for 10 min, thus giving L-PEI/DNA complexes in final volumes of 100, 200 and 400 µl. Complexes in 200 µl 5% glucose were mixed with 200 µl autologous mouse serum and incubated for 10 min.Complexes were injected in the tail vein (n = 3). Organs were collected 24h later and luciferase activity in homogenates was determined as described in Materials and Methods.

Figure 7 .

 7 Figure 7. Systemic gene delivery leads to scattered expression profiles.L-PEI/DNA complexes were prepared in 400 µl of 5% glucose with 50 µg pCMVLuc (N/P=10). For tail vein injection, see Materials and Methods. For portal vein injection, mice were anaesthetised by intramuscular injection of ketamine hydrochloride (500 µg/20 g of body weight). The mesenteric vein was surgically exposed by a 1.5 cm abdominal midline incision and complexes diluted to 1 ml with 5% glucose were injected in the portal vein over at least one minute, using a 1/2 inch 30-gauge needle and a 1 ml syringe (n=3). For intracardiac injection, mice were anaesthetised as described above. A median sternal incision of the skin exposed the left thorax. A left ventricular puncture was performed between the 4th and 5th intercostal space on the left clavicular midline using a 1/2 inch 30-gauge needle and a 1 ml syringe; complexes were injected over 30s (n=3). Organs were collected and processed as described in Materials and Methods.

  Tail vein Portal vein Left ventricle

Figure 6 .

 6 Figure 6. Surface charge (zeta potential, left axis) of L-PEI/DNA complexes and percentage of cationic polymer present in complexes formed in 5% glucose (right axis) as a function of the PEI to DNA ratio (N/P).In a nonionic glucose solution, the surface charge of the complexes reaches a limiting cationic value for N/P of 2-3. Accordingly, the amount of PEI in the complexes should then remain constant, the extra amount of PEI being free polymer in solution. This was roughly verified, e.g. for N/P=10, 30% of PEI is complexed, i.e. in the complexes, N/P has reached its limiting value of 3.

Journal of Gene Medicine Preprint 2:(2) 2000 © 2000 John Wiley & Sons, Ltd

ACKNOWLEDGEMENTS

We thank Dr. Pierre Bischoff (Institut d'Hématologie, Strasbourg) for providing animal facilities. The work was supported by grants from the Association pour la Recherche contre le Cancer (ARC) and the Ligue contre le Cancer. SZ and PE are recipient of postdoctoral fellowships from the Ministère des Affaires Etrangères and the ARC, respectively.


Ben Fatma

Guefrech

Florent Berthaut

Patricia Plénacoste

Yvan Peter

Laurent Grisoni

Revealable Volume Displays: 3D Exploration of Mixed-Reality Public Exhibitions

Keywords:

In this paper, we present a class of mixed-reality displays which allow for the 3D exploration of content in public exhibitions. The shared experience of the exhibition and the preservation of artworks are two very important aspects of these contexts, in particular for museum exhibits. The use of display cases as a protection tool is substantially accepted. It decreases the risks of damages to artworks and cultural materials hosted in museums. In addition, the transparent panels create a reflection of the visitors inside the display case. This reflection can be used to augment and interact in 3D with the exhibited content, by coupling Spatial Augmented-Reality and Optical Combiners. We call such a combination a Revealable Volume Display (RVD). It allows visitors to reveal information placed freely inside or around protected artefacts, visible by all, using their reflection in the panel. However, it may also suffer from unfamiliar gestures and disrupted depth perception cues, making 3D exploration of content difficult. In this paper, we first discuss the implementation of RVDs, providing both projector-based and mobile versions. We then present a design space that describes the interaction possibilities that they offer. Drawing on insights from a field study during a first exhibition, we finally propose and evaluate techniques for facilitating 3D exploration with RVDs.

INTRODUCTION

Mixed-reality has known in the past years impressive technological progress which democratised its use in our daily lives. It is now common in several aspects of human activity (games, distant social interaction, virtual tourism, ...). However, current approaches are still mostly limited to situations that are compatible with quite fragile and expensive (even though more and more accessible) devices. Consequently, when envisaging public situations, unless users come with their personal equipment, it is still difficult in practice to reasonably imagine to actually use such mixed-reality systems. Such interaction scenarios, where visitors are focused on their personal devices and interactions, also strongly limit a collectively shared visit experience.

In public situations where mixed-reality is aimed to combine digital information with existing (tangible) artefacts, one will also wish * fatma.benguefrech@univ-lille.fr † florent.berthaut@univ-lille.fr ‡ patricia.plenacoste@univ-lille.fr § yvan.peter@univ-lille.fr ¶ laurent.grisoni@univ-lille.fr a b c to protect the artefacts from the visitors (for various reasons, including protection from stealing, or degradation). Indeed, some specific public exhibitions (such as museums) have even more constraints on the preservation of artefacts, such as preserving sensitive artefacts from temperature, humidity and pollutants and the risks of accidental damage. Glass display cases are a common mean to present fragile, rare or valuable objects coming from cultural or artistic heritage in a safe way. In addition, well-lit display cases allow for showing each artefact in its full potential. The challenge faced by mediators is then to find a way to transform the closed glass display cases into smart and attractive exhibitions that can catch the visitors' attention. Looking at these multiple constraints, it appears that public exhibitions would benefit from novel mixed-reality technologies which would engage the visitors while preserving the physical and social aspects of the exhibition ecosystem.

In this paper, we present a class of Mixed-Reality displays that we call Revealable Volume Displays (RVDs), which are particularly suited for this context of public exhibitions. They rely on users actions to reveal 3D virtual content inside or around exhibited physical objects, as shown on Figure 1, using the reflection in the glass panel which protects them. RVDs ensure that the visitor's attention is focused on the actual exhibits, avoiding the "heads-down" effect of mobile mixed-reality; they entirely rely on the user's interaction to display content, maximising the engagement with the exhibition; they provide shared augmentations, unlike mixed-reality headsets, correctly perceived by all visitors, hence opening social interactions on projected digital information; they leave physical artefacts safely protected and unaltered by the display.

Our contribution is four-fold :

• We describe the concept and implementation of RVDs • We propose a design space that shows the augmentation and interaction possibilities that they offer • We investigate visitors' responses to RVDs with an in-situ experiment on a first prototype • We evaluate techniques and propose design guidelines to facilitate the use of such displays

RELATED WORK

In the following section, we present related work on the use of mixed-reality in public exhibitions, especially in museums. We then describe other augmented and mixed reality display technologies that focus on public / shared virtual content. Finally we focus on studies that aim at guiding the exploration of digital content, in various contexts.

Mixed-Reality for Mediation

In the context of mediation (museums, products exhibition, ...), Augmented (AR) and Mixed-Reality (MR) technologies offer the opportunity to combine digital information with exhibited physical objects. We can cite as an example mobile AR applications for museums which offers visitors a device to explore exhibits. For example, Augmented Reality Stories at Acropolis Museum presented by Keil et al. [START_REF] Keil | The House of Olbrich An Augmented Reality tour through architectural history[END_REF] shows how mobile devices bring augmented colours and stories to art pieces at the Acropolis Museum through AR developed by the CHESS project (Cultural Heritage Experiences through Sociopersonal interactions and Storytelling). These applications exploit a smartphone back camera to integrate virtual objects on the physical space or to display data providing useful information for visitors . In these cases, AR technology might make finding information in the museum easier and enrich the visitors' experience. Bekele and al. recently realised a survey of Augmented, Virtual and Mixed Reality for Cultural Heritage (CH) [START_REF] Bekele | A survey of augmented, virtual, and mixed reality for cultural heritage[END_REF]. They classified projects according to the technologies used in CH projects for tracking and registration, virtual environment modelling, devices and interaction interfaces as well as the purpose of the project : education, exhibition enhancement, exploration, reconstruction, and virtual museums. The former categories provides the design dimensions of an AR application. Ibáñez & Delgado-Kloos have realised a review of Augmented Reality for STEM learning on the period 2010 -2017 [START_REF] Ibáñez | Augmented reality for stem learning: A systematic review[END_REF]. Their study is not restricted to the use of AR in cultural heritage sites and museums but it shows that the AR applications tend to use more than one type of media to convey information. Text and 2D images are the most used media but animations, 3D objects and videos are also quite common. The type of activities can be classified in three categories : exploration, simulation and game. In this paper, we focus mostly on the exploration aspect, with displays dedicated to this activity.

Liarokapis et al. [START_REF] Liarokapis | Augmented reality interface toolkit[END_REF] state that AR exhibition success is highly related to the level of realism achieved. Especially, with AR for museum exhibition, visitors have high expectations that the visualised information will be naturally presented in an entertaining manner. According to Boisvert et al. [START_REF] Boisvert | The relationship between visitor characteristics and learning-associated behaviors in a science museum discovery space[END_REF] two necessary steps are required to ensure visitors engagement in museums: the attractiveness of the interface and the holding power. Attraction is defined as the measurement of visitors who stop at the exhibit while the holding power is time spent by visitors in the exhibit. The study by Yahya [START_REF] Yahya | Museum Learning, The Museum Visitor, The Museum Visit An[END_REF] indicates that attraction and holding power are important variables for understanding the learning environment of the museum. In addition, holding power affects the visitor participation during visiting the exhibition. These information are important indicators of museums' ability to engage visitors' attention and maintain their interest. Even though AR and MR technologies can motivate users to interact in new ways with exhibits, according to Hsi [START_REF] Hsi | A study of user experiences mediated by nomadic web content in a museum[END_REF], they may suffer from a "heads-down" effect where the interaction with the device takes most of the attention of the visitor, hindering the experience as well as the interaction with other visitors [START_REF] Chang | Development and behavioral pattern analysis of a mobile guide system with augmented reality for painting appreciation instruction in an art museum[END_REF][START_REF] Hsi | A study of user experiences mediated by nomadic web content in a museum[END_REF][START_REF] Mayr | Potentials and Challenges of Mobile Media in Museums[END_REF]. Another dimension of the engagement of museum visitors is collaboration and social experience. As stated by vom Lehn et al., social interaction and collaboration are becoming increasingly important to the design and development of exhibitions [START_REF] Vom Lehn | Video-based Field Studies in Museums and Galleries[END_REF]. Indeed, discussion between visitors fosters exchanging point of views and elaborating the reasons for their opinion on the exhibits which leads to learning and a more thorough experience [START_REF] Heath | Interaction and interactives: collaboration and participation with computer-based exhibits[END_REF].

In this paper, we present a new class of mixed-reality displays that take these constraints into account, i.e. where user engagement is ensured by relying on their interaction for the display of virtual content and where the augmentations are displayed directly on the physical objects, visible by all visitors.

Mixed-reality displays for public contexts

In this section we consider MR displays that enable multiple users to collectively perceive virtual content augmenting physical objects. Kajita et al. propose SkyAnchor a glass-free mixed-reality solution that overlaps virtual content with a physical object [START_REF] Kajita | Skyanchor: Optical design for anchoring mid-air images onto physical objects[END_REF]. Their solution enables a zero-latency imaging while the object moves by attaching the light source to the object. The tracking is based on the surface on which the object is (capacitive markers), so one can not lift it. The size of the image is also dependant on the size of the system. Karnik et al. describe Mustard [START_REF] Karnik | Mustard: a multi user see through ar display[END_REF], a multiviewer see-through display to provide information about objects in a glass cabinet. The system is based on two liquid crystals panels between which the objects are placed. The front panel is used to display information while the back panel serves as a hole-mask that restricts what a user can view at one position. This enables the systems to have location specific views for different users. The user does not have the means to interact with the system to change the information displayed. Ridel et al. propose another way to explore information by the controlled projection of 3D visualisation on top of previously scanned artefacts [START_REF] Ridel | The revealing flashlight: Interactive spatial augmented reality for detail exploration of cultural heritage artifacts[END_REF]. The user can drive the projection using a revealing flashlight. This interactive device provides three characteristics : spot (what to show), distance (size and illumination) and angle (intensity of the augmentation). Projected information is however limited to the surface of the exhibited objects and might damage fragile artefacts. The same authors also describe EgoSAR, an augmented reality display that superimposes personalised virtual content for different viewers around a physical object [START_REF] Ridel | Egosar: Towards a personalized spatial augmented reality experience in multi-user environments[END_REF]. The system is based on retro-reflective material combined with multiple light sources. The different views are however associated to specific locations unless the user is equipped with a portable projector in an alternative setting. Martinez et al. explore an idea similar to what we present in this paper [START_REF] Martinez Plasencia | Through the combining glass[END_REF]. They use it as an example of how optical combiners can be used to augment physical spaces. However, they do not evaluate the interaction with the system or provide guidelines on the design of augmented museum cabinets using such a system.

Guiding exploration of virtual content

Exploration of virtual content in public exhibitions can be made difficult by the amount of accessible information and the type of display technology used, and can therefore benefit from guiding. Presenting 3D visualisations in public spaces is in fact identified as an important challenge by Besançon et al. [START_REF] Besançon | The state of the art of spatial interfaces for 3d visualization[END_REF]. Ynnerman et al. [START_REF] Grisoni | Exploranation: A new science communication paradigm[END_REF] propose the term exploranation as the merging of exploratory and explanatory visualisation. It offers new possibility for mediation in public settings, through adaptive interfaces, sensitive guidance to enhance the experience of exploration.

Another technique is to have other users demonstrate gestures which need to be performed, in order to generate a shared knowledge. The study by Cash et al. [START_REF] Cash | Investigating design: A comparison of manifest and latent approaches[END_REF] involves the gestures repetition as a tool to build shared understanding. Yasui et al. [START_REF] Yasui | Collaborative idea construction: Repetition of gestures and talk in joint brainstorming[END_REF] demonstrate that gesture repetition helps building a shared knowledge within participants.

Another possibility is to provide visual guides. In the context of 2D interfaces, Burigat et al. [START_REF] Burigat | Visualizing locations of offscreen objects on mobile devices[END_REF] propose a technique to display large information spaces on the limited screens of mobile devices by indicating the direction of Points of Interest (PoI) outside the displayed part of a map. In the context of 3D Virtual Environments, according to Chen et al. [START_REF] Chen | Guiding exploration through three-dimensional virtual environments: A cognitive load reduction approach[END_REF], including navigation aids to guide the exploration process provides a significant learning effect. Prior studies [START_REF] Galyean | Guided navigation of virtual environments[END_REF] also confirm that guiding the user in a Virtual Reality Museum experience with continuous inputs in space and time stimulates an improved experience.

In the context of Mobile Augmented Reality, Schinke et al. [START_REF] Schinke | Visualization of off-screen objects in mobile augmented reality[END_REF] propose a technique that embeds arrows pointing at nearby PoI. Their study confirms that 3D arrows help users estimate the position of objects more precisely than a mini-map. In addition, according to Carmo et al. [START_REF] Carmo | PoI awareness, relevance and aggregation for augmented reality[END_REF], the integration of PoI allows for a better knowledge of the environment and a better user guidance through a collection of clues on off-screen objects and a 2D map with a radar view. Recently, the study of Galatis et al. [START_REF] Galatis | Mobile augmented reality guides in cultural heritage[END_REF] suggests KnossosAR as an outdoor AR tool for the visitors of cultural heritage sites. This mobile application deals with the occlusion problem and how to handle occluded PoI. Their user evaluation trials confirms that guides stimulate the interest of participants to physically explore the archaeological site. In the context of Spatial Augmented-Reality, Sodhi et al. [START_REF] Sodhi | Lightguide: Projected visualizations for hand movement guidance[END_REF] explore how on-body projected visual hints could be used for movement guidance. Schmidt and al. [START_REF] Schmidt | Floor-Projected Guidance Cues for Collaborative Exploration of Spatial Augmented Reality Setups[END_REF] explore a spatial augmented reality environment through a floor projected guidance user interface. Their aim is to support the storytelling process by guiding users to regions of interest and ideal viewpoints. In this paper, we investigate similar techniques to guide users through the exploration of RVDs.

REVEALABLE VOLUME DISPLAYS : IMPLEMENTATION AND DESIGN SPACE

In this section, we first describe the principle of Revealable Volume Displays, their implementation and the content and techniques that they enable.

General approach

RVDs aim at satisfying a number of constraints for mixed-reality public exhibitions, which are not all taken into account by existing MR displays:

1. They preserve the exhibited objects by leaving them behind protective panels and not projecting directly on them. This configuration also protects the exhibited artefacts from the visitors' hands 2. They keep visitors focused on the physical objects, not on an external screen 3. They encourage the exploration [START_REF] Grisoni | Exploranation: A new science communication paradigm[END_REF] of the museum exhibits as the virtual content is only visible when visitors are interacting with it, unlike with AR headsets 4. They allow all visitors, i.e. not only the ones interacting as it would be the case with an AR headset, to perceive the augmentations consistently, which encourages personal trajectories and collaborations [START_REF] Benford | From interaction to trajectories: Designing coherent journeys through user experiences[END_REF].

As shown in Figure 2.a, RVDs utilise the glass cabinet that protects exhibited objects. When a physical surface, such as a hand, body part or handheld object is placed and moved in front of the cabinet, its reflection is perceived as moving through the volume inside the cabinet, intersecting the exhibited objects. We use this principle to reveal virtual content placed inside the cabinet by re-projecting the intersections between the reflected surface and the virtual content onto the surface. For example, in Figure 2 RVDs draw inspiration from Slicing Displays [START_REF] Berthaut | Revgest: Augmenting gestural musical instruments with revealed virtual objects[END_REF][START_REF] Cassinelli | Volume slicing display[END_REF], AR displays which use Optical Combiners [START_REF] Martinez Plasencia | Through the combining glass[END_REF], swept-volume displays [START_REF] Sun | An Improved Design of 3D Swept-Volume Volumetric Display[END_REF] and Spatial Augmented reality [START_REF] Bimber | Spatial Augmented Reality Merging Real and Virtual Worlds[END_REF]. Like swept-volume displays, RVDs display voxels, i.e. elements composing virtual content placed in 3D in the physical space. In doing so, they provide depth cues such as stereopsis without requiring head-tracking or a stereoscopic technology. Unlike swept-volume displays, but like slicing displays, the entire display of information relies on visitors actively exploring the content. If the content is not actively revealed by a user, it is not visible. Unlike swept-volume displays and slicing displays, RVDs also allow for augmenting physical objects with virtual content placed inside or around them. Like Spatial AR displays, the content displayed is visible by all users in the physical space, although only when looking through the same panel, but RVDs also allow for placing content in mid-air, i.e. not necessarily on a pre-existing projection surface in the target space. Finally, like Optical Combiners based displays, due to the flatness of the optical combiner (here the glass cabinet) as explained by Martinez and al. [START_REF] Martinez Plasencia | Through the combining glass[END_REF], the virtual content is perceived consistently (at the same position) by all visitors independently from their point of view. Unlike many other Optical Combiners based displays however , such as "Pepper's Ghost"-type displays, RVDs allow for displaying virtual content placed anywhere in a volume, not on a fixed surface.

Implementation

RVDs allow one to place 3D meshes (primitive shapes, arbitrary 3D models) at any position within the volume inside the glass cabinet. These meshes will hold the different types of content (solid or gradient colours, text, 2D and 3D textures, video textures). The tracking and display can be implemented in two ways. Our most common implementation relies on a depth camera and a projector. In this case, the software part of our system resembles the augmented reality pipeline described by Berthaut et al. [START_REF] Berthaut | Revgest: Augmenting gestural musical instruments with revealed virtual objects[END_REF], with additional filtering possibilities described below. The slicing process is entirely done on the GPU in three rendering passes. It requires that the projector and camera are calibrated to retrieve the projection and view matrices that will be used to render the 3D scene of virtual content. A 3D mesh is created from the depth image captured by the camera and rendered to a first depth texture from the projector point of view. The second pass selects all pixels of the back faces of the virtual objects which are behind pixels of the depth texture and renders them to a second texture. In the third pass, pixels of the front faces are finally selected for rendering if they are located before pixels in the depth texture and if they match pixels of the same object in the second texture. Their colour is then computed based on the type of content contained in their corresponding 3D mesh and on the 3D coordinates of the depth texture pixel relative to the mesh.

Because our system aims at displaying text annotations and images, we added a filtering technique that reduces both temporal and spatial noise due to the low resolution (640x480 at 30fps or 320x240 at 60fps) of the depth camera (Asus Xtion or a similar device based on projected IR pattern). To that extent, we combine a temporal filter using a 1e filter [START_REF] Casiez | 1 C filter: a simple speed-based low-pass filter for noisy input in interactive systems[END_REF] that removes the small jitter from the depth image while preserving fast movements of the physical surface, and a spatial filter that averages the depth positions to flatten captured physical surfaces. Both filters are computed on the CPU and passed as an alternative depth image to the slicing pipeline. The first pass then selects each pixel between the last depth image from the camera and the filtered depth image depending on a difference threshold. In practice, this leads to very stable depth meshes when the physical surfaces are relatively motionless and accurate displacement when they are moving. Compared to other approaches to real-time depth image filtering such as using the albedo [START_REF] Wu | Real-time shading-based refinement for consumer depth cameras[END_REF], our approach only relies on the depth image and is therefore less dependent on the lighting conditions of exhibitions.

In addition to this projector / depth camera combination, a second simpler and preliminary prototype displayed in Figure 2.c shows how RVDs can be implemented using a mobile device. Here a web page can be served to the mobile device of visitors containing an application written in Javascript and WebGL. It uses the Aruco [START_REF] Ramirez | Speeded up detection of squared fiducial markers[END_REF] marker-based tracking library to obtain the position of the virtual camera relatively to a marker board placed inside the museum cabinet. This camera is configured as an orthographic camera with a very close near plane, effectively resulting in only a slice of the virtual content being shown on the screen.

Design space

In this section, we propose a set of dimensions which describe the design opportunities offered by a Revealable Volume Display.

Role of virtual content

RVDs allow virtual content to play various roles with respect to augmented physical objects. Although these are similar to roles available with other types of Augmented Reality Displays, RVDs present the content in a unique way : perceptually aligned with physical objects for all the users in front of the display, but only as a fraction of the entire virtual content. This content can therefore be used with one or a combination of the following roles :

• to reveal the inside of the physical object, e.g a slice of layers inside the bird house show on Figure 1.a. • to annotate parts of a physical object to explain or direct the user to other information, as shown on Figure 1.b.

• to extend a physical object with virtual content, e.g. completing a partial archaeological artefact with a virtual reconstruction of the remainder. • to highlight the contour / surface of the physical object, as shown on Figure 1.a on the contours of the bird house. • to complement the object with additional content, e.g. a video which presents the usage context of an artefact.

Use of depth in the virtual content

In RVDs, depth is a very specific dimension since the XY plane acts as a 2D screen representing a slice of content visible by all. Depth can therefore have the following uses :

• Spatial when one moves through layers in a 3D texture, such as shown on Figure 1.c. • Temporal when one moves through positions (e.g. frames) in a video or animation such as seen on Figure 1.a. • Semantic when one moves through semantically organised content (e.g. the chronology of evolution of an object across history)

Exploration guidance

Because RVDs heavily rely on exploration of virtual content, how the user is guided through this exploration is an important part of the design. This exploration can be :

• Spontaneous when user moves freely to explore the virtual scene • Guided when the exploration is triggered and guided by e.g.

annotations or sounds which allow for proposing a trajectory

Implementation dependent interactions

Depending on the implementation chosen for the RVD, various interaction possibilities arise. The first implementation uses the projector + depth camera system, with the user holding a unprepared opaque panel or using their body or clothes to reveal the content. The second implementation also uses projector + depth camera together with prepared opaque panels featuring printed tags, such as shown in Figure 4.a. The third implementation also relies on a projector + depth camera setup combined with a transparent (e.g. acrylic) panel to which is attached a piece of paper, on which one can write. The revealed content is then visible by transparency on the other side of the panel and can be traced or annotated. This version is shown in Figure 1.c . Finally the fourth implementation is the mobile display shown in Figure 2.c in which the user can also access the touchscreen of the smartphone/tablet. We provide in Figure 3 the possible interactions for each implementation described above. These interactions are :

• Exploration of content, in which users simply reveal the static virtual content by moving the device through it • Triggering events, in which users can activate dynamic content (e.g. animations, videos, sounds ...) • Thematic selection, in which users can select between a set of virtual content placed at the same physical position, for example to allow for multiple information regarding the same physical object. An example of that is used in our preliminary study in Section 4.1 where visitors can access information related to either the habitat or food of naturalised birds. • Content manipulation, in which users can interact with the content in a more advanced way than only triggering and filtering, e.g. scrolling through a map, manipulating data diagrams. • Content creation refers to the possibility of adding content, for example leaving messages or annotation on the exhibited objects, through the mobile touchscreen for example. • Content capture refers to the possibility of capturing the revealed content, either manually by tracing it on the transparent panel implementation, or by taking a picture and screenshot with the mobile implementation. The last design dimension in RVD which influences interaction possibilities is the side of the glass cabinet through which objects are viewed. Exhibited artefacts will be either perceived mostly through a vertical side, such as seen in Figure 2.a or through the top side, from above as seen in Figure 2.b. In the case of vertical sides, artefacts can be placed at any distance from the surface, i.e. on the reflected axis, which may lead to more time finding the content as this reflected axis seems more difficult to control, as explained in more details in the following experiments. With the horizontal top side, objects are usually closer to the panel, requiring less adjustments on the reflected axis and making the exploration of content potentially faster. Horizontal sides may however make the content less visible since visitors can be placed all around the case, not necessarily facing the panels used to reveal the content, and not all in front of it as with vertical sides.

FACILITATING 3D EXPLORATION WITH A REVEALABLE VOLUME DISPLAY

In this section we describe two experiments that investigate the appropriation of RVDs by users and how to facilitate exploration with them. Both of them were conducted before the COVID-19 pandemic, in the summer and fall of 2018. These experiments use the projector + camera + opaque panel implementation. However the other RVDs implementations suffer from the same perceptual issues and should therefore benefit from the proposed techniques.

Preliminary study

The purpose of this first study was to investigate information exploration by individuals and to evaluate the appropriation of our prototype in an ecological situation. Due to time constraints of conducting the study in a museum setting, we focused on gathering qualitative data.

The prototype was trialed in a regional natural history museum. It was deployed in a showcase containing naturalised birds (see Figures 4.a and 4.b). For each bird we provided their name, diet, living environment and number of laid eggs. 30 participants (15 groups of 2 participants each) were recruited for the experiment in two sessions in the museum. Participants included 15 males and 15 females with a mean age of 27.3 (sd=12.37, min=8, max=52). None of them had previously participated in similar studies or used our system but some had experience with augmented reality (AR experience rated between 1-5 with mean=2.3, sd=1.34). They all participated voluntarily and signed an informed consent form. The duration of the experiment was between 10 and 15 minutes.

The experiment involved pairs of users (to allow for a collaborative task) each facing half of the showcase (see figures 4.a and 4.b). Before each task, subjects were allowed to spend enough time to get familiar with the device. The experiment was divided into two steps :

• In the first step, participants were introduced to the prototype and told how to interact with it. They were given time to experiment until they felt accustomed to it. • The second step was dedicated to the trial of navigation guides to help users find relevant information around the exhibits. The users were informed that starting from the head of the animals, they would find a guide to follow to find information. They were asked to find information about the number of eggs laid by each animal and to identify the one having the more (resp. less) eggs. Three kinds of guides were evaluated. This aspect of our system will be described in more details in the second experiment, as no significant results were obtained. Each pair of users had the same guides and we alternated between groups.

The trials were recorded for later analysis of the users' interactions with the device and comments. After each step, each user passed a questionnaire with 5 levels Likert items to assess usability and acceptance of the system. Results from the questionnaires in the first and the second step are shown in Figure 5.

Although the feedback from participants of this first experiment was very positive as shown by the results of our questionnaires, the observation of participants in an ecological situation revealed that exploration with the system remains difficult, see Figure5. In particular they seemed to have trouble finding the virtual content around the exhibited objects. We believe this is due to the reflected interaction, which reverses displacements along the z-axis (towards or away from the glass panel), and to the limited visual depth cues. In fact, visual cues for depth perception such as parallax, occlusions, shading and shadows are lost with the RVD. This causes difficulties of perception and navigation for users and may hinder their experience in the museum. We therefore decided to investigate how individual exploration can be facilitated with a second, more controlled experiment.

Controlled experiment

This controlled experiment seeks to answer the following questions:

• Does having the contour of physical objects virtually highlighted improve the positioning around the physical objects ? • Do animated guides make it easier to find to which object or part of an object virtual content is attached, compared to static links (3D lines) ? • Does highlighting depth contours for the virtual content help users discover this content ? 20 participants (16 males, 4 females) were recruited for the experiment (aged mean=31.10 SD=11.17 min=18 max=61). None of them had previously participated in similar studies. They participated voluntarily after signing and informed consent form. Results from a 21st participant were removed because they had trouble adjusting to the system, resulting in very low performance compared to the other participants. The experiment was performed in a quiet, illuminated laboratory room. Before each task subjects were allowed to spend enough time to get familiar with the device.

The experiment lasted around 30 minutes. For each task participants were holding an A5 piece of white foam board with their two hands to drive the projection. The glass cabinet contained three physical objects. We used an Optoma 1080E projector with a 1920x1080 resolution at 60fps, combined with an Asus Xtion providing a 320x240 depth image at 60fps. The experimental setup is shown in Figure 4.c.

The experiment followed a within-subject design. It was divided into three tasks : CONT OURS, GUIDED, CONT ENT . We added a fourth task named CONT ENT 2, based on initial results, that the participants performed in a second session. Each task had two conditions. Between each condition participants filled a subjective questionnaire with 5 levels Likert items to gather their impressions about the task. At the end of each task, they also selected their preferred condition. CONT ENT 2 was designed to refine the results from CONT ENT , as post task interviews revealed an effect of the two conditions that was not visible in the data. It was conducted between 2 and 7 days after the first experiment on only 12 of the participants of CONT ENT (11 males, 1 female, aged of mean=30.58, SD=11.46, min=18, max=61).

Contours task

The goal of this task was to evaluate whether highlighting the physical objects with their virtual contour helps users in their exploration, i.e. if they better perceive where the reflection of the panel they are holding is, relatively to the physical objects. At the beginning of each trial, participants were asked to reveal a virtual sign indicating around which object a virtual stationary red sphere would be placed and where around the object it would be (e.g. "above to the left"). As soon as the sign disappeared they had to find the sphere as fast as possible and remain on it until it disappeared (after 2 seconds). The sphere was placed near one of the 3 objects and at one of 4 positions around the object (right, left-behind, left-top-behind, rightbehind) (at ∼50mm from the contour of the object). Each participant therefore performed 12 trials (3*4) per condition and the trials were randomised. Two conditions were evaluated: no contours for the

Guides task

We seek to guide users from a specific part of a physical object to a piece of information, in order to facilitate annotations of the physical objects with "floating" virtual content. For that, we trialed animated guides (GUIDE_DY NAMIC) that give a clue of the direction to follow with a series of 3D cones moving towards the target and static guides (GUIDE_STAT IC) that link the object and the information with a single 3D cone. These two guides are depicted in Figures 7.c 7.d, and the result when revealing both the name and red sphere with both guides is shown in Figures 6.c and 6.d. We chose not to test any guide which would require the system to know the position of the user, e.g. 3D arrows in perspective, in order to maintain the public and shared aspects of RVDs.

Each trial started with a virtual sign indicating the object around which a virtual red sphere was going to be. As soon as the virtual sign disappeared, they had to move to the designated object, reveal its name (which was placed in the centre of the object) and then find the stationary sphere as quickly as possible. They then had to remain on the sphere until it disappeared (after 2 seconds) and then move back to the sign for the next trial. The sphere was placed around one of the three objects in two possible directions (right+above, left+above) and two planes (same depth as the object / closer to the user). This resulted in 12 trials (3*4) per condition for each subject and the order of trial was randomised.

Content task

With this task, we investigate how to improve depth perception during the exploration of virtual content especially when the content does not change much along the depth axis (few images, text, compared to 3D text). In the task, participants were asked to first remain on a virtual sign with instructions. As soon as this sign disappeared, they had to find a word placed in front of or behind the virtual sign as quickly as possible and say the word out loud (without necessarily remaining on the word). After this, the word disappeared and they moved back to the virtual sign for the next trial. Two conditions were evaluated. In FRAME_FLAT the word was surrounded by a simple box, shown in Figure 7.a. In FRAME_DEPT H, the shape of the frame surrounding the word quickly increased from 0% to 95% of the box size at both extremities, and then increased slowly along the depth to reach 100% in the middle of the box. This shape is depicted in Figure 7.b. The FRAME_DEPT H shape was designed so as to show almost no visual difference with the FRAME_FLAT shape, in order to preserve the maximum space for virtual content placed inside. This absence of difference is visible in Figures 6.e and 6.f. Both shapes were of 100mm in width and 100mm in height.

Between trials, we varied direction of the frame (i.e. behind / before of panel), the distance of the frame to the panel (100mm and 150mm) and the depth of the frame (60mm and 150mm) together with the depth of the word placed at the centre of the frame (occupying a third of the depth of the frame, so respectively 20mm and 50mm). Each participant therefore performed 8 trials (2 directions * 2 distances * 2 depths) per condition and the trials were randomised. We measured the number of errors (finding and loosing the word), and the time taken to find the word once inside the frame.

Content2 task

The initial Content task was modified to require more precision from the participants. To that extent, the thickness of the word that participants had to find was reduced from 50mm and 20mm to 20mm and 10mm. We also varied the depth of the frame separately between 60mm and 150mm. Each participant therefore performed 16 trials (2 directions x 2 frame depth x 2 frame distance x 2 word thickness) per condition and the trials were randomised. In addition, they were asked to find the word as fast as possible and more importantly to remain on it until it disappeared (after 2 seconds). The same conditions were evaluated as in the Content task (i.e. FRAME_FLAT and FRAME_DEPT H).

Results

Here we give all statistically significant differences observed between conditions. They are also depicted in Figure 8. The implications of these results are discussed in Section Guidelines. We provide both frequentist and bayesian analyses, these were performed using JASP 0.13.1 1 . We used a Student t-test if the normality was verified with a Shapiro-Wilk test, otherwise we used a Wilcoxon signed rank non-parametric test. Bayesian statistics were performed to provide a finer grained analysis of the effects (e.g. weak, moderate, strong ...), using the interpretation of Bayes factors from JASP, in turn adapted from Jeffrey's scheme [START_REF] Ly | Harold jeffreys's default bayes factor hypothesis tests: Explanation, extension, and application in psychology[END_REF].

Contours task

Before analysing the results, we removed the outliers which were trials that lasted for more than 20s, correlated with the participant forgetting the instruction, i.e. around which object the target was. Adding a virtual contour around the objects (CONT OUR_Y ES) resulted in less errors (mean1.528, sd = 0.768) that without them (CONT OUR_NO, mean = 1.870, sd = 0.65). A Student's t-test (t(19) = -2.316, p = 0.032) confirms that the difference is statistically significant. The corresponding Bayes factor (BF 10 = 1.985) shows a weak evidence for a difference. Regarding completion time, a Bayesian t-test reveals a moderate evidence for an absence of difference between the conditions (BF 10 = 0.232). There is also a statistically significant difference (t(19)=3.214, p=0.005) when comparing task completion time (in milliseconds) between the first condition participants had and the second one. This duration reduces (mean = 3978.776ms, sd = 1129.721) between the first condition (no matter which one it was) and the second (mean = 3191.554mssd = 1188.005). The corresponding Bayes factor (BF 10 = 9.908) also shows a strong evidence for a difference. We also compared the results between participants who had CONT OUR_Y ES first (n=11) and participants with CONT OUR_NO first (n=9), which leads to the following observations :

• In terms of errors, there is a statistically significant difference depending on whether the users started with CONT OURS_Y ES or CONT OURS_NO (t(18) = -2.618, p = 0.017). The errors increased when they passed from CONT OURS_Y ES to CONT OURS_NO (mean = 0.114, sd = 0.728). The errors decreased in the reverse condition (mean = -0.620, sd = 0.460). A Bayesian independent t-test confirms that there is moderate evidence for a difference (BF 10 = 3.485). • There is also a statistically significant difference in terms of reentries in objects (t(18) = -2.311, p = 0.033) but with only a weak evidence in Bayesian analysis (BF 10 = 2.247). The number of reentries increased when passing from CONT OURS_Y ES to CONT OURS_NO (mean = 0.202, sd = 1.909) and decreased in the reverse order (mean = -1.415, sd = 0.947).

Guides task

The results do not show statistically significant differences in completion time or errors between the two kinds of guides. Bayesian analysis shows a moderate evidence for an absence of difference in errors (BF 10 = 0.242) and completion time (BF 10 = 0.274). However we can note a statistically significant difference (t(19) = 2.783, p = 0.012) in reentries in guides (i.e. leaving the guide and entering it again) with less reentries when using GUIDE_DY NAMIC (mean = 4.4, sd = 1.7) compared to GUIDE_STAT IC (mean = 5.612, sd = 1.488). This is confirmed by a moderate evidence for a difference (BF 10 = 4.447). Participants finally expressed a preference towards dynamic guides (13 against 7).

Content 1 and 2 tasks

Data from Content 1 does not show any statistically significant difference either in terms of completion time or errors. However a majority of participants reported differences that could indicate a change in depth perception, although they could not tell what the difference between the conditions was. Among the 20 participants: 8 perceived no difference between the two conditions; 3 felt that the FRAME_DEPT H condition was easier; the remaining 9 perceived a difference related to distance perception : 5 users felt that the word was closer in FRAME_DEPT H condition, 3 felt that the word were thinner in FRAME_DEPT H and 1 that they could reach the word faster in FRAME_DEPT H.

The results from the second iteration of the task Content2, which required more precision (due to thinner words), show a statistically significant difference in word finding time (t(11) = -2.840, p=0.016) with lower duration for FRAME_DEPT H (mean=1385.208 ms, sd=241.669) than for FRAME_FLAT (mean=1668.667 ms, sd=357.446). This is confirmed by a Bayesian t-test showing a moderate evidence for a difference (BF 10 = 4.003).

DISCUSSION AND GUIDELINES

Even though we found a number of statistically significant differences between the techniques in each task, these remain quite small : less than 1 second for the completion time and only one or two additional errors. However, these were obtained on very short and guided tasks, i.e. participants knew what they were supposed to find and they were only looking for one information at a time. We believe that these small differences might accumulate and become more problematic in ecological conditions where visitors do not always know precisely what they are looking for, where there are more objects and virtual content and where visitors might get frustrated and stop using the system if they feel like they are not able to achieve the task fast enough. In the following, we propose guidelines based on our results for designing interaction with RVDs to optimise the user experience. Although we only tested the projector + camera + opaque panel implementation, we believe they can ve applied to all implementations of RVDs.

Progressive complexity

Results of the Contours task indicate that there is a short learning curve for RVDs. In fact participants became a lot more confident and accurate over time. We therefore believe that in order to facilitate the use of RVDs, a trajectory could be defined that would start with a cabinet featuring only a few simple virtual annotations on a single object and increase complexity and diversity of the content progressively as spectators progress into the exhibitions. Another possibility is to use the thematic selection that we presented in Section 3.3 to filter out some of the content for novices who would use a panel with a special AR tag.

Dynamic 3D Guides

In order to attach virtual content such as annotations around exhibited objects, a method for guiding visitors to the content needs to be added. Instead of relying on text indications ("move upwards") or arrows which require the system to know which content visitors are looking for, we proposed two types of virtual guides simply created by placing 3D shapes in the physical space between the physical object and the annotation. Even though there were no differences in performance times, results of our study show that participants mostly preferred the dynamic guides and that they lost them less often than when following the static guides. It therefore seems that dynamic guides with an animation showing the direction of the virtual content are to be picked in priority. In addition, dynamic guides led to a strategy of slowly following the guides (without loosing them), while static guides led to a strategy where users would scan the volume with fast and large movements. We believe that slower movements should be encouraged as they will help visitors explore more complex scenes of virtual content, where large movements would make them miss some of the content, especially with tracking and projection latency issues. Our recommendation is therefore to rely mostly on dynamic guides.

Highlighting contours of physical objects

When interacting with an RVD, depth perception cues such as occlusions, shading and shadows are lost. It is therefore difficult for visitors to precisely perceive where the reflection of the surface they are holding is, relative to the exhibited objects. Our study suggests that less errors were made in finding virtual content of a physical object when the contours of that object were highlighted, and the same contours led to a stronger reduction of errors over time. This might indicate that participants had a better perception of position within the display case / around the object when given those contours. They might therefore serve as an anchor in the exploration around physical objects. The presence of contours also led participants to use slower movements in order to keep the contour visible at all times. We believe this induced behaviour would also improve the overall experience of RVDs, since visitors will move with more precision and stability through the virtual content when exploring. Implementing this contours technique however implies that all exhibited objects are scanned and the obtained 3D meshes are aligned with them.

Highlighting depth inside virtual content

Another effect of the impaired depth perception in RVDs is that it can be difficult for visitors to know their position along the z-axis when revealing virtual content. Findings from our study seem to indicate that amplifying this displacement with changes in the content or of a frame surrounding the content leads to faster selection of content along the z-axis, even if the changes are not consciously perceived by the users. Although the time difference remains small ( 300ms) in our task with a single word that had to be revealed, it could lead to much larger differences when accumulated during the exploration of more complex content. We recommend using depth highlighting frames, i.e. shapes that change contour when one moves forward and backward. A very small change such as the one used in our study seems to already restore a sense of depth. We believe that stronger changes with for example spherical frames, depth layered images or 3D textures would also help visitors to better perceive their displacement inside the museum cabinet.

CONCLUSION

In this paper, we presented the concept of Revealable Volume Displays, mixed-reality displays for 3D exploration in public exhibitions. We described how they can be implemented and what interaction possibilities they afford. Following an in-situ experiment on their appropriation by museum visitors, we conducted a controlled experiment to investigate techniques for facilitating their use. Our results suggest that virtual contours, dynamic guides and amplified depth movements can help reduce exploration time and errors and lead users to more stable and precise content exploration. The projector + camera implementation with added contours and guides has been used on several occasions since the experiment, and is being integrated in upcoming exhibitions. Among the main perspectives, the other RVDs implementations presented will be investigated further to see if additional guiding techniques are required.

Figure 1 :

 1 Figure 1: Revealable Volume Displays enable various content and interactions with mixed-reality exhibitions : (a) Image layers inside a physical object with highlighted contours, (b) Text annotations attached with 3D guides (d) Exploring and annotating a revealed MRI scan inside a head using a transparent panel and paper

Figure 2 :

 2 Figure 2: Implementation of Revealable Volume Display : (a) Projector + depth camera implementation with a visitor revealing the name of an exhibited bird, (b) Horizontal configuration with objects below the glass panel and text annotations revealed using a sheet of paper, (c) Mobile implementation of RVD

Figure 3 :

 3 Figure 3: Possible interactions with virtual content depending on the implementation of RVDs

Figure 4 : 2 WFigure 5 :

 425 Figure 4: Setup for the two experiments: (a) Experiment 1 : Introduction phase where participants reveal the names of the birds, (b) Experiment 1 : Exploration phase in which the average number of eggs laid by each bird is attached to it with a 3D guide. (c) Experiment 2

Figure 6 :Figure 7 :

 67 Figure 6: Experiment 2 : Conditions for the three tasks. Contours Task : CONT OUR_NO (a) and CONT OUR_Y ES (b). Guides Task : GUIDE_STAT IC (c) and GUIDE_DY NAMIC (d). Content Task : FRAME_FLAT (e) and FRAME_DEPT H (f)

Figure 8 :

 8 Figure 8: Plots of the statistically significant results in the Contours, Guides and Content tasks.