Anne Rodallec

Guillaume Sicard

Raphaelle Fanciullino

Sébastien Benzekry

Bruno Lacarelle

Gerard Milano

Dr Joseph Ciccolini
email: joseph.ciccolini@univ-amu.fr

TURNING COLD TUMORS INTO HOT TUMORS: HARNESSING THE POTENTIAL OF TUMOR IMMUNITY USING NANOPARTICLES

Keywords: Immunotherapy, nanoparticles, drug delivery, immunogenicity, pharmacokinetics, cancer

Introduction: Immune checkpoint inhibitors have considerably changed the landscape in oncology.

However apart world-acclaimed success stories limited to melanoma and lung cancer, many solid tumors failed to respond to immune checkpoint inhibitors, due to limited immunogenicity, unfavorable tumor micro-environment (TME), lack of infiltrating T lymphocytes or increase in Tregs.

Areas Covered: Combinatorial strategies is foreseen as the future of immunotherapy, and using cytotoxics or modulating agents is expected to boost the efficacy of immune checkpoint inhibitors. In this respect, nanoparticles displaying unique pharmacokinetic features such as tumor targeting properties, optimal payload delivery and long-lasting interferences with TME, are promising candidates for such combinations. This review covers the basis, expectancies, limits and pitfalls of future combination between nanoparticles and immune check point inhibitors.

Expert Opinion: Nanoparticles allow optimal delivery of variety of payloads in tumors while sparing healthy tissue, thus triggering immunogenic cell death. Depleting tumor stroma could further help immune cells and monoclonal antibodies to better circulate in the TME, plus immune-modulating properties of the charged cytotoxics. Finally, nanoparticles themselves present immunogenicity and antigenicity likely to boost immune response at the tumor level.

Current achievements and Limits of immunotherapies

The use of immune checkpoint inhibitors has profoundly changed the evolution profile of cancer patients with advanced disease [START_REF] Temel | Keeping Expectations in Check With Immune Checkpoint Inhibitors[END_REF]. Antibodies targeting the T cell inhibitory receptors cytotoxic Tlymphocyte antigen 4 (CTL A 4) and the programmed death receptor 1 (PD 1) (or its ligand PDL 1) are now clinically recognized with FDA approvals in melanoma, non-small-cell lung cancer (NSCLC), renal cell carcinoma and head and neck cancers to name but a few [START_REF] Spranger | Impact of oncogenic pathways on evasion of antitumour immune responses[END_REF]. Checkpoint inhibitors have a different toxicity profile than chemotherapy with a lower incidence of pancytopenia or digestive toxicities which are the most frequent adverse events with chemotherapeutic agents. However initial over-optimism about checkpoint inhibitors has been tempered by the limited number of patients exhibiting spectacular response rates, or the issue of emerging specific toxicities [START_REF] Temel | Keeping Expectations in Check With Immune Checkpoint Inhibitors[END_REF]. Another limitation is the fact that baseline biomarker studies have shown that checkpoint blockade therapy is mainly active through reactivation of antigen specific T cells present within tumor microenvironment (TME) [START_REF] Spranger | Impact of oncogenic pathways on evasion of antitumour immune responses[END_REF]. It is a matter of fact that the majority of solid tumors do not exhibit a productive T cell infiltrate and can thus be considered as non-T cell-inflamed [START_REF] Spranger | Impact of oncogenic pathways on evasion of antitumour immune responses[END_REF]. Thus turning cold tumors into hot tumors is currently one of the major goals to improve treatment of solid tumors with checkpoint inhibitors.

Turning cold tumors into hot tumors: an ongoing challenge

To achieve transforming cold tumors into hot ones, the wide range of possible combinations between immunotherapeutic agents and other drugs has to be appraised. A recent review by Lafolla and coworkers [START_REF] Iafolla | Rational design and identification of immuno-oncology drug combinations[END_REF] covered a total of 410 combination trials involving two or more than two immuneoncology drugs, mostly in skin and genitourinary malignancies. Gene expression data from TCGA were investigated and put into evidence 178 targets in 9089 tumors from 19 cancer-types. This allowed several promising new drug combinations to be identified. Above all, the impact of a combination between checkpoint inhibitors with cytotoxics or targeted agents to reshape tumor milieu in order to get immune infiltrate compatible with an expected activity of the checkpoint inhibitors is getting more and more attention [START_REF] Mellman | Cancer immunotherapy comes of age[END_REF]. The main contributor for a replenishment of cytotoxic T cells is the tumoral neo-epitope production [START_REF] Chabanon | Mutational Landscape and Sensitivity to Immune Checkpoint Blockers[END_REF]. This neo-epitope production can be stimulated in different way, such as co-administration of agents, the most efficient ones being standard cytotoxic drugs [START_REF] Chabanon | Mutational Landscape and Sensitivity to Immune Checkpoint Blockers[END_REF]. Modulation of the immune response through checkpoint inhibition may be increased by cytotoxic chemotherapy not only through the potential for neo-antigen crosspresentation but also by inhibiting the ratio of cytotoxic lymphocytes to T regulatory (Tregs) lymphocytes [START_REF] Roselli | Effects of conventional therapeutic interventions on the number and function of regulatory T cells[END_REF] or blocking the STAT6 pathway to upregulate dendritic-cell activity [START_REF] Lesterhuis | Platinum-based drugs disrupt STAT6-mediated suppression of immune responses against cancer in humans and mice[END_REF].

Nanoparticles at a glance

Nanoparticles are small (i.e., <200 nm) entities designed to cargo anticancer agents throughout the body to the tumor site. Beyond canonical liposomes, a wide range of supports and scaffolds have been made available now, ranging from dendrimers, squalene derivatives, nanospheres, to inorganic carriers such as silica beads or gold nanoparticles [START_REF] Jurj | The new era of nanotechnology, an alternative to change cancer treatment [Internet][END_REF]. Despite their heterogeneity (Figure 1), all these nanoparticles share common features, such as ability to passively target tumor tissue and to accumulate preferentially in the tumor micro-environment, through the Enhanced Permeation and Retention effect (EPR effect). Indeed, provided that their size is below 200 nm, nanoparticles will pass through gaps and loose tight junctions of the tumor neovessels, thus enabling a preferential extravasation in tumor neighborhood. In addition, the latest generation of nanoparticles such as immunoliposomes or conjugated nanoparticles can further target actively tumor antigens or tumor micro-environment [START_REF] Fanciullino | Challenges, expectations and limits for nanoparticles-based therapeutics in cancer: A focus on nano-albumin-bound drugs[END_REF]. Overall, nanomedicines in oncology all aim at improving the efficacy/toxicity balance of cytotoxics through an optimized biodistribution profile sparing healthy tissues and targeting tumor cells (Figure 2) [START_REF] Rodallec | Seek and destroy: improving PK/PD profiles of anticancer agents with nanoparticles[END_REF]. In addition to carrying drugs, nanoparticles can be further used in theranostic applications such as iron-oxyde constructs [START_REF] Tseng | Cetuximab-conjugated iron oxide nanoparticles for cancer imaging and therapy[END_REF], or radiosensitizing agents [START_REF] Liu | Metal-based NanoEnhancers for Future Radiotherapy: Radiosensitizing and Synergistic Effects on Tumor Cells[END_REF], highlighting how the carrier itself could have therapeutic applications. This per se efficacy could come from intrinsic properties of inorganic particles, such as antibody-dependent cell cytotoxicity (ADCC) -even when < 200 nm, nanoparticles are large enough to trigger immunogenic response, especially when they are composed of inorganic materials [START_REF] Shah | Immunological effects of iron oxide nanoparticles and iron-based complex drug formulations: Therapeutic benefits, toxicity, mechanistic insights, and translational considerations[END_REF].

How nanoparticles could help to preserve patient's innate immunity while delivering cytotoxics.

Cancer patients are generally heavily treated by a wide variety of drugs but standard cytotoxics remain the backbone of most regimens in solid tumors. The canonical and common drug-related toxicity of most cytotoxics is pancytopenia, leading to sepsis and sometimes lethal outcome in the most dramatic cases [START_REF] Kurtin | Myeloid Toxicity of Cancer Treatment[END_REF]. This side-effect is the direct consequence of the non-specific mechanism of action of the vast majority of cytotoxics, which have all been selected to interfere with rapidlydividing cells such as cancer cells. Because hematopoietic progenitors are also rapidly-dividing cells, they are doomed to be affected by most chemotherapy regimen, regardless of the pharmacological class (e.g., antimicrotubules, antimetabolites, or alkylating agents to name but a few). In the light of the critical importance of innate immunity against cancer, one can assume that affecting lymphocytes count or other cells implicated in the immune response will have deleterious effects on the clinical outcome first, and will impair the efficacy of any immunotherapy-based strategy next, thus triggering innate resistance. For instance, several studies have suggested that elevated neutrophil-to-lymphocyte ratios (i.e., NLR >5) or diminished absolute lymphocyte count are associated with poor clinical outcome in patients treated with immune check point inhibitors [START_REF] Sacdalan | Prognostic utility of baseline neutrophil-to-lymphocyte ratio in patients receiving immune checkpoint inhibitors: a review and meta-analysis[END_REF][START_REF] Putzu | Blood cell count indexes as predictors of outcomes in advanced non-small-cell lung cancer patients treated with Nivolumab[END_REF].

It has already been shown that prior treatment with cytotoxics and history of drug-related lymphopenia are associated with poor prognosis of various cancer types [START_REF] Liu | The Prognostic Value of Treatment-Related Lymphopenia in Nasopharyngeal Carcinoma Patients[END_REF][START_REF] Wild | The Association Between Chemoradiation-related Lymphopenia and Clinical Outcomes in Patients With Locally Advanced Pancreatic Adenocarcinoma[END_REF] and considered now as a possible unfavorable factor in patients treated with immune check point inhibitors [START_REF] Yarchoan | Relationship between lymphopenia and objective response rate with programmed death-1 (PD-1) inhibitor therapy: A single-center retrospective analysis[END_REF]. This hypothesis is however controversial and other studies have failed to confirm this relationship, at least during phase-I studies [START_REF] Sun | Baseline lymphopenia should not be used as exclusion criteria in early clinical trials investigating immune checkpoint blockers (PD-1/PD-L1 inhibitors)[END_REF]. Indeed, transient lymphopenia can be an opportunity for reshaping the repertoire of immune cells [START_REF] Zitvogel | Immunological aspects of cancer chemotherapy[END_REF], thus explaining why an in-depth understanding of the duration and level of chemo-induced lymphopenia plus full characterization of the kinetics of T-cells recovery is critical to better picture the impact of lymphopenia on homeostatic proliferation and efficacy of immune check-point inhibitors. As seen before, one of the most remarkable achievement of nanoparticles is their ability to target tumor tissue, thus sparing, at least partly, healthy cells and reducing thereby canonical cytotoxics-related side effects such as hematological toxicities. Several experimental data showed how the biodistribution of anticancer agents shifts from totally nonspecific when used as free drugs to a more tumor-directed distribution when used as nanoparticles.

For instance, tumor VS. healthy tissue comparative biodistribution studies showed that paclitaxel is more concentrated in tumors and less in liver, spleen or kidneys when administered as nabpaclitaxel, a 120 nm conjugated form of paclitaxel linked to a bioengineered albumin that targets SPARC and other glycoproteins of the TME [START_REF] Desai | Increased antitumor activity, intratumor paclitaxel concentrations, and endothelial cell transport of cremophor-free, albumin-bound paclitaxel, ABI-007, compared with cremophor-based paclitaxel[END_REF]. As a direct consequence of this increase in tumortargeting properties, several studies have shown how nanoparticles led to reducing side effects in patients scheduled for chemotherapy. Because one of the canonical treatment-related severe toxicity is pancytopenia, sparing hematopoietic progenitors could help to maintain patient's innate immune system functional. For instance, liposomal doxorubicine or liposomal vincristine show both reduced toxicities as compared with free doxorubicine or free vincristine [START_REF] Rafiyath | Comparison of safety and toxicity of liposomal doxorubicin vs. conventional anthracyclines: a meta-analysis[END_REF][START_REF] O'brien | High-Dose Vincristine Sulfate Liposome Injection for Advanced, Relapsed, and Refractory Adult Philadelphia Chromosome-Negative Acute Lymphoblastic Leukemia[END_REF]. In a non-clinical study, it has been demonstrated that stealth liposomal 5-FU was less likely to trigger severe leucopenia than standard 5-FU [START_REF] Fanciullino | In Vitro and In Vivo Evaluation of Lipofufol, a New Triple Stealth Liposomal Formulation of Modulated 5-Fu: Impact on Efficacy and Toxicity[END_REF]. Similarly, severe neutropenia appeared less in patients treated with nab-paclitaxel drug conjugate, as compared with standard paclitaxel [START_REF] Gradishar | Phase III Trial of Nanoparticle Albumin-Bound Paclitaxel Compared With Polyethylated Castor Oil-Based Paclitaxel in Women With Breast Cancer[END_REF], and model-informed studies have further confirmed how paclitaxel nanoparticles were less toxic against blood cells [START_REF] Ait-Oudhia | Meta-analysis of nanoparticulate paclitaxel delivery system pharmacokinetics and model prediction of associated neutropenia[END_REF].

More recently, in the Napoli-1 trial, liposomal irinotecan associated with LV-5-FU led to 18% of severe hematological toxicities, a figure markedly lower than the incidence of this toxicity when standard irinotecan is associated with 5-FU, such as the FolFiri regimen [START_REF] Adiwijaya | Population Pharmacokinetics of Liposomal Irinotecan in Patients With Cancer[END_REF]. Finally, by sparing hematologic progenitors, nanoparticles can be indirectly further helpful in patients scheduled for immunotherapy, by reducing the need to use antibiotics in patients. Indeed reducing toxicities, especially the neutropenia-related infections, will lead to cutting broad-spectrum antibiotherapy, whom administration has been repeatedly associated with reduced survival in immunotherapytreated patients [START_REF] Derosa | Negative association of antibiotics on clinical activity of immune checkpoint inhibitors in patients with advanced renal cell and non-small-cell lung cancer[END_REF], much probably because of the disruption of gut microbiome necessary to the immune surveillance, e.g. by enhancing the function of dendritic cells and elevating recruitment and function of T cells interacting with these later [START_REF] Yi | Gut microbiome modulates efficacy of immune checkpoint inhibitors[END_REF]. Overall, because they are better tolerated, using nanoparticles to deliver anticancer agents could be a promising strategy when a patient is scheduled next for immunotherapy because it is more protective towards patient's immunity, both by reducing the incidence of hematological toxicities and by limiting antibiotics whom use has been proven to be deleterious on survival.

How Nanoparticles could increase neo-antigens burden and modulate actors of immune response.

Standard anticancer agents are expected to boost immunotherapy at least in two distinct ways: by triggering immunogenic cell death and by adding effects on cancer and normal host cells in the TME.

Delivering immunogenic agents.

Lack of suitable neo-antigens and disruption in antigen processing or presentation is usually associated with impaired immune response to cancer [START_REF] Schumacher | Neoantigens in cancer immunotherapy[END_REF]. Consequently, promoting immunogenic cell death prior introducing immune check-point inhibitors should help improving efficacy, supporting the hypothesis that cytotoxics should be associated indeed with immunotherapy [START_REF] Kepp | Molecular determinants of immunogenic cell death elicited by anticancer chemotherapy[END_REF][START_REF] O'donnell | Resistance to PD1/PDL1 checkpoint inhibition[END_REF].

Immunogenic response induced by cytotoxics probably involves the purinergic receptor P2RX7 or the pattern recognition receptor toll-like receptor-4 (TLR-4) [START_REF] Green | Immunogenic and tolerogenic cell death[END_REF]. In addition, intrinsic tumor immunogenicity can be enhanced with cytotoxics by upregulating tumor antigens such as CEA or by increasing tumor antigen presentation and recruitment of antigen presenting cells (APCs), i.e. through the overexpression of MHC class-I molecules. Using nanoparticles should further trigger immunogenic response, because they frequently exhibit higher cytotoxicity as compared with standard drugs. For instance, liposomal 5-FU shows greater antitumor properties, both in vitro and in vivo, as compared with free 5-FU. In particular, stealth liposomal 5-FU induces deeper TS inhibition in colorectal cell lines, thus triggering Fas-mediated apoptosis because of the thymineless stress in cancer cells [START_REF] Fanciullino | In vitro and in vivo reversal of resistance to 5fluorouracil in colorectal cancer cells with a novel stealth double-liposomal formulation[END_REF]. In a quite similar way, liposomal gemcitabine proved to perform better than standard gemcitabine in pancreatic cancer models [START_REF] Bornmann | A new liposomal formulation of Gemcitabine is active in an orthotopic mouse model of pancreatic cancer accessible to bioluminescence imaging[END_REF], and nab-paclitaxel exhibited higher antiproliferative efficacy than free paclitaxel in a variety of solid tumors [START_REF] Desai | Increased antitumor activity, intratumor paclitaxel concentrations, and endothelial cell transport of cremophor-free, albumin-bound paclitaxel, ABI-007, compared with cremophor-based paclitaxel[END_REF]. The superior antiproliferative efficacy of nanoparticles over standard cytotoxics has been confirmed at bedside as well, since liposomal vincristine was found to be more effective than standard vincristine [START_REF] O'brien | High-Dose Vincristine Sulfate Liposome Injection for Advanced, Relapsed, and Refractory Adult Philadelphia Chromosome-Negative Acute Lymphoblastic Leukemia[END_REF] whereas the combo daunorubicine + cytarabine given as a liposomal formulation improved response rates as compared with standard combination in patients with myelodysplastic syndromes [START_REF] Kim | Daunorubicin and Cytarabine Liposome in Newly Diagnosed Therapy-Related Acute Myeloid Leukemia (AML) or AML With Myelodysplasia-Related Changes[END_REF]. In addition to the selective delivery of cytotoxics likely to trigger immunogenic cell death, nanoparticles can be further used to deliver antigens or adjuvants to specifically dendritic cells (DCs). For instance, lymph node-targeting nanoparticle-conjugate vaccines (i.e., TAA-NP and CpG-NP) proved to induce stronger cytotoxic CD8+ T-cell responses, higher antiproliferative efficacy and extended survival in mice bearing melanoma [START_REF] Jeanbart | Enhancing efficacy of anticancer vaccines by targeted delivery to tumor-draining lymph nodes[END_REF].

Modulating TME Immune cells.

In addition to promoting cancer immunogenicity by increasing neo-antigens burden, nanoparticles carrying cytotoxics or cytokines could modulate as well immune response through a variety of mechanisms, ranging from upregulating the expression of MHC Class I molecules to which the antigens bind, upregulating of co-stimulatory molecules and PD1/PDL1 expression, to downregulating co-inhibitory molecules such as PD-L1/B7-H1 or B7-H4, thus enhancing the strength of effector T cell activity. For instance, canonical 5-FU can abrogate Myeloid Derived Suppressive Cells (MDSCs) and Treg activity, while additionally it makes T cell-mediated lysis more effective through Fas-dependent mechanisms [START_REF] Chen | Chemoimmunotherapy: reengineering tumor immunity[END_REF][START_REF] Emens | The interplay of immunotherapy and chemotherapy: harnessing potential synergies[END_REF][START_REF] Katsuya | Expression of programmed death 1 (PD-1) and its ligand (PD-L1) in thymic epithelial tumors: Impact on treatment efficacy and alteration in expression after chemotherapy[END_REF]. Cisplatin and cyclophosphamide both proved to decrease Tregs expression and to increase CD8+ T cell activity [START_REF] Ercolini | Recruitment of latent pools of high-avidity CD8(+) T cells to the antitumor immune response[END_REF]. In addition to modulating T lymphocytes, chemotherapy, especially when administered with low dose such as following metronomic regimen, can modulate as well DCs phenotype and function. For instance vincaalkaloids, taxanes and cyclophosphamide proved to enhance the function of DCs, much probably through an IL-12 dependent mechanism [START_REF] Kaneno | Chemomodulation of human dendritic cell function by antineoplastic agents in low noncytotoxic concentrations[END_REF][START_REF] Shurin | Chemotherapeutic agents in noncytotoxic concentrations increase antigen presentation by dendritic cells via an IL-12-dependent mechanism[END_REF]. Much interestingly, the very distribution profile of nanoparticles, which are expected to nest in the TME and start releasing their payload regularly and over a long period of time, could meet this metronomic-like exposure required to enhance DC. One of the common features of all drug-carriers is indeed to stay longer in the tumor site, whereas free drugs enter massively before being rapidly cleared out of the tumor tissue. Consequently pharmacodynamic properties of standard cytotoxics are best described by the Hill equation, i.e. the higher the dose, the higher the effect on tumor cells or TME, with no lag compartment when modeling the effects. In contrast, the complex interplay between carrier's PK, payload release, carrier's interactions with TME, makes the PK/PD profiles of nanoparticles more likely to ensure a sustained and constant release of cytotoxics at the tumor site [START_REF] Rodallec | Seek and destroy: improving PK/PD profiles of anticancer agents with nanoparticles[END_REF]. In addition and because of the complete lack of specificity of anticancer agents, the high dose regimen required to trigger immunogenic cell death will have deleterious impact on bone marrow cells (see previous paragraph), with subsequent lack of clonal expansion of T cells. This calls for using nanoparticles to redefine optimal dosing and scheduling of cytotoxics, so as to limit their hematological toxicities while preserving immunogenic cell death at the tumor level. In addition, drastic tumor debulking with cytotoxics leading to minimal residual disease may mitigate the negative impact of tumor burden on the efficacy of immune check point inhibitors. In this context, using nanoparticles once again could help to solve this once contradictory issue, because PK/PD relationships of nanoparticles allow sparing healthy cells while exerting maximal antiproliferative efficacy at the tumor level [START_REF] Rodallec | Seek and destroy: improving PK/PD profiles of anticancer agents with nanoparticles[END_REF].

Although published data remain sparse, it has been recently confirmed that nanoparticles could achieve higher antiproliferative efficacy when combined to immune check point inhibitors because of their immunomodulating properties. For instance after having demonstrated how topoisomerase-I inhibitors such as irinotecan could enhance T-cell-mediated cytotoxicity of melanoma tumors in vitro, liposomal irinotecan was associated with anti-PDL1 in tumor-brearing-mice. Results confirmed greater efficacy of this association, much probably because of the higher cytotoxic functionality observed with CD8+ T cells in the combination group [START_REF] Mckenzie | The Effect of Topoisomerase I Inhibitors on the Efficacy of T-Cell-Based Cancer Immunotherapy[END_REF].

Triggering immunogenic response.

Nanocarriers themselves can exert some kind of immunomodulation on their own, due to their physico-chemical composition. Despite being < 200 nm, nanoparticles remain foreign objects likely to be recognized by the MPS. When circulating in the body, this could be an issue associated with lack of stability and different strategies can be undertaken to increase nanoparticle stealthness, such as surface pegylation [START_REF] Rodallec | Pharmacokinetics variability: Why nanoparticles are not just magic-bullets in oncology[END_REF]. However once the TME is reached, immunogenicity and antigenicity of nanoparticles could help to boost immune response against cancer [START_REF] Ilinskaya | Understanding the immunogenicity and antigenicity of nanomaterials: Past, present and future[END_REF]. Generating antibodies against nanoparticles can be obtained through a thymus-dependent pattern. Bioengineered nanoparticles will activate DC's producing cytokines that activate T-helper cells. These T-helpers will recognize next antigens from APCs and finally prompt B cells to proliferate and to differentiate against the antigenic nanoparticles. In addition, B-cell activation can be achieved by repetitive elements in the antigen without T-cell involvement [START_REF] Sauerborn | Immunological mechanism underlying the immune response to recombinant human protein therapeutics[END_REF]. Of note, beyond inorganic scaffolds such as metal-based nanoparticles [START_REF] Zolnik | Nanoparticles and the immune system[END_REF], it has been shown that antibodies can be raised even against 100% biocompatible lipid carriers such as liposomes [START_REF] Banerji | Antibodies against liposomes in normal and immune-defective mice[END_REF][START_REF] Neun | Understanding the Role of Anti-PEG Antibodies in the Complement Activation by Doxil in Vitro[END_REF]. Of note, even surface pegylation, a common strategy in nanomedicine to increase stealthness and mask nanoparticles from scavengers and phagocytic cells, can show some immunogenicity as anti-PEG IgM have been already described in non-clinical models [START_REF] Shimizu | Transport of PEGylated liposomes from the splenic marginal zone to the follicle in the induction phase of the accelerated blood clearance phenomenon[END_REF]. Nanoparticles immunogenicity could be therefore mostly based upon antibody-dependent cell cytotoxicity (ADCC)-like mechanism, the antibodies being capable to recognize either surface groups or core components. Because in oncology nanoparticles are expected to preferentially accumulate and concentrate in tumors, this immunogenicity could contribute to attract in TME new actors of the immune response. However, to what ADCC could contribute to a better efficacy when using next immune check point inhibitors must be clearly investigated, as some nanoparticles showed inconsistent results. For instance, silicon nanoparticles encapsulated with sorafenib and coated with anti-CD326 antibodies showed increased efficacy in various breast cancer models. However, concentration-dependent ADCC was evidenced in MCF7 cells, but not in the MDA-MB231 model [START_REF] Shahbazi | A prospective cancer chemo-immunotherapy approach mediated by synergistic CD326 targeted porous silicon nanovectors[END_REF], whereas elsewhere a complete lack of ADCC mechanism was reported with gold nanoparticles [START_REF] Ahmed | Lack of in vivo antibody dependent cellular cytotoxicity with antibody containing gold nanoparticles[END_REF]. Despite this discrepancy in ADCC properties, several strategies have been successfully tested to boost patient's immunity. For instance, inorganic nanoparticles (i.e., polymeric or gold) with surface modifications were used to target Tumor Associated Macrophages (TAM) thanks to PEG-sheddable, mannose-modified constructs [START_REF] Zhu | Targeting of tumor-associated macrophages made possible by PEG-sheddable, mannose-modified nanoparticles[END_REF]. Elsewhere, artificial APCs for direct CTL activation were achieved using iron-oxide nanoparticles coated with dextran and quantum dots coated with avidin led to an increase in T-cells and successful tumor rejection in melanoma-bearing mice [START_REF] Perica | Nanoscale artificial antigen presenting cells for T cell immunotherapy[END_REF].

Helping immune cell and therapeutic monoclonal antibodies to better trafficking into tumor micro-environment

Tumor-associated immune factors and chemokines in TME are heterogeneous and play a critical role in determining clinical outcome in cancer patients treated with immune checkpoint inhibitors. As early of the early 2000's it was found that TME was associated with higher turnover of Tlymphocytes, as compared with non-cancerous tissue, suggesting that targeting TME could be of importance in the era of immunotherapy because of its immunosuppressive properties [START_REF] Whiteside | Apoptosis of immune cells in the tumor microenvironment and peripheral circulation of patients with cancer: implications for immunotherapy[END_REF]. By affecting TME cytotoxics may enhance the efficacy of tumor-educated lymphocytes. The higher the amount of cytotoxics in tumor surrounding, the more effective will be the action on tumor stroma, desmoplastic cells, fibroblasts and eventually tumor cells. However, it seems that the balance between direct cytotoxicity (and subsequent immunogenic cell death), depleting effect on TME, and immunomodulating properties of cytotoxics all depends on the dosing and the scheduling of the regimen, and should be finely tuned as it is probably drug-dependent. It has been demonstrated, long before immunotherapy was on the rise, that neo-adjuvant paclitaxel could ease new intratumoral immune cell infiltrates with subsequent higher apoptotic response and clinical efficacy in breast cancer [START_REF] Demaria | Development of tumor-infiltrating lymphocytes in breast cancer after neoadjuvant paclitaxel chemotherapy[END_REF]. Much interestingly, paclitaxel can be delivered either as a cremophor formulated drug, or as a conjugated nanoparticle, nab-paclitaxel. Nab-paclitaxel proved to deplete tumor stroma, reducing density and disrupting tumor micro-environment, in addition to direct effect on tumor cells microtubules. Because one of the main causes for immune escape is unfavorable TME [START_REF] Pitt | Resistance Mechanisms to Immune-Checkpoint Blockade in Cancer: Tumor-Intrinsic and -Extrinsic Factors[END_REF], such additional mechanism of action is likely to make hot an initially cold tumor, thus further easing the infiltration of dendritic cells and antigen-experienced T cells as compared with free paclitaxel. Indeed, tumor-specific CD8 T-cells subsequently differentiated into effector T-cells require trafficking to the TME prior to kill cancer cells expressing neoantigens. By depleting tumor stroma, nanoparticles could actually both enhance the infiltration in TME of T cells, but as well that of therapeutic monoclonal antibodies such as immune checkpoint inhibitors which are usually molecules too large to properly reach solid tumors because of high stroma density and binding-site barrier issues [START_REF] Jain | Barriers to drug delivery in solid tumors[END_REF]. Although to date no experimental data are available to support this hypothesis because little is known on the tumor distribution of immune check-point inhibitors in vivo, one can speculate that depleting effect on TME by nanoparticles should additionally increase drug delivery of these large therapeutic antibodies next.

Conclusion: How nanoparticles could be best combined with immunotherapy.

Overall, nanoparticles can be used to boost immunotherapy in several ways. Optimizing delivery at the tumor site of a variety of payloads ranging from cytotoxics triggering immunogenic cell death, cancer vaccine or therapeutic RNAs is an appealing strategy. Interestingly, nanotechnologies offer today such a wide sort of scaffolds that there is no limit but the imagination of the researchers to achieve this goal (Figure 2). For instance gold nanoparticles have been designed to deliver anti-VEGF siRNA both to TAMs and to lung cancer cells, achieving a synergistic efficacy in tumor-bearing mice [START_REF] Conde | Dual targeted immunotherapy via in vivo delivery of biohybrid RNAi-peptide nanoparticles to tumour-associated macrophages and cancer cells[END_REF]. Elsewhere, dendrimers loaded with paclitaxel conjugated with mAbK1 antibodies proved to be effective in ovarian cancer mice models [START_REF] Jain | The development, characterization and in vivo anti-ovarian cancer activity of poly(propylene imine) (PPI)-antibody conjugates containing encapsulated paclitaxel[END_REF]. Nontoxic core-shell nanoparticles encapsulating photosensitizer pyrolipid proved to increase the efficacy of anti-PDL1 in metastatic breast cancer models, partly by increasing tumor mutational burden after photodynamic therapy [START_REF] Duan | Photodynamic Therapy Mediated by Nontoxic Core-Shell Nanoparticles Synergizes with Immune Checkpoint Blockade To Elicit Antitumor Immunity and Antimetastatic Effect on Breast Cancer[END_REF]. In breast cancer model again, liposomal nanoparticle coated with tumor-targeting peptide and coanti-PI3K drug plus a specific agonist of therapeutic T cells, proved to act synergistically when CAR-T cells were administered next. Increase of immune effector cells such as CD8+ lymphocytes and invariant natural killer cells were evidenced, and increase in efficacy was further confirmed in a mouse model of human glioblastoma [START_REF] Zhang | Nanoparticles That Reshape the Tumor Milieu Create a Therapeutic Window for Effective T-cell Therapy in Solid Malignancies[END_REF]. Elsewhere nanogels carrying interleukin-15 were successfully developed to boost mouse T cell and CAR-T cell therapy in a melanoma model [START_REF] Tang | Enhancing T cell therapy through TCR-signaling-responsive nanoparticle drug delivery[END_REF]. In addition to drug delivery properties or tumor micro-environment reshaping, nanoparticles can also exhibit directly some immunogenic properties. Size, composition, surface properties, electric charge, all probably matter and call for a comprehensive understanding of the immunogenicity and antigenicity of nanoparticles. For instance, cobalt oxide nanoparticles coated with PMIDA and conjugated to a lysate antigen triggered increase in IgG and CD4+ response in mice [START_REF] Chattopadhyay | Metal based nanoparticles as cancer antigen delivery vehicles for macrophage based antitumor vaccine[END_REF]. In breast cancer models, manganese dioxide liposomal nanoparticles combined with doxorubicin increased the infiltration of CD8+ T cells in TME, thus boosting antitumor efficacy as compared with free doxorubicine alone. Much interestingly, naïve tumor-bearing mice transplanted with splenocytes from mice previously treated by this combination were able to control tumor growth, whereas splenocytes from control mice or from doxorubicine-treated mice had no antiproliferative effect, thus highlighting how immunomodulating properties of nanoparticles were at the origin of the observed efficacy [START_REF] Amini | Combining Tumor Microenvironment Modulating Nanoparticles with Doxorubicin to Enhance Chemotherapeutic Efficacy and Boost Antitumor Immunity[END_REF]. Although only experimental, all these studies pave the way for future combinations with immune check point inhibitors. However, there are several pitfalls in developing combinatorial strategies between nanoparticles and immunotherapy. First, the most immunogenic nanoparticles such as the ones based upon metal, silica nanosphere of other inorganic or polymeric components, have all unaddressed issues in terms of nanosafety, because little is known about their possible long-term effects once injected in the body [START_REF] Meng | Walking the line: The fate of nanomaterials at biological barriers[END_REF]. In addition, being too immunogenic may expose nanoparticles to early recognition by macrophages and scavenging cells before the nanoparticles even reach the tumor site. Second, apart long-studied liposomes, little is known about the exact pharmacokinetic profile of the most recent nanoparticles, because their unique features differ to that of standard drugs [START_REF] Rodallec | Seek and destroy: improving PK/PD profiles of anticancer agents with nanoparticles[END_REF][START_REF] Rodallec | Pharmacokinetics variability: Why nanoparticles are not just magic-bullets in oncology[END_REF]. Extensive PK/PD modeling is therefore much awaited to better understand how and when nanoparticles should be best combined with immune check-point inhibitors. For instance, increasing mutational burden and achieving drastic tumor debulking is more likely to be observed when high concentrations of cytotoxics reach tumor cells, whereas immumodulating properties on DCs, Tregs or effector T-cells have been conversely reported with metronomic regimen leading to sustained and continuous exposure to low levels of drugs [START_REF] Kareva | A Combination of Immune Checkpoint Inhibition with Metronomic Chemotherapy as a Way of Targeting Therapy-Resistant Cancer Cells[END_REF]. The era of trial-and-error strategies, inherited from the 20 th century medicine, is gone and with respect to the possible number of combinations made available now, strong pharmacometric support is mandatory to ensure successful development of immuno-nano-therapeutics.

Expert Opinion:

Beyond the initial frenzy sparked by the successful stories in melanoma and lung cancer, immunotherapy seems to have reached a glass-ceiling in oncology, at least as single-agents. Poor trafficking of activated T lymphocytes to the tumors, massive recruitment of Tregs or MDSCs in the tumor micro-environment, low mutational burden, are the most frequent reasons explaining the innate resistance of most solid tumors towards immune checkpoint inhibitors. Pre-treating patients with agents likely to boost immune response is therefore an appealing strategy to optimize the efficacy of immunotherapy. Nanoparticles present a wide range of characteristics such as optimized drug delivery in the tumor surroundings and immunogenic properties that make them suitable candidates for combinatorial strategies with immune checkpoint inhibitors. In addition, nanoparticles can cargo a limitless number of payloads such as cytotoxics, cytokines, vaccines or nucleotides, all likely to modulate tumor immunity or to trigger immunogenic cell death. However, a current bottleneck in the clinical development of nanoparticles remains scale-up issues and pitfalls when shifting from bench-size batches to clinical-size batches. Apart from liposomes and immunoliposomes for which proof-of-concepts of successful industrial production has already been made, the complexity to produce large and reproducible batches of sophisticated scaffolds has to be taken into account and could be a limitation to future bedside applications. Nanosafety issues and emerging tight regulations regarding manufacturing and handling of nanoparticles also has to be taken into account as a possible limitation, especially with inorganic materials [START_REF] Warheit | Hazard and risk assessment strategies for nanoparticle exposures: how far have we come in the past 10 years?[END_REF]. In addition, there is another possible safety risk when administrating highly immunogenic nanoparticles in combination with immune check point inhibitors, because of a possible potentiation of immune-related adverse effects (IRAEs) reported with immunotherapy. In addition, immunogenic nanoparticles can also be recognized by scavenging cells or macrophages when trafficking in the blood, spleen or liver, thus having little chance to reach the tumor. Stealthness is a common strategy to limit spleen or liver uptake -however designing a nanoparticle smart enough to be stealth in healthy tissue to limit IRAEs while triggering immunogenicity only once malignant tissue are reached is particularly challenging.

Finally, lessons have to be learned from previous recent failures when trying to combine empirically immune check-point inhibitors with other treatments such as metronomic chemotherapy, radiotherapy or anti-angiogenics. In-depth knowledge of nanoparticles PK/PD profiles, ideally using model-informed approaches, is urgently needed to better picture the optimal modality to combine nanoparticles with immunotherapy. The complexity in the multiple and possibly contradictory effects on immune cells, especially when using encapsulated cytotoxics, require finely tuned protocols in terms of dosing, sequencing and treatment duration. With respect to the number of treatment modalities and scheduling, extensive mathematical resources are required to help determining in silico the strategy that is the most efficient to yield synergistic effect between nanoparticles, immune check-point inhibitors, and possibly other drugs likely to boost efficacy such as anti-angiogenics.

Should the optimal conditions be met, because nanoparticles are more protective towards hematopoietic progenitors and therefore less likely to trigger lymphopenia, not to mention stroma-targeting properties and boosting tumor immunogenicity, they could be of great value for awakening tumor immunity and help once resistant solid tumors to respond to immune check-point inhibitors?

Article highlights

• Immunotherapy is currently limited by cold tumors displaying unfavorable immunogenic profile because of low mutational burden, harsh tumor micro-environment, plus possible deleterious impact of prior chemotherapy on patient's innate immunity.

• Combinatorial therapy is seen as the future of immunotherapy, and associated treatments are all expected to turn cold tumors into hot tumors, so as to boost the efficacy of immune check-point inhibitors.

• Nanoparticles present promising features, making them particularly suitable candidates to be associated with immune check-point inhibitors.

• Improved pharmacokinetics, immunogenic properties and ability to cargo a wide variety of anticancer drugs or immunomodulating agents provide nanoparticles with unique properties.

• Determining the optimal modality of such combination is challenging because of the multiple and complex interplays between nanoparticles, patient's immunity, tumor microenvironment and tumor cells.

Disclosures:

Figure 1 :

 1 Figure 1: Different scaffolds and nanoparticles available to cargo payloads to the tumor site. NP: nanoparticle.

Figure 2 :

 2 Figure 2: Schematic comparison of the biodistribution profiles of xenobiotics administered as free drugs (A) or as nanoparticles (B).

Figure 3 :

 3 Figure 3: Representation of the possible multiple interplays between nanoparticles, cancer cells, and immune cells in the TME. TAM: Tumor-Associated Macrophages. MDSC: Myeloid Derived Suppressive Cells, APC: Antigen-Presenting Cells.

Anne Rodallec received a grant from French charity La Ligue Nationale contre le Cancer (# GB/MA/CD/Ep-1204) as part of her Ph.D. studies. All authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. This article was not funded.


B Zhang
email: bzhang@pasteur.fr

Jalal M Fadili

Jean-Luc Starck

Jean-Christophe Olivo-Marin

M J Fadili

J.-L Starck

J.-C Olivo-Marin

Multiscale Variance-stablizing Transform for Mixed-Poisson-Gaussian Processes and its Applications in Bioimaging

Keywords: variance stabilizing transform, Mixed-Poisson-Gaussian process, wavelet, fluorescence microscopy

published or not. The documents may come   L'archive ouverte pluridisciplinaire

INTRODUCTION

Fluorescence microscopy is a widely used technique to image biological specimens. The resulting images are corrupted by photon and camera readout noises. The stochastic data model is thus a Mixed-Poisson-Gaussian (MPG) process. For many applications such as denoising and deconvolution, it would be rather complicated to directly deal with such processes since every sample exhibits an infinite Gaussian mixture distribution. A commonly used technique is to first apply a variance stabilizing transform (VST), e.g., the generalized Anscombe transform (GAT) [START_REF] Starck | Image Processing and Data Analysis[END_REF], to Gaussianize the data so that each sample is near-normally distributed with an asymptotically constant variance. The VST allows to apply standard denoising and deconvolution methods on the transformed data.

Then, the final estimate is obtained by inverting the VST on the processed data.

In this paper, we propose a new VST to Gaussianize a low-pass filtered MPG process. This transform can be considered as a generalization of the GAT and a recently proposed VST for Poisson data [2]. Then, this VST is combined with the isotropic undecimated wavelet transform (IUWT) [START_REF] Starck | Image Processing and Data Analysis[END_REF] leading to a multiscale VST (MS-VST). The usefulness of MS-VST is demonstrated for image denoising and spot detection in fluorescence microscopy. In the first case, we detect significant Gaussianized wavelet coefficients under the control of a false discovery rate (FDR) [START_REF] Benjamini | The control of the false discovery rate in multiple testing under dependency[END_REF]. A sparsity-driven iterative scheme is proposed to properly reconstruct the final estimate. In the second case, we show that a slight modification of the denoising algorithm leads to a fluorescent-spot detector, where the false positive rate of the detection in pure noise can be controlled. Experiments show that the MS-VST approach outperforms the GAT in denoising, and that the proposed detection scheme allows efficient spot extraction from complex background.

VST FOR A FILTERED MPG PROCESS

A MPG process x := (X i ) i∈Z d is defined as:

X i = αU i + V i , U i ∼ P(λ i ), V i ∼ N (µ, σ 2 ) (1)
where α > 0 is the overall gain of the detector, U i is a Poisson variable modeling the photon counting, V i is a normal variable representing the readout noise, and all (U i ) i and (V i ) i are assumed mutually independent. Given a discrete filter h, we note a filtered MPG process as Y i := j h[j]X i-j . We will use X and Y to denote any one of X i and Y i respectively. We further denote by τ k the quantity i (h[i]) k for k ∈ N * .

To simplify the following analysis we assume that λ i = λ within the support of h. It can be verified that the variance of Y (Var [Y ]) is an affine function of the Poisson intensity λ. To stabilize Var [Y ], we seek a transformation Z := T (Y ) such that Var [Z] is (asymptotically) constant, irrespective of the value of λ. We define: Lemma 1 indicates that the square-root transform (2) is indeed a VST for stabilizing and Gaussianizing a low-pass filtered MPG process.

Lemma 1 (square root as VST [START_REF] Zhang | Wavelets, Ridgelets and Curvelets for Poisson Noise Removal[END_REF]) If τ 1 = 0, then we have:

T (Y ) -b • sgn(τ 1 ) |τ 1 |αλ D -→ λ→+∞ N 0, αb 2 τ 2 4|τ 1 | (3)
This result holds for any c ∈ R. However, the convergence rate in (3) varies with the value of c (b is only a normalizing factor), and we want to determine its optimal value.

Optimal parameter of the VST

Without loss of generality, suppose that τ 1 > 0, then Pr(Y + c > 0) can be made arbitrarily close to 1 as λ → +∞. So in our asymptotic analysis below, we will essentially consider the VST in the form

T (Y ) = bT 0 (Y ) = b √ Y + c. Expand- ing T 0 (Y ) by Taylor series about the point Y = E [Y ]
up to the 4th order term, and by applying the expectation one can calculate the asymptotic expectation and variance of T (Y ):

E [b 1 T 0 ] ≈ √ λ + 4τ 1 (τ 1 µ + c) -τ 2 α 8τ 2 1 α C E λ -1/2 (4) Var [b 2 T 0 ] ≈ 1+ 8τ 2 1 τ 2 (σ 2 -αµ) -4τ 1 α(2τ 2 c + τ 3 α) + 7τ 2 2 α 2 8α 2 τ 2 1 τ 2 C Var λ -1 (5)
where

b 1 = (τ 1 α) -1 2 and b 2 = 2( τ 1 ατ 2 ) 1 2
. These settings normalize respectively the asymptotic expectation and variance to √ λ and 1, both values being independent of the filter h. Then the optimal c is found by minimizing the following biasvariance tradeoff (controlled by η):

c * := arg min c∈R E η (c) := ηC 2 E + (1 -η)|C Var |, η ∈ [0, 1] (6)
With no prior preference for either bias or variance, η can be set to 1/2. Note that C E is squared to give an equivalent asymptotic rate for the tradeoff terms in ( 4) and ( 5). It can be shown that ( 6) admits a unique solution, which can be explicitly derived out as a function of τ k , µ, σ, α and η. This VST reduces to the GAT if h = Dirac filter δ and η = 0.

In practice, if µ, σ, and α are unknown a priori, they can be estimated by matching the first four cumulants of X with the k-statistics [START_REF] Rose | Mathematical Statistics with Mathematica, chapter 7.2C: k-Statistics: Unbiased Estimators of Cumulants[END_REF] of the samples in a uniform image region. This follows from the property that the k-statistics are the minimum variance unbiased estimators for cumulants.

IMAGE DENOISING USING MS-VST

Isotropic structures are often presented in biological fluorescent images due to micrometric subcellular sources. Toward the goal of image denoising, we will combine the proposed VST with the IUWT. Indeed, since IUWT uses isotropic filter banks, this transform adapts very well the isotropic features in images. The left side of [START_REF] Yamada | The hybrid steepest descent method for the variational inequality problem over the intersection of fixed point sets of nonexpansive mappings[END_REF] gives the classical IUWT decomposition scheme, and by applying the VST on the (low-pass filtered) approximation coefficients at each scale, we obtain a MS-VST scheme shown on the right side:

a j = h↑j-1 ⋆ a j-1 d j = a j-1 -a j ⇒ a j = h↑j-1 ⋆ a j-1 d j = T j-1 (a j-1 ) -T j (a j ) (7)
Here h is a symmetric low-pass filter, a j and d j are respectively the approximation and the wavelet coefficients at scale

j, h ↑j [l] = h[l] if l/2 j ∈ Z and 0 otherwise, h[n] = h[-n]
and "⋆" denotes convolution. The filtering of a j-1 can be rewritten as a filtering of the original MPG data x = a 0 :

a j = h (j) ⋆ a 0 , where h (j) = h↑j-1 ⋆ • • • ⋆ h↑1 ⋆ h for j ≥ 1
and h (0) = δ. T j is the VST operator at scale j (cf. (2)):

T j (a j ) = b (j) sgn(a j + c (j) )|a j + c (j) | 1/2
The constants b (j) and c (j) are associated to h (j) , and c (j) should be set to c * . Theorem 1 shows that (7) transfers the asymptotic stabilized Gaussianity of the a j 's to the d j 's:

Theorem 1 (d j under a high intensity assumption) Setting b (j) := sgn(τ

(j) 1 )/[α|τ (j)
1 |] 1/2 , we have:

d j D -→ λ→+∞ N 0, τ (j-1) 2 4τ (j-1) 1 2 + τ (j) 2 4τ (j) 1 2 - h (j-1) , h (j) 2τ (j-1) 1 τ (j) 1
where τ (j)

k := i h (j) [i]
k , and •, • denotes inner product.

This result shows that the asymptotic variance of d j depends only on the wavelet filter bank and the current scale, and thus can be pre-computed once h is chosen.

Detection of significant coefficients by FDR

Wavelet denoising can be achieved by zeroing the insignificant coefficients while preserving the significant ones. We detect the significant coefficients by testing binary hypothesis: ∀ d, H 0 : d = 0 vs. H 1 : d = 0. The distribution of d under the null hypothesis H 0 is given in Theorem 1. Thus, a multiple hypothesis testing controlling the FDR can be carried out [START_REF] Benjamini | The control of the false discovery rate in multiple testing under dependency[END_REF]. The control of FDR offers many advantages over the classical Bonferroni control of the Family-Wise Error Rate, i.e., the probability of erroneously rejecting even one of the true null hypothesis. For example, FDR usually has a greater detection power and can handle correlated data easily. The latter point is important since the IUWT is over-complete.

Sparsity-driven iterative reconstruction

After coefficient detection, we could invert the MS-VST [START_REF] Yamada | The hybrid steepest descent method for the variational inequality problem over the intersection of fixed point sets of nonexpansive mappings[END_REF] to get the final estimate:

a 0 = T -1 0 [T J (a J ) + J j=1 d j ]
, but this solution is far from optimal. Indeed, due to the non-linearity of the VST and the over-completeness of IUWT, the significant coefficients are not reproducible when IUWT is applied once more on this direct inverse, implying a loss of important structures in the estimation. A better way is to find a constrained sparsest solution, as sketched below (see [START_REF] Zhang | Wavelets, Ridgelets and Curvelets for Poisson Noise Removal[END_REF] for details).

We first define the multi-resolution support [START_REF] Starck | Image Processing and Data Analysis[END_REF] M := {(j, l) | d j [l] is significant}, which is determined by the set of the detected significant coefficients. The estimation is then formulated as a constrained convex optimization problem in terms of wavelet coefficients: where W is the wavelet analysis operator, and R its synthesis operator. Clearly by doing so, we minimize a sparsitypromoting ℓ 1 objective function [START_REF] Donoho | Optimally sparse representation in general (nonorthogonal) dictionaries via ℓ 1 minimization[END_REF] within the feasible set C := S 1 ∩ S 2 . The set S 1 requires that the elements of d preserve the significant coefficients; the set S 2 assures a modelconsistent estimate since

E [X i ] = αλ i + µ ≥ µ.
Gradient descent method such as the hybrid steepest descent (HSD) iterations [START_REF] Yamada | The hybrid steepest descent method for the variational inequality problem over the intersection of fixed point sets of nonexpansive mappings[END_REF] can be used to solve (8):

d (k+1) := T C d (k) -β k+1 sgn T C d (k) (9)
where the step length β k satisfies: (i) lim k→∞ β k = 0, (ii)

k≥1 β k = +∞, (iii) k≥1 |β k -β k+1 | < +∞. The oper- ator T C is defined as T C := P S 1 • Q S 2 ,

and

P S 1 d := Wx in M d otherwise ; Q S 2 d := WP µ Rd (10)
where P µ is the projector onto the set {x|x i ≥ µ}. It is worth noting that compared with the direct reconstruction, every iteration of (9) involves a projection onto the set S 1 that restores all the significant coefficients. Therefore, important structures are better preserved by the iteratively reconstructed solution.

Results

We first test our denoising approach on a simulated isotropicsource grid (pixel size = 100 nm) shown in Fig. 1. From the leftmost to the rightmost column, the source radii increase from 50 nm to 350 nm. The image is then convolved with a 2D Gaussian function with a standard deviation σ g = 116 nm, which approximates the point spread function of a typical fluorescence microscope [START_REF] Zhang | Gaussian approximations of fluorescence microscope PSF models[END_REF]. Fig. 1(a) shows the sources with amplitudes λ i,j ∈ [0.05, 83.5], 1 ≤ i ≤ 18, 1 ≤ j ≤ 10.

After adding a MPG noise, we obtain Fig. 1(b). Fig. 1(c) and (d) respectively show the denoised images using the GAT and the MS-VST. To give a fair comparison, we have set η = 0 so that our VST parameter is derived using the same criterion as for GAT. We can see that MS-VST is more sensitive than GAT since more faint sources are restored. In terms of the L 1 loss, the MS-VST-denoised image is also more accurate ( err. L 1 = 3.09) than the GAT result ( err. L 1 = 3.34).

SPOT DETECTION USING MS-VST

A slight modification of the denoising algorithm can serve as a fluorescent-spot detector. Since wavelets are band-pass filters, background information is mostly encoded in the approximation band. Therefore, if we zero the approximation band at the last iteration of (9), the background will be largely suppressed from the final estimate and, consequently, only detail (spot) structures are reconstructed. Then, a positive threshold can be easily found to binarize the result and all connected components are extracted as putative bright spots. With this approach, the false spot-detection rate in pure noise can be controlled: Proposition 1 Suppose that the FDR of wavelet coefficient detection is controlled, i.e., FDR ≤ γ. Then the probability of erroneously detecting spots in a spot-free homogeneous MPG noise (λ i = λ) is upper bounded by γ.

Results

Fig. 3 shows the detection of endocytic vesicles of COS-7 cells in a wide-field microscopy image. Although the original image exhibits a highly nonuniform background (Fig. 3(a)), the detection (Fig. 3(b)) is very effective as most spots are well extracted while the background is canceled.

CONCLUSION

We have designed a VST to stabilize and Gaussianize a lowpass filtered MPG process. The VST is then combined with the IUWT yielding the MS-VST. We have shown the MS-VST approach to be very effective in fluorescent image denoising and spot detection. Our future work will apply the MS-VST in deconvolution and super-resolution detection.

  min d∈C J(d) := d 1 where C := S 1 ∩ S 2 S 1 := {d|d = Wx in M} and S 2 := {d|Rd ≥ µ} (8)

Fig. 2 Fig. 1 .

 21 Fig. 1. Simulated source denoising. h = 2D B 3 -Spline filter, η = 0, FDR = 0.01, and 10 (a) simulated sources (amplitudes λ i,j ∈ [0.05, 83.5]; background = 0.05); (b) MPG noisy image (α = 20, µ = 10, and σ = 1); (c) GAT-denoised image ( err. L 1 = 3.34); (d) MS-VST-denoised image ( err. L 1 = 3.09)

Fig. 2 .

 2 Fig. 2. Denoising of a 3D confocal image of a drosophila melanogaster ovary. h = 3D B 3 -Spline filter, FDR = 0.05, η = 0.5, and 10 iterations. Observed image: (a) z = 22µm; (b) z = 26µm; MS-VST-denoised image: (c) z = 22µm; (d) z = 26µm.

T (Y ) := b • sgn(Y + c)|Y + c| 1/2 , b = 0, c ∈ R(2)

ACKNOWLEDGMENT

The authors would like to thank M. M. Mhlanga (Institut Pasteur) for providing the image of drosophila melanogaster ovary, and A. Hémar (CNRS UMR 5091) for providing the COS-7 cell image.

* This work is funded by CNRS and Institut Pasteur of France.


A Manchon

C Ducruet

L Lombard

S Auffret

B Rodmacq

B Dieny

S Pizzini

J Vogel

V Uhl

M Hochstrasser

G Panaccione

Analysis of anisotropy crossover due to oxygen in Pt/Co/MOx trilayer

Keywords: numbers: 75.47.-m, 75.75.+a, 72.25.Ba, 73.20.At, 73.40.Rw, 75.70.Cn X-ray photoelectron spectroscopy, X-ray absorption spectroscopy, Magnetic tunnel junctions, Perpendicular magnetic anisotropy 1

Extraordinary Hall effect and X-ray spectroscopy measurements have been performed on a series of Pt/Co/MOx trilayers (M=Al, Mg, Ta...) in order to investigate the role of oxidation in the onset of perpendicular magnetic anisotropy at the Co/MOx interface. It is observed that varying the oxidation time modifies the magnetic properties of the Co layer, inducing a magnetic anisotropy crossover from in-plane to out-of-plane. We focused on the influence of plasma oxidation on Pt/Co/AlOx perpendicular magnetic anisotropy. The interfacial electronic structure is analyzed via X-ray photoelectron spectroscopy measurements. It is shown that the maximum of out-of-plane magnetic anisotropy corresponds to the appearance of a significant density of Co-O bondings at the Co/AlOx interface.

I. INTRODUCTION

The recent demonstration of current-induced magnetization excitations in magnetic tunnel junctions 1,2 and its potential application in magnetic random access memories 3 (MRAM) has shown the necessity to fabricate high quality low resistance magnetic tunnel junctions (MTJ). Since the first publication of room temperature tunnelling magnetoresistance 4 (TMR), the development of deposition and oxidation techniques allowed research groups to reach very high TMR ratios up to 100% in AlOx-based MTJs 5 and 500% in MgO-based MTJs 3 . However, controlling the oxidation process in micronic devices imposes to achieve heavy lithographic processes. In-situ TMR characterisation would be of great interest to get rid of this microfabrication step.

To do so, Worledge et al. 6 proposed an original way to estimate the junctions' TMR from current-in-plane measurements, without using any lithographic patterning of these junctions. Unfortunately, this technique becomes harder to use when the MTJ resistancearea product becomes smaller. Rodmacq et al. 7,8 recently proposed another technique to control the oxidation of an insulating barrier, correlating the oxygen-induced perpendicular magnetic anisotropy (PMA) of a trilayer composed of Pt/Co/MOx (M is a metal -Al, Mg, Ta...) with the maximum of TMR of the corresponding Co/MOx/Co MTJ as a function of the barrier oxidation.

However, the role of oxygen in this "anisotropy crossover" has not yet been fully understood. In this article, we propose an investigation of the role of oxygen in the enhancement of the interfacial magnetic anisotropy of a thin cobalt layer sandwiched between a platinum layer and a non-magnetic oxide. Magnetic measurements are correlated to results of X-ray Absorption Spectroscopy (XAS) and X-ray Photoelectron Spectroscopy (XPS).

The paper is organized as follows : after a brief introduction on the role of interfacial states in PMA and TMR (section II), section III presents the conditions of sample preparation. The magnetic properties are described in section IV and an analysis of X-ray measurements is presented in section V. We conclude the article in section VI. Systems consisting of F/MOx interfaces (F is a ferromagnet and M is a diamagnetic metal, MOx being a non-magnetic oxide) have attracted much interest in the scientific community from both magnetic and electronic points of view 9,10 . Tsymbal et al. 10,11 have theoretically demonstrated that in magnetic tunnel junctions, interfacial densities of states, which determine the amplitude of TMR, can be strongly influenced by localized interfacial atoms. Oleinik et al. 12 have theoretically studied the role of interfacial oxygen in Co/AlOx interfaces in different configurations (see also Ref. 13). When AlOx is under-oxidized, the interface termination is composed of Co-Al-O bondings: a charge transfer occurs between Co and O through Al and reduces the magnetic moment of Co, reducing the TMR amplitude. When the AlOx barrier is optimally oxidized 14 , the interface termination is Co-O-Al: hybridization between Co 3d and O 2p bands strongly modifies the band structure of both Co and O, so that O behaves like a magnetic insulator, enhancing the TMR amplitude. Belaschenko et al. 14 predicted that "adsorbed" oxygen near the Co/AlOx interface leads to positive spin polarization of the tunnelling current. These theoretical results underline the dominant role of oxygen atoms in the band structure of Co at the Co/AlOx interface: the precise chemical composition of the interface controls the electrical properties of the MTJ.

Telling et al. 15,16 have experimentally demonstrated the close relation between AlOx oxidation, Co magnetic moment and TMR, showing that a maximum of TMR is reached when the Co magnetic moment is maximum, upon optimal oxidation. The authors then proved that the study of the magnetic properties of Co can give information on the electronic band structure at the interfaces, and thus monitoring the Co magnetic properties can be used to control the barrier oxidation.

B. Perpendicular Magnetic Anisotropy

The discovery of perpendicular magnetization anisotropy (PMA) in Pd/Co, Pt/Co and Au/Co multilayers 17 has opened an exciting field of research questioning the fundamental origin of such PMA and the role of interfacial orbital hybridization 18,19 . Ferromagnetic materials usually used in these studies are Fe, Co and Ni whose atomic structure is 3d n 4s 2 (n=6, 7, 8 resp.). The localized 3d electrons being responsible for the spontaneous magnetization of these ferromagnets, the source of PMA is related to the anisotropy of these orbitals. In a bulk ferromagnet, the 3d orbitals are degenerate in a first approximation (the chemical environment is roughly spherical). The presence of an interface breaks this quasi-spherical symmetry so that the energy of the 3d orbitals pointing towards the interface (d xz , d yz and d z 2 ) is different from the energy of the 3d orbitals with planar symmetry (d xy and d x 2 -y 2 ).

This gives rise to a crystalline field ∆ c = E(yz, zx, z 2 ) -E(xy, x 2 -y 2 ). Bruno 20 showed that the magnetic anisotropy energy ∆E is strongly influenced by this parameter and can be written as:

∆E = G H ξ 4µ B (m ⊥ orb -m || orb ) (1)
where m

⊥(||)

orb is the out-of-plane (in-plane) orbital moment, ξ is the spin-orbit coupling parameter and G/H are band-structure dependent coefficients. The magnetic anistropy arises from the coupling between the anisotropy of the 3d orbital moment -→ l and the magnetic moment -→ s of the ferromagnetic atoms through the spin-orbit coupling ξ -→ s .

-→ l . However, spin-orbit coupling is rather difficult to estimate, its energy being of the order of a few meV. Daalderop et al. 18 have studied the magnetic anisotropy energy (MAE) in a crystalline Co monolayer. The authors demonstrated that in this case, the MAE changes its sign as a function of the filling of the Co valence band. This was confirmed by Kyuno et al. 21 on Co/X (X=Pt, Pd, Cu, Ag, Au) interfaces. This band filling can be tuned by hybridization between the Co 3d orbitals and the orbitals of layer X. For example, in the case of Co/Pt interface, Nakajima et al. 19 demonstrated that the 3d bands of cobalt are hybridized with the 5d bands of platinum.

Furthermore, Weller et al. 22 showed that in the case of strong spin-orbit coupling (like in the case of Co/Au interfaces), this spin-orbit coupling is sufficiently large to align the large spin moment parallel to the small orbital moment. Then the MAE can be modified by the band splitting of the 3d orbitals due to the coupling with the local environment, and by the amplitude of the spin-orbit coupling itself.

Consequently, at the Co/AlOx interfaces, one can expect that the predicted charge transfer 10,12,14 between Co and O in optimally oxidized Co/AlOx interfaces increases the asymmetry of the Co 3d bands, reducing the energy of the 3d orbitals responsible for the out-of-plane anisotropy and creating a splitting between in-plane and out-of-plane d orbitals (parameter ∆ in Bruno's theory 20 ). Thus, in spite of the weak spin-orbit coupling of O atoms, this strong band splitting could lead to measurable PMA.

III. SAMPLE PREPARATION

In order to study the influence of oxidation on the Co magnetization, Pt(3 nm)/Co(0.6 nm)/M+Oxidation (M=Al, Ta, Mg, Ru) were deposited on a thermally oxidized silicon wafer by conventional dc magnetron sputtering with a base pressure of 10 -8 mbar. Two oxidation techniques were used: some of the samples were oxidized naturally under a pressure of 160 mbar during 30 minutes. These samples were generally annealed at 150 • C during 30 minutes. Other samples were oxidized using an oxygen rf plasma with a partial pressure of 3 × 10 -3 mbar and a power of 10 W. These samples were generally thermally stable so that no annealing was needed.

To vary the oxidation state of the metallic layer M, we either varied the thickness of this layer (in the case of natural oxidation) or the oxidation time (in the case of plasma oxidation), keeping the thickness of layer M constant at 1.6 nm in the last case. In the case of natural oxidation, a capping layer of Pt was deposited on top of these trilayers in order to prevent them from further oxidation. In the case of plasma oxidation, the capping layer was not necessary because the oxidation state was particularly stable due to the thick M layer (1.6 nm).

IV. MAGNETIC PROPERTIES A. Natural oxidation

The magnetic characterization of these samples was performed by extraordinary Hall effect (EHE) in a standard four-probes Hall geometry 7,25 . This technique gives direct information on the perpendicular component M z of the sample magnetization. Figure 1 shows the hysteresis loops obtained at room temperature for a Pt(3 nm)/Co(0.6 nm)/Al(d)+Ox/Pt(2 nm) stack with natural oxidation for various Al thicknesses d.

For thick Al layers (d ≥0.8 nm), the magnetization loops show no hysteresis and zero remanence. Magnetization saturates at high external field (-H-¿1 kOe), where the perpendicular component of the Co magnetization M z no longer depends on H and the residual slope comes from the ordinary Hall contribution 7 . This demonstrates that the out-of-plane direction is a hard axis.

For d=0.6 nm and d=0.4 nm, the magnetization loop exhibits a square hysteresis with sharp magnetization reversal and a coercive field of 200 Oe. These features indicate that the out-of-plane direction becomes an easy axis. Finally, at d=0.2 nm, the magnetization loop shows no hysteresis, zero remanence and its amplitude has decreased, which can be attributed to partial oxidation of the Co layer.

These results demonstrate that, due to oxidation of the Al layer, the magnetic anisotropy of the Co layer is strongly modified. To correctly understand the EHE measurements, one should remind that three sources contribute to the magnetic energy of the thin Co layer: (i) the interfacial anisotropies (Pt/Co and Co/AlOx), (ii) the shape anisotropy, (iii) the stress induced anisotropy. The magnetization state of the Co layer arises from the competition between these three quantities.

The initial slope of the loop measured by EHE gives information about the resulting outof-plane anisotropy. For thick and thin Al layers (d ≥0.8 nm and d ≤0.2 nm), the slope at zero field is small and the out-of-plane direction is a hard axis. For intermediate thicknesses (0.8 nm ¿d¿0.2 nm), the EHE signal is a square loop, the interfacial Co/AlOx and Pt/Co PMA dominate the other anisotropy sources and the initial slope is nearly infinite. At his stage, we cannot determine accurately the magnetization state of the samples with thick (≤0.8 nm) or thin (≥0.2 nm) Al layers, since the EHE signals due to in-plane magnetization or to out-of-plane magnetization forming domain structures have the same shape (no remanence).

These results are summarized in Fig. 2. The initial slope and the magnitude of the EHE hysteresis loops are displayed as a function of the oxide thickness d, for various metallic layers M: Al, Mg, Ta, Ru. In all these samples, the initial slope (open squares) can be decomposed in three zones: for thick layers, the slope is small, for intermediate thicknesses (the range of which depends on the metal) the slope increases until a maximum, and finally decreases again for thin layers. For M=Al, Ta, Mg, the magnetization loop has a square hysteresis when the slope reaches a maximum, which indicates that the magnetization is out-of-plane. Concerning M=Ru, the slope decreases with decreasing thickness. This means that the maximum of perpendicular magnetic anisotropy appears for Ru layers thicker than 1.2 nm.

Another characteristic reported on Fig. 2 is the amplitude of the EHE resistance, associated with the magnitude of the M z component. This amplitude shows a plateau for thick layers and decreases when the layers become thinner. The onset of decrease of the signal amplitude generally corresponds to the maximum of the initial slope, except for M=Mg where the magnitude begins to decrease before the anisotropy reaches its maximum. This decrease in EHE signal indicates a reduction of the thickness of the magnetic layer: the oxygen atoms penetrate in the bulk Co after the anisotropy has reached a maximum. The case of Mg is more difficult to analyse since in this case, the decrease in EHE amplitude is less abrupt, probably due to an inhomogeneous oxidation.

B. Plasma oxidation

To understand the origin of this anisotropy enhancement, we prepared Pt(3 nm)/Co(0.6 nm)/Al(1.6 nm)+Ox(t) samples. These samples were deposited as described in section III and oxidized in a O 2 plasma, for various oxidation times t. Fig. 3 presents the EHE signal as a function of the oxidation time. For samples with t ≤25s, the magnetization loops show no hysteresis and nearly zero remanence. Magnetization saturates at high field (-H-¿1 kOe, see Fig. 5(a)). This demonstrates that the out-of-plane direction is a hard axis. Note however that the magnetization loops for samples with t =20s and t =25s are open which clearly indicates that the magnetization is out-of-plane forming a domain structure.

For 30s≤ t ≤40s, the samples show a square hysteresis loop with sharp magnetization reversal and a non-zero coercivity. Finally, from t ≥45s, the EHE measurements show no hysteresis and zero remanence. The magnitude of EHE decreases for t ≥45s, which indicates that the effective thickness of the magnetic layer decreases. This is interpreted as due to the oxidation of the Co layer, as previously discussed. Initial slopes and signal amplitudes are displayed in Fig. 4. They show the same trends as in Fig. 2: the magnetization loops for a decreasing thickness of Al (natural oxidation) possess the same characteristics than the magnetization loop for an increasing oxidation time (plasma oxidation).

To determine the magnetic state of the Co layer, we performed EHE measurements applying the external magnetic field in the plane of the layers. Fig. 5 shows the EHE signal for small (t=15s), intermediate (t=30s) and long oxidation time (t=45s), for perpendicular 8 (left figures) and in-plane (right figures) external field. In this last configuration, the sample plane is misaligned from the direction of the external field by a small angle α (of the order of 1 or 2 • ), in order to induce a coherent rotation of the magnetization. For these measurements, the samples have been previously saturated in positive perpendicular field (when the remanent magnetization is M z = 1) or demagnetized (when the remanent magnetization is less than 1 and when the magnetization loop is open). The Hall resistance is then measured in increasing field from 0 to 12 kOe, the maximum available field, then normalized to the amplitude of the magnetization loop measured in the perpendicular configuration, in order to obtain the magnitude of M z . For t=15s, the variation of M z as a function of the planar applied field is very small (see Fig. 5(d)), indicating that at zero field, the magnetization lies in the plane of the sample. This is confirmed by SQUID measurements (not shown).

For t=30s, the magnetization is perpendicular with a 100% remanence at zero field. In this case, the M z component decreases from 1 to 0 under the external planar field and saturates for applied field of 8 to 9 kOe (see Fig. 5(e)).

Finally, at t=45s, the M z component, initially zero, reaches a maximum at H ≈ 2 kOe (see Fig. 5(f)), corresponding to a projection H z = H sin α ≈ 50 Oe, which coincides with the saturation field H s , measured in Fig. 5(c) in perpendicular applied field. This demonstrates that the magnetization structure at zero field corresponds to an out-of-plane magnetic domain structure. The same behaviour was observed for the samples oxidized during t =15s and t=60s. The magnetic behaviour strongly depends on the oxidation degree and can be summarised as follows:

• t ≤15s: the magnetization of the Co layer lies in the plane • 20s≤ t ≤25s: the magnetization of the Co layer lies out-of-plane, forming a domain structure

• 30s≤ t ≤40s: the magnetization of the Co layer lies out-of-plane, forming a single domain structure

• 45s≤ t: the Al layer is over-oxidized, oxygen penetrates in the Co layer (the thickness of the magnetic layer decreases). The balance between the anisotropy field and the demagnetizing field induces the magnetization of the Co layer lying out-of-plane in a domain structure Fig. 6 presents the out-of-plane anisotropy field measured by EHE with in-plane external field. Setting M x = 1 -M 2 z , one can determine the field at which the M x magnetization saturates at M x = 1, corresponding to the anisotropy field H an . Similarly to the initial slope in Fig. 4, the anisotropy field reaches a maximum of 8-9 kOe for an oxidation time of the order of 30-40s.

In a recent publication, Lacour et al. 26 studied the effect of oxidation on similar samples grown on a Ta buffer layer. The authors obtained out-of-plane Co magnetization even without oxidation and they only observed the second part of the anisotropy crossover: from the out-of-plane monodomain magnetic state to a superparamagnetic state. The superparamagnetic state they obtain for long oxidation times is consistent with our out-of-plane domain structure. However, they claimed strong differences between their results and ours. The fact that they obtain 100% remanence (out-of-plane magnetization) even for zero oxidation time shows that their samples exhibit a larger PMA than ours, probably because of the Ta buffer layer. Furthermore, the authors focused on the coercive field of their samples and the lack of anisotropy measurements doesn't allow one to draw any definite conclusions about the variation of the magnetic anisotropy with oxidation time.

V. CHEMICAL ANALYSIS USING X-RAY SPECTROSCOPY

The above EHE measurements (Fig. 3 and Fig. 5) show a clear dependence of the magnetization behaviour on oxidation, but do not allow us to comment on the oxidation state of the Al and Co layers. For t ≥45s, the amplitude of the EHE signal decreases and this can be attributed to the penetration of O atoms in the Co layer. However, nothing can be said about the smaller oxidation times.

To understand the role of oxygen in the modification of the magnetic anisotropy, we performed X-ray Absorption Spectroscopy (XAS) and X-ray Photoelectron Spectroscopy (XPS) experiments at the Advanced Photoelectric-effect Experiments (APE) beamline of the ELETTRA synchroton in Trieste (Italy). The aim of these element-specific measurements is to correlate the changes in macroscopic magnetic properties with changes in average chemical composition at the Co/Al interface. For the Co thickness used here (0.6 nm), XAS gives information on the chemical composition averaged over the Co layer, while XPS provides information on the electronic structure of Co at the Co/AlOx interface mainly (the collected intensity is exponentially decreasing with a penetration depth of the order of 0.5-0.6 nm, so that we expect that the main contribution comes from the top Co-Al bondings and Co-Co bondings between the first and second Co monolayers).

A. X-ray Absorption Spectroscopy

XAS measurements were performed scanning the X-ray energy around the Co L 2,3 absorption edges (760-800 eV). Absorption spectra were obtained for perpendicular incidence, by measuring the drain current via the sample holder. Figure 7 shows the XAS spectra of the Pt/Co/AlOx trilayers for different oxidation times. Pure Co and CoO XAS spectra are given for reference in Fig. 7(a) and (c), respectively.

For t ≤25s (Fig. 7(a)), the spectra resemble the ones of pure Co. For 30s≤ t ≤40s (Fig. 7(b)), shoulders appear in the absorption spectra indicating the presence of small contributions due to CoO. For 45s≤ t ≤60s (Fig. 7(c)), these shoulders become very clear and the absorption spectra strongly resemble the ones of pure CoO. Fig. 7(c,d,f) shows the corresponding derivatives of the XAS spectra which reveal more clearly the presence of CoO in the Co layer. The vertical lines indicate the energy of the peaks of the derivative, due to Co (solid line) and CoO (dashed line). For small oxidation time (Fig. 7(d)), only peaks corresponding to pure Co are present. For intermediate oxidation times (Fig. 7(e)), a small peak appears at 777 eV, which corresponds to a small contribution of CoO. Finally, for long oxidation times (Fig. 7(f)), two CoO peaks are present, but still smaller than the pure Co peaks. Note the energy shift of the derivative peaks as a function of the oxidation time, attributed to the modification of the chemical environment. This clearly indicates that the Co layer is free from oxygen atoms for small oxidation times, then a small oxidation occurs, probably near the interface, and finally, oxygen deeply penetrates inside the Co layer.

B. X-ray Photoelectron Spectroscopy

In order to have a more quantitative estimate of the chemical composition of Al and Co, we performed X-ray photoelectron spectroscopy on the 3p levels of Al and the 2p levels of Co. The beamline energy is set at hν = 765eV to probe the Al 3p levels. The inset of Fig. 8 shows a typical spectrum of Al 3p in a sample oxidized during 30s. We observe two peaks located at 72.67 eV (corresponding to pure Al) and at 76. pure AlOx). Actually, we expect two peaks located at 72.9 eV and 72.5 eV, corresponding to the Al 3p 1/2 and 3p 3/2 levels respectively. However, the energy resolution of the beamline and the width of these peaks do not allow separating the two peaks.

The ratio of AlOx present in the layer is determined as R = A AlOx /(A AlOx + A Al ), where A AlOx(Al) is the estimated area of the AlOx (Al) peaks (the areas are estimated by gaussian fits -see inset of Fig. 8). Fig. 8 shows that aluminium is not fully oxidized, even after 60s of plasma oxidation. However this measurement is restricted to the first half of the Al layer (the aluminium thickness is 1.6 nm, whereas the estimated escape length is smaller than 1 nm) and indicates that nearly 70% of the first nanometer of the aluminium layer is oxidized during the first 15s of plasma oxidation. For longer oxidation times, the oxygen penetrates deeply inside the Al layer towards the Co/AlOx interface.

To obtain information on the chemical composition of the Co layer, XPS is performed setting the beamline energy at hν = 1130eV to probe the Co 2p levels. Fig. 9 shows Co 2p spectra obtained for the ten samples. The spectrum of the t=60s sample corresponds to pure CoO 28 : one can distinguish two main peaks, corresponding to CoO 2p 1/2 and CoO 2p 3/2 core levels (lying at 796.3 eV and 781.1 eV respectively) and two satellite peaks (denoted S and lying at 803.3 eV and 786.7 eV resp.) which arise from the charge transfer between O 2p and Co 3d. This charge transfer allows the coexistence of two types of transitions: 2p 6 3d 7 → 2p 5 3d 7 + e -(satellite peaks) and 2p 6 3d 8 L → 2p 5 3d 8 L + e -(main peaks), where L is a hole brought by oxygen 28 . The main peaks are shifted towards larger binding energies with respect to Co metal peaks, due to oxygen environment (similarly to AlOx peak). The spectrum of the t=15s sample is similar to that of pure cobalt 29 , with Co 2p 1/2 and Co 2p 3/2 lying at the binding energies of 792.5 eV and 778.1 eV respectively.

It clearly shows that Co is unoxidized for t=15s. At t=20s and t=25s, a small contribution of Co-O bondings appears. Between t=30s and t=40s, both Co-O and Co-Co bondings coexist and for oxidation times longer than 45s, the Co contribution completely desappears. The ratio of CoO in the Co layer has been estimated in Fig. 10, from gaussian fits of the XPS spectra (not shown).

The spectra clearly show that the Co/AlOx interface composition changes from quasi-pure Co to pure CoO during the oxidation process. Since XPS measurements are surface sensitive, the spectra give information about the Co chemical composition averaged over the Co layer, with a dominant contribution from the topmost Co monolayer (top Co-Al bondings, bottom and lateral Co-Co bondings). For samples oxidized during t ≤15s (in-plane anisotropy), the spectra have mainly a pure Co character. For t=20s and t=25s, the relatively high amount of estimated CoO (40% to 60%), compared to XAS results (undetectable CoO), suggests an inhomogeneous presence of oxygen atoms limited to the interface, which only allow small enhancement of the magnetic anisotropy (see Fig. 6). For t=30s to t=40s, the spectra present both Co and CoO characters, showing that most of the Co located at the Co/AlOx interface possesses Co-O bondings (Co-Al bondings are replaced by Co-O bondings), while the small contribution of pure Co in the XPS spectra arises from the remaining deep Co-Co bondings. The estimated ratios (¿80%) show that the interface is nearly fully oxidized. Finally, from t=45s to t=60s, the Co character disappears which indicates that the first Co monolayer (at least) is totally oxidized (Co-Al and Co-Co bondings are replaced by Co-O bondings) and the CoO ratio reaches 100%.

VI. CONCLUSION

The above results strongly suggest that the onset of PMA is related to the appearance of a significant density of interfacial Co-O bondings at the Co/AlOx interface. These results are summarized in Fig. 11. For small oxidation times (Fig. 11(a)), the Al layer is only oxidized at 70% and the magnetization of the Co layer lies in the plane. At 20s ≤ t ≤ 25s (Fig. 11(b)), the perpendicular magnetic anisotropy increases due to the progressive appearance of oxygen at the Co/AlOx interface and the magnetization lies out-of-plane forming a domain structure.

For intermediate (or optimal) oxidation times (Fig. 11(c)), 80% of the Al layer is oxidized and an important amount of oxygen atoms have reached the Co/Al interface, so that most of the Co-Al bondings are replaced by Co-O bondings. This indicates a non homogeneous oxidation: oxygen atoms diffuse through the grain boundaries of the Al layer 30 (AlOx is amorphous but Al is polycrystalline), so that they reach the Co/AlOx interface before Al is fully oxidized. In this case, the magnetic anisotropy at Co/AlOx interface reaches a maximum: the Co/Pt and Co/AlOx PMA overcome the other anisotropy sources and the magnetization of the Co layer lies out-of-plane in a single domain structure.

Finally, for long oxidation times (Fig. 11(d)), the Co layer is being oxidized through the grain boundaries 30 . The penetration of oxygen atoms in this way decreases the exchange coupling between the magnetic grains in the Co layer, reducing the energy of the magnetic domains. Consequently, the Co magnetization breaks into perpendicular magnetic domains, pointing upward or downward, probably forming labyrinthic or stripe domain structures 31 .

In conclusion, we have shown that interfacial oxidation of Co in Pt/Co/AlOx trilayers is at the origin of strong perpendicular magnetic anisotropy. Perpendicular anisotropy crossover induced by annealing in such structures has also been studied 32 . Ab-initio calculations, aiming at clarifying the role of Co-O hybridization in magnetization anisotropy energy, are presently underway.

  band structure effects at the Co/AlOx interface

Figure 1 :

 1 Figure 1: Hall resistance as a function of the applied field for Pt/Co/Al(d)+Ox/Pt samples, for various Al thickness d. Oxidation is carried out in a 160 mbar O 2 atmosphere for 30 min. The field is applied perpendicular to the plane of the trilayers.

Figure 2 :

 2 Figure 2: Initial slope and amplitude of the hysteresis loops measured by EHE for Pt/Co/MOx/Pt samples under natural oxidation, with M=Al, Ta, Mg, Ru. The dotted and solid lines are guides for the eyes.

Figure 3 :

 3 Figure 3: Hall resistance as a function of the applied field for Pt/Co/Al(t)+Ox/Pt samples under plasma oxidation, for various oxidation times. The field is applied perpendicular to the plane of the trilayers.

Figure 4 :

 4 Figure 4: Initial slope -logarithmic scale -and amplitude of the hysteresis loops measured by EHE for Pt/Co/AlOx samples under plasma oxidation as a function of the oxidation time. The dotted and solid lines are guides for the eyes.

Figure 5 :

 5 Figure 5: Extraordinary Hall effect with perpendicular field for three characteristic oxidation times: t=15s (a), t=30s (b) and t=45s (c); Extraordinary Hall effect with planar field for three characteristic oxidation times: t=15s (d), t=30s (e) and t=45s (f).

Figure 6 :

 6 Figure 6: Out-of-plane anisotropy field of Pt/Co/AlOx samples, measured by EHE, as a function of the oxidation time.

Figure 7 :

 7 Photon energy (eV)

Figure 8 :

 8 Photon energy (eV)

Figure 9 :

 9 Figure 9: XPS spectra of Co 2p for Pt/Co/Al(t)+Ox samples as a function of the oxidation time t.

Figure 10 :

 10 Figure 10: Ratio of oxidized cobalt estimated from Co 2p X-ray photoelectron spectra, as a function of the oxidation time.

Figure 11 :

 11 Figure 11: Schematics of the influence of the oxidation on the magnetic properties of the Co layer in a Pt/Co/AlOx trilayer: (a) for very small oxidation times, the magnetization lies in the plane; (b) for small oxidation times, the magnetization lies out-of-plane forming domain structure; (c) for optimal oxidation times, the magnetization lies out of plane in a single domain structure; (d) for long oxidation times, the magnetization lies out-of-plane forming domain structure.

Acknowledgments

The authors acknowledge very fruitful discussions with C. Lacroix, A. Bergmann and M. Chschiev. We are also grateful for the experimental support and assistance of B. Pang, L. Ranno, O. Fruchart and P. Torelli.