Olivier Lévêque
email: olivier.leveque@institutoptique.fr

Caroline Kulcsár

Hervé Sauer

Antony Lee

Pierre Bon

Laurent Cognet

François Goudail

Can phase masks extend depth-of-field in localization microscopy?

Keywords: binary phase masks, depth-of-field extension, single-molecule localization microscopy, co-designed optical system, wavefront coding, image processing, optimization

In localization microscopy, the position of isolated fluorescent emitters are estimated with a resolution better than the diffraction limit. In order to image thick samples, which are common in biological applications, there is considerable interest in extending the depth-of-field of such microscopes in order to make their accuracy as invariant as possible to defocus. For that purpose, we propose to optimize annular binary phase masks placed in the pupil of the microscope in order to generate a point spread function for which the localization accuracy is almost invariant along the optical axis. The optimization criterion is defined as the localization accuracy in the plane expressed in terms of the Cramér-Rao bound. We show that the optimal masks significantly increase the depth-of-field of single-molecule imaging techniques relatively to an usual microscope objective.

INTRODUCTION

Single-molecule localization microscopy is used to observe biological subcellular structures. This super-resolution imaging technique makes it possible to estimate very accurately the position of an isolated fluorescent emitter in the field of the camera beyond the diffraction limit. [START_REF] Hess | Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy[END_REF][START_REF] Betzig | Imaging Intracellular Fluorescent Proteins at Nanometer Resolution[END_REF] A lot of work has been devoted to determine the precision limit of this technique. [START_REF] Chao | Fisher information theory for parameter estimation in single molecule microscopy: tutorial[END_REF][START_REF] Ram | A Stochastic Analysis of Performance Limits for Optical Microscopes[END_REF][START_REF] Ober | Localization Accuracy in Single-Molecule Microscopy[END_REF] Globally, the fundamental limit on localization standard deviation depends on the numerical aperture (NA) of the microscope and on the signal-to-noise ratio. [START_REF] Ober | Localization Accuracy in Single-Molecule Microscopy[END_REF] Most of these works have assumed that the microscope is perfectly focused on the observed object.

Over the last decades, various biological applications have focused on imaging thicker samples in which the objects of interest are generally not confined within the two dimensions of the focus plane. Several approaches have therefore been designed to extend the super-localization concept to the third dimension. [START_REF] Hajj | Accessing the third dimension in localization-based super-resolution microscopy[END_REF] These new approaches make it possible to estimate the 3D position of the fluorescent emitters in the natural depth-of-field (DoF) of the high NA objectives used in single-molecule microscopy. In order to further extend the DoF in 3D localization microscopy, sophisticated point spread function (PSF) engineering designs using pupil wavefront coding have been studied but they all inherently require significant photon spread. [START_REF] Von Diezmann | Three-Dimensional Localization of Single Molecules for Super-Resolution Imaging and Single-Particle Tracking[END_REF][START_REF] Zahreddine | Simultaneous quantitative depth mapping and extended depth of field for 4D microscopy through PSF engineering[END_REF][START_REF] Pavani | High-efficiency rotating point spread functions[END_REF] This leads to considerably reduce the localization performance in the low signal conditions encountered for instance in fast single particle tracking or deep tissue imaging. Another approach consists in extending the DoF by making the PSF of the microscope invariant along the imaging axis to generate volumetric images consisting of 2D projections of the 3D imaging volume. In this work, we present a methodology to apply this optimization concept not to the shape of the microscope PSF but directly to its localization performance, which is the real parameter of interest.

A similar issue is encountered in classical imaging systems where the DoF can be improved by placing a phase mask in the pupil of the objective and by restoring the image with an appropriate deconvolution filter. [START_REF] Dowski | Wavefront coding: jointly optimized optical and digital imaging systems[END_REF][START_REF] Harvey | Digital image processing as an integral component of optical design[END_REF][START_REF] Diaz | Comparison of depth-of-focus-enhancing pupil masks based on a signal-to-noise-ratio criterion after deconvolution[END_REF] The phase function of the mask can be optimized to maximize the quality of the deconvolved image. [START_REF] Harvey | Digital image processing as an integral component of optical design[END_REF][START_REF] Diaz | Increase in depth of field taking into account deconvolution by optimization of pupil mask[END_REF][START_REF] Sitzmann | End-to-End Optimization of Optics and Image Processing for Achromatic Extended Depth of Field and Super-Resolution Imaging[END_REF][START_REF] Elmalem | Learned phase coded aperture for the benefit of depth of field extension[END_REF] This approach consisting in optimizing an imaging system while taking into account the image formation model, the properties of the optical system and the method of information extraction is called "co-design". [START_REF] Cathey | New paradigm for imaging systems[END_REF][START_REF] Robinson | Joint Design of Lens Systems and Digital Image Processing[END_REF] Our purpose is to apply this approach to DoF enhancement of localization microscopy. The optimization criterion is different from that used in classical imaging since it is needed to optimize a location accuracy instead of a restored image quality, leading to possibly different optimal masks. This present work proposes to explore two perspectives described in our previous work. [START_REF] Lévêque | Codesigned annular binary phase masks for depth-of-field extension in single-molecule localization microscopy[END_REF] The first one is to characterize DoF-enhancing phase masks when the measurements are corrupted by signal-dependent photon noise, which is a more realistic model than additive Gaussian noise. The second one is to propose different optimization criteria based on the Cramér-Rao bound (CRB) to extend DoF of localization microscopy. This paper is organized as follows. Section 2 describes the 2D localization problem and the loss of localization accuracy when the observed particle is out-of-focus. We introduce the Fisher information matrix and remind the concept of the fundamental limit of localization accuracy based on the CRB. In section 3, we present a co-design approach to improve the localization accuracy of defocused imaging systems and introduce the annular binary phase masks that will be used for that purpose. In section 4, we optimize these masks, present and discuss their performance. Conclusions and perspectives are drawn in section 5.

OUT-OF-FOCUS LOCALIZATION ACCURACY

Within thick samples, the fluorescent particles of interest may be located outside the best focus range of the imaging system. In this case, the PSF of the emitter varies significantly as a function of its axial position. In this section, we describe the 2D localization problem and the loss of localization accuracy when the observed fluorescent particle is out-of-focus. We introduce the Fisher information matrix and recall the concept of the fundamental limit of localization accuracy based on the CRB.

Optical model for defocus

In the Fresnel approximation, the generalized pupil phase function corresponding to an out-of-focus object is

Φ(r) = 2πψr 2 λ , r ∈ [0, 1] (1)
where r is the normalized radial distance in the polar coordinates of the exit pupil, λ is the wavelength of the collected light and ψ is the wavefront error at the edge of the pupil considered as the traditional parameter for quantifying defocus. [START_REF] Born | Principles of Optics: Electromagnetic Theory of Propagation, Interference and Diffraction of Light[END_REF] This parameter (an optical path difference) is defined as

ψ = NA 2 ∆z 2n (2)
where NA denotes the object numerical aperture of the imaging system; ∆z is the longitudinal distance between the observed particle and the nominal in-focus plane in the object space; n is the refractive index of the immersion object space.

The image of the emitter in the sensor plane is the translated PSF of the system:

f ψ,θ0 (x, y) = f ψ (x -M x p , y -M y p ) ( 3
)
where M is the lateral magnification of the imaging setup, and f ψ (x, y) is the 2D spatial distribution of irradiance in the image space when the single-molecule is located at θ 0 = (x p , y p ) t in the object plane. The superscript t denotes transposition. The 2D spatial distribution of irradiance is normalized so that its integral is equal to 1 (i.e. f ψ,θ0 (x, y) dx dy = 1) and is proportional to the squared modulus of the Fourier transform of the generalized pupil function exp[iΦ(r)], if r < 1, 0 otherwise with Φ(r) defined in Eq. ( 1).

We assume that we observe a 2D pixelized square image of a single-molecule with a width of 2K + 1 pixels. To take into account the signal-dependent photon noise, we define s ij , the number of photo-electrons observed at pixel (i, j) ∈ N 2 with |i| and |j| ≤ K, as a Poisson distributed random variable of mean

µ ψ,θ0 ij = N 0 (i+ 1 2 )∆xy (i-1 2 )∆xy (j+ 1 2 )∆xy (j-1 2 )∆xy f ψ,θ0 (x, y) dx dy (4)
where N 0 denotes the total number of photo-electrons expected in the whole image and ∆ xy denotes the width of the square pixels.

The ψ-dependence of the PSF is illustrated in Fig. 1, where we present 3 particle image simulations obtained for 3 different defocus parameters: ψ = {0, 0.5λ, 1λ}. When ψ = 0, the object is in focus and the PSF is described by the classical Airy pattern whose size is defined by the object NA of the microscope objective. When ψ increases, the PSF progressively gets wider.

Due to the circular symmetry of the PSF, another way to illustrate its ψ-dependence is presented in Fig. 2.a. We have indeed represented the variation of the PSF profile along the x-axis as a function of the defocus parameter ψ for an example of microscope configuration. Only the positive values of ψ are illustrated because PSF is identical for ψ and -ψ (i.e. on either side of the focus point). We note that as defocus increases, the PSF spreads out and its central lobe gets fainter.

We therefore perceive that the more ψ increases, the more difficult it is to accurately estimate the position of an emitter because the PSF deteriorates.

Fundamental localization accuracy limit

To quantify the loss of location accuracy with defocus, we introduce the well known concept of fundamental limit of localization accuracy. [START_REF] Ober | Localization Accuracy in Single-Molecule Microscopy[END_REF] This limit can be obtained from the Fisher information matrix defined by This probability is defined as

I(θ 0 ) = K i=-K K j=-K E      ∂ ln p s ij |µ ψ,θ ij ∂θ θ=θ0     ∂ ln p s ij |µ ψ,θ ij ∂θ θ=θ0   t    (5
p s ij |µ ψ,θ0 ij = µ ψ,θ0 ij sij exp -µ ψ,θ0 ij (s ij ) ! ( 6
)
where the symbol (.) ! denotes the factorial function.

Due to the circular symmetry of the PSF f ψ,θ0 (x, y) for any value of ψ, the off-diagonal terms of the Fisher information matrix are zero and the square root of Cramér-Rao bound, denoted RCRB, is the square root of the inverse of the diagonal elements of the Fisher information matrix. The RCRB along the x and y-axis are equal and its expression is

RCRB =   N0 K i=-K K j=-K (j+ 1 2 )∆xy (j-1 2 )∆xy f ψ,θ0 i -1 2 ∆ xy , y -f ψ,θ0 i + 1 2 ∆ xy , y dy 2 (i+ 1 2 )∆xy (i-1 2 )∆xy (j+ 1 2 )∆xy (j-1 2 )∆xy f ψ,θ0 (x, y) dx dy    -1 2 . ( 7
)
We note that the fundamental limit on localization is inversely proportional to the square root of the total number of photo-electrons √ N 0 expected in the image and depends on the spatial sampling of the PSF.

In Fig. 2.b, we have represented the variation of RCRB as a function of defocus for an example of microscope configuration. Two y-axis are displayed: the left one uses an absolute nanometric unit in the object space, and the second one uses an arbitrary unit (a.u.) chosen such as RCRB(ψ = 0) = 1. We have represented RCRB only over the positive values of ψ since it is symmetric. It is observed that RCRB increases slowly until ψ = λ/4, then the increase gets much sharper. The range |ψ| < λ/4 corresponds to the Rayleigh criterion: it is classically considered that defocus is not critical for this range. It is noticed that for a defocus parameter of ψ = 1λ, RCRB is 2.9 times larger than for a focused particle (i.e. ψ = 0).

Since we have chosen a signal-dependent photon noise model, the value of RCRB is inversely proportional to √ N 0 , as it appears in Eq. 7. Thus, the absolute value of the RCRB in Fig. 2.b depends on N 0 , but the global shape of the curve does not. So we will adopt for the next plots the normalized RCRB.

CO-DESIGN APPROACH

As we just showed in the previous section, the lateral resolution in localization microscopy depends on the defocus. Our objective is to make the location accuracy of a single-molecule as invariant as possible to defocus. In other words, we want to extend the DoF of the localization microscope. We therefore propose to co-design annular binary phase masks in order to generate a PSF which achieves this goal.

Annular binary phase masks

Annular binary phase masks are static spatial phase modulating optical elements (see Fig. 3.a) defined by the normalized outer radius of their concentric rings (see Fig. 3.b), where each ring introduces a phase modulation of 0 or π radians at a nominal wavelength λ. For example, we have represented in Figs. 3 a four-ring annular binary phase mask defined by the parameter vector of normalized radius ρ = (ρ 1 , ρ 2 , ρ 3 ) t , with 0 ≤ ρ 1 ≤ ρ 2 ≤ ρ 3 ≤ 1, where ρ n is the outer normalized radius of the nth ring. The binary phase function defined by the mask is Φ mask (r, ρ).

In consequence, the pupil phase function corresponding to an out-of-focus object, previously defined in Eq. ( 1), becomes

Φ(r, ρ) = 2πψr 2 λ + Φ mask (r, ρ), r ∈ [0, 1] . (8)
An interesting property of annular binary phase masks with π-phase modulation is that the PSF is symmetrical on either side of the focus point. [START_REF] Falcón | Performance limits of binary annular phase masks codesigned for depth-of-field extension[END_REF] So we continue to represent the plots for positive defocus values only.

Co-design optimization criteria

Co-optimization for DoF extension consists in optimizing optical parameters, here the mask parameters ρ, in order to obtain a "good" estimated molecule position. But what does "good" mean? And how to perform this optimization?

A first reasonable criterion for phase mask optimization is to minimize the value of the fundamental limit of localization accuracy for the worst defocus parameter. [START_REF] Lévêque | Codesigned annular binary phase masks for depth-of-field extension in single-molecule localization microscopy[END_REF] In mathematical terms, the optimized binary phase mask parameters, called ρ opt , are defined by

ρ opt (ψ max ) = arg min ρ max ψ∈[0,ψmax] RCRB(ρ, ψ) (9)
where [0, ψ max ] characterize the defocus range on which we want localization to be accurate.

This minimax criterion makes it possible to ensure a certain localization accuracy by limiting the maximal RCRB value. However, for some applications, it might be preferable to optimize the average of the RCRB over the desired DoF range, rather than its maximal value. Thus, we propose to compare in the next section, the minimax criterion with a second co-design optimization criterion called minimean criterion and defined as

ρ opt (ψ max ) = arg min ρ mean ψ∈[0,ψmax] RCRB(ρ, ψ) . ( 10
)

ANNULAR BINARY PHASE MASK OPTIMIZATION

By applying the optimization criteria defined in the previous section, we optimize annular binary phase masks for various defocus ranges. Since these optimization criteria are not simple and present several local minima, we start by restricting our attention to two-ring mask co-optimization, since this problem involves only a single parameter. We then consider co-optimization of masks with more than two rings.

Two-ring phase masks

Since a two-ring phase mask is defined by a single parameter ρ = ρ 1 , the optimization of this parameter can be easily performed by exhaustive search. Figure 4.a compares the two co-optimization criteria by plotting, as a function of the defocus range ψ max , the values of RCRB obtained with and without the optimal mask of parameter ρ opt . The blue line corresponds to the maximal value of the RCRB over the defocus range, called RCRB max , obtained with an aberration-free optical system without phase mask and can be used as a reference for comparison. The orange and purple areas correspond respectively to the set of RCRB values obtained for all values ψ ∈ [0, ψ max ], using a mask optimized with the minimean and the minimax criterion. Figure 4.b gives the value of the optimal mask parameter ρ opt as a function of the defocus range.

The main conclusion that can be drawn from these graphs is that optimal binary masks significantly improve localization accuracy for all the defocus ranges such that ψ max > 0.3λ whatever the optimization criterion used. For instance, when the defocus range is equal to ψ max = 0.7λ, the two-ring binary mask optimal for the minimax criterion yields a RCRB max 2 times smaller than the one obtained without mask. For the mask optimal for the minimean criterion, it is 1.5 times smaller. For small defocus ranges such that ψ max < 0.3λ, the optimal mask parameter is not null but it leads to the same RCRB as no mask at all, so that binary phase masks do not improve localization accuracy for these ranges.

In Fig. 4.c, we have represented the evolution of the normalized RCRB as a function of defocus for an optical system using a two-ring binary phase mask optimized for ψ max = 0.7λ. We note that depending on the criterion used, the compromise on the values of RCRB is not the same. Indeed, the ratio between the RCRB obtained with minimax criterion and the RCRB obtained with the minimean criterion is equal to 1.32 for ψ = 0 and to 0.81 for ψ = ψ max = 0.7λ. The minimean criterion promotes good localization accuracy for low DoF values, while the minimax criterion seeks to limit the maximum value of the RCRB over the defocus range. The choice of the optimization criterion is therefore an important component of the phase mask optimization problem and depends of the desired final application. We have presented in this section two criteria of co-optimization but of course, others are possible such as for example a criterion based on a linear combination of the RCRB obtained for different defocus parameters. In the next section, we will only focus on the minimax criterion.

Multi-ring phase masks

In this section, we address the following question: is it possible that binary phase masks with more rings, and therefore more degree of freedom in optimization, further improve localization accuracy? We will discuss the performance limits of multi-ring binary phase masks for depth-of-field extension in localization microscopy as it has been investigated for conventional imaging systems [START_REF] Falcón | Performance limits of binary annular phase masks codesigned for depth-of-field extension[END_REF] or for localization microscopy when the measurement noise is normally distributed. [START_REF] Lévêque | Codesigned annular binary phase masks for depth-of-field extension in single-molecule localization microscopy[END_REF] To answer this question, we optimize the minimax criterion defined in Eq. ( 9) with a larger number of rings. Figure 5.a represents the maximal value of the normalized RCRB over the defocus range obtained with the optimal mask parameter set ρ opt , defined as

RCRB max = max ψ∈[0,ψmax] RCRB(ρ opt , ψ) . ( 11
)
This value is plotted as a function of the defocus range until ψ max = 2λ. The blue line is obtained with an optical system without mask. Similarly to Fig. 4.a, the RCRB increases slowly until ψ = λ/4, then increases more rapidly, steps down from ψ max 0.7λ, then increases again sharply from ψ max 1λ, then has a series of successive steps until ψ max = 2λ. These steps reflect the fact that an increase in ψ max may not always lead to an increase of RCRB max . The red line is obtained using a two-ring mask optimized for the corresponding defocus range. As pointed out in the previous section, for ψ max > 0.3λ, the use of such a phase mask makes it possible to reduce the RCRB max and therefore increases the DoF of the optical system. For instance, for ψ max = 2λ the use of an optimized two-ring binary phase mask reduces by half the RCRB max . The curve with yellow dots is obtained using an optimized multi-ring binary phase mask. Since it is no longer possible to use an exhaustive search to achieve the multi-ring non-convex optimization defined in Eq. ( 9) with a larger number of rings, we use the particle swarm optimization algorithm. We performed this phase mask optimization for discrete values of ψ max spaced by 0.1λ from 0 to 2λ. We can see that using a multi-ring mask further reduces the RCRB max . In other words, these additional degrees of freedom in the optimization lead to obtain more efficient phase masks. For example, for ψ max = 2λ, the use of an optimized multi-ring binary phase mask makes it possible to divide by 3 the RCRB max . However we note again that for ψ max < 0.3λ, phase masks, whatever their number of rings, are useless since the obtained performance is identical to that obtained without mask.

Figure 5.b represents the parameters of the optimal multi-ring masks whose localization performance is given by the curve with yellow dots in Fig. 5.a. The outer radii of the optimized annular mask rings are displayed as a function of the defocus range ψ max . As highlighted previously, the additional degrees of freedom in the optimization make it possible to obtain more efficient phase masks. In fact, we notice that the number of rings of the optimal mask increases with the defocus range ψ max . Indeed, from ψ max = 0 to 0.4λ the optimal annular binary phase masks have two rings, from ψ max = 0.4λ to 0.8λ three rings, from ψ max = 0.8λ to 1.2λ four rings, and for ψ max > 1.2λ at least four rings. Similar results have already been obtained in classical imaging systems where the DoF is improved by placing a binary phase mask in the pupil of the objective and by restoring the image with an appropriate deconvolution filter system. [START_REF] Falcón | Performance limits of binary annular phase masks codesigned for depth-of-field extension[END_REF] In addition, it is seen in Fig. 5.b that the radii of the rings of the optimal masks vary almost continuously with the defocus range, and "split" into two values when one more ring is necessary. The few discontinuities, illustrated by dotted curves, correspond to different local minima in the optimization problem.

CONCLUSION

We have investigated the problem of DoF extension in localization microscopy with a Poissonian noise model (see our previous work [START_REF] Lévêque | Codesigned annular binary phase masks for depth-of-field extension in single-molecule localization microscopy[END_REF] for more details in the Gaussian case) in order to answer our initial question: Can phase masks extend depth-of-field in localization microscopy? This work is different from that presented in our previous work [START_REF] Lévêque | Codesigned annular binary phase masks for depth-of-field extension in single-molecule localization microscopy[END_REF] where the measurement noise is normally distributed.

We have shown that placing an optimized multi-ring annular binary phase mask in the exit pupil of the localization microscope allows to significantly increase the localization performance within the required defocus range. For example for a defocus range of ψ max = 2λ, the optimized annular binary phase mask reduces the RCRB by a factor 3 compared to a standard system. Additionally, we also noticed that the larger the desired DoF extension, the more rings the optimal mask needs.

The framework developed in this article makes it possible to optimize and compare the performance of any type of DoF-enhancing mask, since it is based on an explicit and general information theoretical criterion. The presented results take into account a signal-dependent photon noise model. An interesting perspective of this work is to extend this approach to other mask models, and to compare their performance in terms of DoF extension.

Another perspective is to use a more accurate theoretical description of the PSF of optical localization microscopes. The present work demonstrated the utility of the annular binary phase mask for the DoF extension problem in localization microscopy using a simple imaging model taking into account signal-dependent photon noise and defocus aberration. However, more accurate theoretical description of the PSF exist in the literature [START_REF] Aguet | Super-Resolution Fluorescence Microscopy Based on Physical Models[END_REF] and could be implemented within this framework.

(a) ψ = 0 (Figure 1 .

 01 Figure 1. Particle image simulations sij of an optical localization microscope such as (a) ψ = 0, (b) ψ = ±0.5λ and (c) ψ = ±1λ. Simulation parameters: θ0 = (0, 0) t µm, N0 = 500 photo-electrons, ∆xy = 10 µm, 2K + 1 = 21 pixels, NA = 1.3 (object numerical aperture), λ = 700 nm (wavelength), and M = 60 (magnification).

Figure 2 .

 2 Figure 2. Evolutions of (a) the PSF profile along x-axis and (b) the limit of the localization accuracy, denoted RCRB, as a function of the defocus parameter for an aberration-free microscope objective. Simulation parameters: θ0 = (0, 0) t µm, N0 = 500 photo-electrons, ∆xy = 10 µm, 2K + 1 = 21 pixels, NA = 1.3 (object numerical aperture), λ = 700 nm (wavelength), and M = 60 (magnification).

Figure 3 .

 3 Figure 3. Example of four-ring annular binary phase mask (a) in 3D and (b) in 2D, defined by ρ = (ρ1, ρ2, ρ3) t . Each ring is defined as an annular region with constant phase modulation. Dark gray areas induce a phase of 0 radians and light gray areas induce a phase of π radians at a nominal wavelength λ.

Figure 4 .

 4 Figure 4. (a) Evolution of the maximum (RCRBmax) and minimum (RCRBmin) of the RCRB as a function of the defocus range, characterized by ψmax, of a localization microscope using a two-ring binary phase mask. (b) Evolution of the optimized parameter mask ρopt as a function of the defocus range. (c) Evolution of RCRB as a function of defocus for an optical system using a binary phase mask optimized for ψmax = 0.7λ. Simulation parameters: θ0 = (0, 0) t µm, N0 = 500 photo-electrons, ∆xy = 10 µm, 2K + 1 = 21 pixels, NA = 1.3 (object numerical aperture), λ = 700 nm (wavelength), and M = 60 (magnification).

Figure 5 .

 5 Figure 5. (a) Evolution of the RCRBmax as a function of the defocus range, characterized by ψmax, of a localization microscope using a multi-ring binary phase mask. (b) Evolution of the optimized parameter mask ρ opt as a function of the defocus range ψmax. Simulation parameters: θ0 = (0, 0) t µm, N0 = 500 photo-electrons, ∆xy = 10 µm, 2K + 1 = 21 pixels, NA = 1.3 (object numerical aperture), λ = 700 nm (wavelength), and M = 60 (magnification).


M Maalej

R E Forgione

Dr R Marchetti
email: roberta.marchetti@unina.it

Prof R Lanzetta

Prof A Molinaro

Prof A Silipo

Dr + F Bulteau

Dr M Thøpaut

Prof C Laguri

Prof. F J.-P Simorre

Fieschi

Keywords: carbohydrates, lipopolysaccharides, molecular recognition, NMR spectroscopy, proteins

Human Macrophage Galactose-Type Lectin (MGL) Recognizes the Outer Core of Escherichia coli Lipooligosaccharide

Meriem Maalej + , [a, b] Rosa Ester Forgione + , [a] Roberta Marchetti,* [a] FranÅois Bulteau, [b] Michel ThØpaut, [b] Rosa Lanzetta, [a] Cedric Laguri, [b] Jean-Pierre Simorre, [b] Franck Fieschi, [b] Antonio Molinaro, [a] and Alba Silipo [a] Carbohydrate-lectin interactions intervene in and mediate most biological processes, including a crucial modulation of immune responses to pathogens. Despite growing interest in investigating the association between host receptor lectins and exogenous glycan ligands, the molecular mechanisms underlying bacterial recognition by human lectins are still not fully understood. Herein, a novel molecular interaction between the human macrophage galactose-type lectin (MGL) and the lipooligosaccharide (LOS) of Escherichia coli strain R1 is described. Saturation transfer difference NMR spectroscopy analysis, supported by computational studies, demonstrated that MGL bound to the purified deacylated LOS R1 mainly through recognition of its outer core and established crucial interactions with the terminal Gala(1,2)Gal epitope. These results assess the ability of MGL to recognise glycan moieties exposed on Gram-negative bacterial surfaces.

Bacterial cell surfaces are decorated with highly diverse glycoconjugates, in the form of capsular polysaccharides, peptidoglycans, lipopolysaccharides (LPSs) and other glycolipids, [1] which perform several functions, ranging from structural to protective roles. [2] Bacterial glycans take part in many key biological events, including pathogen recognition, receptor activation, cell adhesion and signal transduction. Additionally, these structures often serve as molecular patterns that are recognised by specific glycan-binding receptors of the host immune system; thus triggering a pathogen-specific immune response.

It is well known that LPSs, the major constituents of the outer membrane of Gram-negative bacteria, [3] are one of the main virulence factors of several feared bacterial strains, including enteropathogenic Escherichia coli, which is implicated in severe foodborne and urinary tract infections. [4] From a structural point of view, LPS is composed of three structural motifs that can be distinguished because they are encoded by different gene clusters. Lipid A, which represents the glycolipid portion, is an acylated bis-phosphorylated glucosamine disaccharide that anchors the LPS to the outer membrane. Lipid A is covalently linked to a core oligosaccharide (OS) that can be further divided into two different portions: the more conserved inner region, which is characterised by the presence of peculiar sugar residues, such as 3-deoxy-d-manno-oct-2-ulopyranosonic acid (KDO), and the more variable outer core. Finally, the O-antigen, which is a polysaccharide composed of several OS repeating units, extends to the extracellular medium and acts as a hydrophilic coating surface. [3,[START_REF] Silipo | Endotoxins: Structure, Function and Recognition[END_REF] However, Gram-negative bacteria can also produce rough-type LPS, namely, lipooligosaccharide (LOS); a truncated version of LPS that lacks the O-antigen.

To date, the receptor complex formed by the toll-like receptor 4 and the small secreted MD2 protein is among the main species involved in bacterial LPS recognition by host immune cells. [6] More recently, it has been shown that LPSs are also intracellularly detected by caspase. [7] In both cases, the glycolipid part seems to be heavily involved in binding and in the followup of signalling.

Given their ability to selectively recognise carbohydrate structures, human lectins have emerged as potential LPS receptors specifically for their carbohydrate moiety. [8] Lectins are ubiquitous oligomeric proteins that have multiple roles in cellcell communication, cellular trafficking and regulation of immune cell functions; therefore, representing validated therapeutic targets. [START_REF] Orozco | Proc. Natl. Acad. Sci[END_REF][START_REF] Napoletano | [END_REF] Within the many different families of lectins described in humans, to date, the C-type lectin (CTL) class is the biggest and most diverse, including both transmembrane and soluble receptors. Host immune cells, such as dendritic cells and macrophages, are known to express a wide range of CTLs that contain at least one carbohydrate recognition domain (CRD), which is responsible, in most cases, for the recognition of glycan structures in a Ca 2 + -dependent manner. In the last decade, the use of glycan arrays has provided valuable details on the ligand specificity of different human CTLs; thus highlighting their ability to recognise carbohydrate epitopes belonging to both mammalian and microbial glycans. [11] However, although it is acknowledged that the recognition of 1 pathogen-associated glycostructures by human CTLs represents a key point of host immune defence, [START_REF] Orozco | Proc. Natl. Acad. Sci[END_REF][START_REF] Denda-Nagai | Irimura in C-Type Lectin Receptors in Immunity[END_REF][START_REF] Geissner | Proc. Natl. Acad. Sci[END_REF] more insights into the molecular basis of the binding mechanisms between these pattern recognition receptors and their bacterial ligands, including the LPS, are still needed.

Within this framework, we have set up a proof of concept study and investigated the ability of the macrophage galactose-type lectin (MGL; also referred to as CLEC10 A or CD301), [START_REF] Suzuki | [END_REF] to recognise the carbohydrate moiety of bacterial LPS. In contrast to several other CTLs, including dendritic cellspecific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) and the macrophage mannose (MR) receptors, which exhibit a preference for mannose-type sugars, human MGL is characterised by an exquisite specificity for terminal GalNAc/Gal residues; thus making it potentially an attractive target for the galactose-containing LOS structure of E. coli R1. [15] Core OS recognition by MGL has been investigated by means of saturation transfer difference (STD) NMR spectroscopy analysis combined with computational studies.

Herein, we used the sequence of isoform 2 of human MGL, which is shorter than that of MGL isoform-1 (also called DC-ASGPR), for cloning and recombinant production. Initially, two groups cloned these two MGL isoforms, firstly from macrophages (this CTL was therefore named macrophage galactose lectin) and then from DCs, leading to the name DC-ASGPR due to similarity to liver ASGPR with regard to selectivity. [START_REF] Suzuki | [END_REF]16] DC-ASGPR is a longer variant of MGL that results from alternative splicing with an insertion of 27 amino acids and a deletion of 3 amino acids in the neck domain. Both MGL and DC-ASGPR possess identical CRDs, which suggests that there is no difference in their carbohydrate recognition specificities, mainly characterised on MGL up to now. The MGL extracellular domain (MGL-ECD; Figure 1 A,B) has been cloned and overexpressed at high levels in E. coli as inclusion bodies (Figure 1 C). Inclusion bodies were isolated and refolded as described herein. Folded functional MGLs have an oligomeric state that allows tight binding, through an avidity-based mechanism, on a GalNAc-agarose column. [17] Elution is performed thanks to the use of ethylenediaminetetraacetic acid (EDTA); thus dem-onstrating that binding is Ca 2 + dependent. This first affinity purification step ensures the correct sorting of functionally folded MGL-ECDs, which are reloaded with Ca 2 + in a second purification step of size-exclusion chromatography. MGL-ECD oligomers, reported as trimers, [17] are then pure and ready to use for biophysical studies (Figure 1 D).

Firstly, the LOS of E. coli R1 (OS R1 ) was extracted, purified and de-acylated to derive a soluble OS, which was, in turn, further characterised by means of NMR spectroscopy (Figure 2 A,B and see the Supporting Information for more details). Once the NMR resonances were assigned, the ability of the MGL-ECD to recognise and bind the core OS was assessed by means of STD NMR; a method that allows the measurement of the transfer of saturation from the receptor to the ligand, and thus, provides a reliable mapping of the ligand epitope in the bound state. [18,19] The observation of STD enhancements in the STD spectrum of the mixture of MGL-ECD/OS R1 clearly revealed the proteinligand interaction (Figure 2 B,C). The great overlap between the NMR resonances of sugar protons hampered an extensive and quantitative study of the STD effects. However, analysis of signals resonating in isolated regions of the spectrum, together with the strong differences in multiplicity and relative intensities of STD signals with respect to the corresponding refer-Roberta Marchetti studied chemistry at the University of Naples "Federico II". In 2013, she received her PhD on NMR studies of molecular recognition processes. Since 2016, she has been a researcher at the Department of Chemical Sciences, University of Naples "Federico II". She is specialised in organic chemistry, with a special focus on the isolation, purification and characterisation of bacterial cell wall glycoconjugates. Her research interests are mainly focused on the comprehension of glycoconjugate-protein interactions through the application of NMR spectroscopy methods. ence, suggested that the outer core was the moiety more engaged in binding with the lectin. Notably, several STD signals belonged to the outer core region. In detail, the strongest STD effects derived from the terminal galactose units (L and K) and, in particular, protons H-2-H-5 of residue L (see the discussion on the computational analysis below). This observation was consistent with the previously reported ability of the human MGL to accommodate not only the N-acetylgalactosamine, but also the galactose moiety, in its binding site. [20] Saturation transfer to the glucose residues, I, G and M, adjacent to the terminal galactose disaccharide, was also detected, although to a lesser extent; thus suggesting lesser involvement in the interaction with MGL. On the contrary, the residues of the inner core region do not seem to be involved in the binding interface, as suggested, for instance, by the absence of STD effects for protons at position 3 of KDO moieties and those of protons at position 2 of the two glucosamine units of lipid A.

To further demonstrate the pivotal role of the terminal galactose moiety in the recognition and interaction process, we investigated the binding of MGL with the deacylated core OS from E. coli R3, the structure of which contains the same inner core as that of OS R1 , but differs in the composition of the outer core by the presence of glucose instead of galactose residues in the terminal region (Figure S4 A). In particular, OS R3 is composed of an a-Glc-(1-2)-a-Glc-(1-2)-[a-GlcNAc-(1-3)]-a-Gal-(1-3)-a-Glc branched pentasaccharide connected to the inner core. Interestingly, the lack of terminal galactose units totally destroyed the binding with MGL, which resulted in the absence of STD NMR signals (Figure S4 B).

The interaction of MGL-ECD with LOS R1 was further characterised by using molecular modelling and computational studies to gain a 3D perspective of the molecular recognition mechanism. Because the crystal structure of the protein has not yet been elucidated, a homology model of the MGL CRD (Cys157-Leu284) was built by using the protein structure characterised by the highest sequence identity (74 %), namely, the CRD of the mammalian asialoglycoprotein receptor (PDB ID: 1DV8; see also Figure S4) as a template. [21] Then, docking calculations of OS R1 within the modelled structure were performed.

As a first step toward understanding the binding mode of the MGL in the interaction with the bacterial OS, the terminal disaccharide Gala(1,2)Gal was docked into the primary binding site of the MGL; thus allowing us to confirm the binding specificity of the protein for the galactose. In accordance with data previously reported by Marcelo et al. on the interaction of MGL with a single galactose unit, [22] && [22] not by Marcelo, change to [22]?&& our results revealed that the terminal galactose residue was the major determinant of the binding process. Indeed, although the second galactose moiety was predicted to be close to the binding site of the protein, it seemed to play a secondary role in the interaction with the MGL (see the Supporting Information).

Considering that, from the results of the STD NMR spectroscopy analysis, the outer core of LOS was the moiety in closest proximity to the MGL binding surface, the galactose-containing branched pentasaccharide, which composed the terminal part of the LOS, was then used for docking calculations (Figure 3). The resulting 3D model of the complex revealed that the terminal Gal moiety was located inside the Ca 2 + -containing sugar binding site of the MGL CRD. Crucial contacts between OH-3 and OH-4 of the galactoside ring and the Ca 2 + ion were indeed observed and confirmed the specificity of the receptor toward galactoside residues (Figure 2). From the analysis of the receptor/ligand complex, relevant polar interactions were found between the terminal Gal OH-3 and OH-4 and the side chains of Asp245, Asn268, Asp269 and Glu256 residues. In addition, hydrogen-bond interactions with the carboxylate group of Glu242 were observed, namely, with OH-2 of the glucose residue (G) and OH-6 of the branched Glc moiety (M; Figure 2 A). Interestingly, in contrast with the results obtained on the disaccharide Gala(1,2)Gal (see the Supporting Information), only one possible binding mode has been predicted for the pentasaccharide docked in the protein binding pocket (Figure S7 B,C). The selection of this binding mode, in which the polar contacts were markedly reinforced by stacking interactions between the aromatic system of the conserved Trp247 and protons H-1 and H-2 of the galactose moiety, could be ascribable to steric hindrance of the pentasaccharide branched structure, which permitted only one orientation of the galactose residue (Figure S7 B,C). STD NMR spectroscopy analysis of the MGL-OS R1 mixture. A) Schematic structure of OS R1 derived from the LOS of E. coli R1. Symbol nomenclature for glycans has been used. [22] Phosphate groups are depicted as white letter P in orange circles. B) STD-derived epitope mapping of the MGL:OS R1 interaction, with colour coding from the highest (red) to lowest (yellow) observed STD effects. C) 1 H NMR reference spectrum (bottom) and 1D STD NMR spectrum (up) of the 1:100 mixture of MGL-OS R1 . Some key proton resonances are labelled. STD NMR spectroscopy analysis indicated that the inner core pointed farther from the surface of the MGL protein, and was mainly involved in the interaction with the terminal region of the OS.

These are not the final page numbers! ÞÞ These are not the final page numbers! ÞÞ The described MGL/pentasaccharide complex was used as a starting point to manually dock the whole OS R1 structure onto the CRD of MGL (Figure 4). The resulting 3D complex clearly indicated that the inner core region of the LOS was not implicated in the interaction with MGL because it pointed far away from the binding site of the receptor model.

Overall, these observations were in good accordance with the previously discussed STD data; thus further suggesting that significant interactions were mainly established between the protein and sugar residues of the outer core region, whereas the inner core did not seem to play a key role in the interaction with the protein.

In conclusion, we have demonstrated that human CTLs were potential binders of bacterial lipopolysaccharide in solution and that, as expected, they bound the LPS carbohydrate moiety. In particular, herein we have provided evidence that the human MGL was able to detect not only tumour-related carbohydrate structures present in mucin-like glycopeptides, but also glycan structures exposed on the bacterial surface. Whereas a wide range of infectious microorganisms were identified as pathogenic ligands of CTLs, such as DC-SIGN and MR, [24,25] glycan microarray profiling on MGL revealed its interaction only with filoviruses and the helminth parasite Schistosoma mansoni. [26] Moreover, only a few papers have been pub-lished, so far, attempting to prove the potential role of the MGL in detecting LPS structures derived from Bordetella pertussis and Campilobacter jejunii. [27,28] Indeed, we provided molecular insights into the structure of the MGL-LOS complex. We proved, by means of STD NMR spectroscopy, that the MGL was able to recognise and bind the sugar part of the LOS of E. coli R1, the major glycoform of which exhibited a terminal Gala(1,2)Gal epitope. Further details of the binding mode of MGL to LOS were obtained by homology modelling and docking calculations. Our results improve, at a molecular level, knowledge of the interaction between human CTLs and bacterial glycans, and may help to better understand the role of MGL in shaping immunity upon bacterial recognition. MGL has already been reported as a good candidate receptor for DC-based cancer immunotherapy, given its well-known ability to endocytose specific tumour-related antigens. [29] The discovery of the ability of MGL to detect an endotoxin component, LOS, of the outer membrane of the Gram-negative bacterium E. coli might offer great potential for the future development of therapeutics for bacterial disease intervention. Further studies will be necessary to investigate if and how structural variations between different LOSs could affect recognition and binding process; thus tailoring the outcome of the host-pathogen interplay. The 3D symbol nomenclature for glycans has been used. [23] B) Specific interactions between binding-site residues and the OS are depicted. Sugar residues are coloured according to the symbol nomenclature for glycans. Yellow dotted lines represent polar contacts. Distinguishing features: Carbohydratelectin interactions intervene in and mediate most biological processes. From results of saturation transfer difference NMR spectroscopy and computational studies, the ability of human macrophage galactose-type lectin (h-MGL) to recognise glycan moieties exposed on the lipooligosaccharide (LOS) of E. coli strain R1 is revealed.

Investigation into human macrophage galactose-type #lectin recognition of #glycan moieties on exposed lipooligosaccharides by STD #NMRspectroscopy (Marchetti @uninaIT) #ChemBioTalents SPACE RESERVED FOR IMAGE AND LINK Share your work on social media! ChemBioChem has added Twitter as a means to promote your article. Twitter is an online microblogging service that enables its users to send and read short messages and media, known as tweets. Please check the pre-written tweet in the galley proofs for accuracy. If you, your team, or institution have a Twitter account, please include its handle @username. Please use hashtags only for the most important keywords, such as #catalysis, #nanoparticles, or #proteindesign. The ToC picture and a link to your article will be added automatically, so the tweet text must not exceed 250 characters. This tweet will be posted on the journals Twitter account (follow us @ChemBioChem) upon publication of your article in its final (possibly unpaginated) form. We recommend you to re-tweet it to alert more researchers about your publication, or to point it out to your institutions social media team.

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Meriem Maalej

Figure 1 .ÝÝ

 1 Figure 1. Cloning and purification of MGL-ECD. A) Domain organisation of MGL. TM: transmembrane domain. Cyto.: cytoplasmic domain. B) Construct for the overproduction of MGL-ECD. Strep: StrepTag II. Xa: factor Xa protease cleavage site. 3G: tri-glycine linker. C) SDS-PAGE analysis of MGL-ECD overexpression. Lane M: PageRuler unstained protein ladder (Fermentas); lane 1: total proteins before induction; lane 2: total proteins after induction. A band at about 28 kDa corresponding to MGL-ECD MW is overexpressed. D) SDS-PAGE analysis of purified MGL-ECD. Lane 1: A unique band at about 28 kDa corresponding to pure MGL-ECD is observed.

Figure 2 .

 2 Figure2. STD NMR spectroscopy analysis of the MGL-OS R1 mixture. A) Schematic structure of OS R1 derived from the LOS of E. coli R1. Symbol nomenclature for glycans has been used.[22] Phosphate groups are depicted as white letter P in orange circles. B) STD-derived epitope mapping of the MGL:OS R1 interaction, with colour coding from the highest (red) to lowest (yellow) observed STD effects. C) 1 H NMR reference spectrum (bottom) and 1D STD NMR spectrum (up) of the 1:100 mixture of MGL-OS R1 . Some key proton resonances are labelled. STD NMR spectroscopy analysis indicated that the inner core pointed farther from the surface of the MGL protein, and was mainly involved in the interaction with the terminal region of the OS.

Figure 3 .

 3 Figure 3. A 3D model of the MGL-pentasaccharide complex. A) Pentasaccharide binding at the MGL binding site. The main amino acid residues involved in binding are labelled. Galactose and glucose residues are depicted in yellow and blue, respectively. B) The binding pocket of MGL in the presence of the pentasaccharide in the bound form, as derived from the Autodock program. Only the amino acid residues involved in the binding process and the Ca 2 + ion are depicted. Green dotted lines represent intermolecular hydrogen bonds.

Figure 4 .

 4 Figure 4. A 3D model of the MGL-OS R1 complex. A) Binding pose of the OS from E. coli R1, OS R1 , docked into the binding pocket of the MGL starting from the 3D model of the MGL-pentasaccharide complex and based on NMR spectroscopy data. The 3D symbol nomenclature for glycans has been used.[23] B) Specific interactions between binding-site residues and the OS are depicted. Sugar residues are coloured according to the symbol nomenclature for glycans. Yellow dotted lines represent polar contacts.

ÝÝ

  ChemBioChem 2019, 20, 1 -6 www.chembiochem.org 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 4 These are not the final page numbers! ÝÝ These are not the final page numbers!

  COMMUNICATIONS M. Maalej, R. E. Forgione, R. Marchetti,* F. Bulteau, M. ThØpaut, R. Lanzetta, C. Laguri, J.-P. Simorre, F. Fieschi, A. Molinaro, A. Silipo && -&& Human Macrophage Galactose-Type Lectin (MGL) Recognizes the Outer Core of Escherichia coli Lipooligosaccharide

  These are not the final page numbers! ÞÞ These are not the final page numbers! ÞÞ

	DOI: 10.1002/cbic.201900087	Communications
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Acknowledgements

For MGL-ECD production, this work used the Multistep Protein Purification Platform (MP3) of the Grenoble Instruct Centre (ISBG; UMS 3518 CNRS-CEA-UJF-EMBL) with support from FRISBI Grant ANR-10-INSB-05-02 and GRAL Grant ANR-10-LABX-49-01 within the Grenoble Partnership for Structural Biology. We thank the French Agence Nationale de la Recherche (ANR) PIA for Glyco@-Alps (ANR-15-IDEX-02) for their support of F.B. and M.M. We acknowledge Prof. Sven Muller-Loennies for providing the E. coli strains used herein (R1 and R3 core types). Finally, we wish to thank the European Union (FSE, PON Ricerca e Innovazione 2014-2020, Azione I.1 "Dottorati Innovativi con caratterizzazione Industriale") for funding a Ph.D. grant to R.E.F.

Conflict of Interest

The authors declare no conflict of interest.


Thomas Cossuet

Estelle Appert

Odette Chaix-Pluchery

Herve Roussel

Laetitia Rapenne

Gilles Renou

Frédéric Sauvage

Vincent Consonni
email: vincent.consonni@grenoble-inp.fr

Hervé Roussel

Epitaxial TiO2 Shell Grown by Atomic Layer Deposition on ZnO Nanowires Using a Double-Step Process and Its Beneficial Passivation Effect

come

INTRODUCTION

ZnO nanowires (NWs) have received an overwhelming interest in the last decade for their integration into a large variety of nanoscale devices in the fields of piezoelectricity, [START_REF] Wang | Towards Self-Powered Nanosystems: From Nanogenerators to Nanopiezotronics[END_REF] optoelectronics, [START_REF] Willander | Zinc Oxide Nanorod Based Photonic Devices: Recent Progress in Growth, Light Emitting Diodes and Lasers[END_REF][START_REF] Tian | Nanoscale Ultraviolet Photodetectors Based on One-Dimensional Metal Oxide Nanostructures[END_REF] photovoltaics, [START_REF] Consonni | Nanowires for Solar Cells: A Comprehensive Review[END_REF] detection, and sensing. [START_REF] Zhu | Room-Temperature Gas Sensing of ZnO-Based Gas Sensor: A Review[END_REF] In most of these devices, the control of the surface properties of ZnO NWs exhibiting a high aspect ratio at nanoscale dimensions is a critical issue, because their performances are largely governed by the different interfaces in the system. Owing to the high density of surface traps lying in the range of 10 12 -5x10 [START_REF] Irannejad | Effect of the TiO 2 Shell Thickness on the Dye-Sensitized Solar Cells with ZnO-TiO 2 Core-Shell Nanorod Electrodes[END_REF] cm -2 6-7 as well as to its chemical instability over a broad range of conditions in solution and gas phases, the covering of ZnO NWs by a TiO2 shell using atomic layer deposition (ALD) has been considered for many devices, specifically in the fields of photovoltaics and photocatalysis. The present combination is also favorable in these devices thanks to the type II band alignment between ZnO and TiO2. [START_REF] Wang | Measurement of Wurtzite ZnO/Rutile TiO 2 Heterojunction Band Offsets by X-Ray Photoelectron Spectroscopy[END_REF] The insertion of a TiO2 shell by ALD in dye-sensitized solar cells made of ZnO NWs has been reported to drastically increase the open-circuit voltage and fill factor, enhancing the photo-conversion efficiency up to 7.0 % by improving the interface with the dye. [START_REF] Law | ZnO-Al 2 O 3 and ZnO-TiO 2 Core-Shell Nanowire Dye-Sensitized Solar Cells[END_REF][START_REF] Wang | Dye-Sensitized Solar Cells Based on Nanoparticle-Decorated ZnO/TiO 2 Core/Shell Nanorod Arrays[END_REF][START_REF] Xu | Multilayer Assembly of Nanowire Arrays for Dye-Sensitized Solar Cells[END_REF][START_REF] Prabakar | TiO 2 Thin Film Encapsulated ZnO Nanorod and Nanoflower Dye Sensitized Solar Cells[END_REF][START_REF] Irannejad | Effect of the TiO 2 Shell Thickness on the Dye-Sensitized Solar Cells with ZnO-TiO 2 Core-Shell Nanorod Electrodes[END_REF][START_REF] Feng | Synthesis of ZnO@TiO 2 Core-Shell Long Nanowire Arrays and Their Application on Dye-Sensitized Solar Cells[END_REF][START_REF] Zhao | ZnO/TiO 2 Core-Shell Nanowire Arrays for Enhanced Dye-Sensitized Solar Cell Efficiency[END_REF][START_REF] Goh | Low Temperature Grown ZnO@TiO 2 Core Shell Nanorod Arrays for Dye Sensitized Solar Cell Application[END_REF] Similarly, Parize et al. showed that the extremely thin absorber solar cells made of ZnO / Sb2S3 core-shell NW heterostructures have an enhanced performance when a TiO2 shell deposited by ALD is inserted, leading to a photo-conversion efficiency of up to 2.3 %. [START_REF] Parize | ZnO/TiO 2 /Sb 2 S 3 Core-Shell Nanowire Heterostructure for Extremely Thin Absorber Solar Cells[END_REF] Cheng et al. showed that the insertion of a TiO2 shell deposited by ALD on ZnO NWs resulted in the increase in the short-circuit current density and photo-conversion efficiency in bulk heterojunction quantum dot solar cells incorporating PbS quantum dots, indicating the improvement of the charge carrier collection. [START_REF] Cheng | Dimension-and Surface-Tailored ZnO Nanowires Enhance Charge Collection in Quantum Dot Photovoltaic Devices[END_REF] The same enhancement has been reported in ZnO NW-based hybrid solar cells with P3HT [START_REF] Greene | ZnO-TiO 2 Core-Shell Nanorod/P3HT Solar Cells[END_REF][START_REF] Lee | Optimization of ZnO Nanorod Array Morphology for Hybrid Photovoltaic Devices[END_REF][START_REF] Atienzar | Control of Photocurrent Generation in Polymer/ZnO Nanorod Solar Cells by Using a Solution-Processed TiO 2 Overlayer[END_REF] and in ZnO NW-based halide perovskite solar cells to reach a photo-conversion efficiency of 14.35 % [START_REF] Son | Effects of Seed Layer on Growth of ZnO Nanorod and Performance of Perovskite Solar Cell[END_REF][START_REF] Wang | Hole-Conductor-Free Perovskite Solar Cells with Carbon Counter Electrodes Based on ZnO Nanorod Arrays[END_REF] by using a TiO2 shell. Additionally, the photocatalytic activity of ZnO / TiO2 core-shell NW heterostructures has been shown to outperform the one of bare ZnO NW arrays. [START_REF] Wang | Visible-Light-Responsive TiO 2 -Coated ZnO:I Nanorod Array Films with Enhanced Photoelectrochemical and Photocatalytic Performance[END_REF][START_REF] Mukhopadhyay | Design and Application of Au Decorated ZnO/TiO 2 as a Stable Photocatalyst for Wide Spectral Coverage[END_REF] Eventually, the much higher chemical stability of TiO2 has been used to combine ZnO NWs with a large number of materials grown in acidic, corrosive conditions in order to benefit from their high aspect ratio. [START_REF] Parize | Synthesis and Properties of ZnO/TiO 2 /Sb 2 S 3 Core-Shell Nanowire Heterostructures Using the Silar Technique[END_REF] Overall, the TiO2 shell grown by ALD on ZnO NWs has been found to be polycrystalline with a varying degree of crystallinity with the purposes of passivating the surface traps together with improving their chemical stability. However, the comprehensive role of the TiO2 shell including the effects of its crystalline phase, its crystallization process, and the related interface properties with ZnO NWs is still under debate despite its primary importance in the performances of the devices.

In order to address that issue, an in situ analysis of the crystallization process of the TiO2 shell grown by ALD on ZnO NWs is achieved by temperature-dependent Raman scattering and

EXPERIMENTAL SECTION

ZnO NW arrays were grown on c-axis oriented polycrystalline ZnO seed layers deposited on (001) silicon wafers, using a two-step chemical synthesis process. First, the polycrystalline ZnO seed layers were deposited by the sol-gel process using dip coating. To form the sol, 375 mM of zinc acetate dihydrate (Zn(CH3COO)2.2H2O, Sigma-Aldrich) and 375 mM of monoethanolamine (MEA, Sigma-Aldrich) were mixed in pure ethanol. The solution was heated and stirred at 60 °C for 12 hours, and then at room temperature for 12 more hours. To form the xerogel film, silicon wafers were dipped in the solution and withdrew at the speed of 3.3 mm/s in ambient atmosphere with a hygrometry smaller than 15 %. The xerogel film was annealed in air on a hot plate kept at 300 °C for 1 hour, and on another hot plate kept at 500 °C for 3 hours. Second, the ZnO seed layer-coated silicon wafers were immersed face down in a sealed beaker containing an equimolar mix of 30 mM of zinc nitrate hexahydrate (Zn(NO3)2.6H2O, Sigma-Aldrich) and HMTA (C6H12N4, Sigma-Aldrich) in deionized water.

The sealed beaker containing the solution was placed in a regular oven heated at 90 °C for 3 hours.

The TiO2 shell was subsequently grown on ZnO NW arrays at 200 °C by atomic layer deposition (ALD) using a F200 Fiji reactor from Cambridge Nanotech. Tetrakis dimethylamino titanium (TDMAT) heated at 75°C as titanium precursor and H2O as oxygen precursor were carried in the chamber by argon gas. Sequential pulses of TDMAT and H2O of 0.1 second followed by a purge of 10 seconds after each pulse were achieved to alternately inject titanium and oxygen precursors, respectively. The chamber pressure was set to 110 mTorr and increased up to 210 mTorr as the pulse proceeded. A complete sequence of 150 cycles was carried out to form an amorphous TiOx shell on ZnO NW arrays. Post-deposition thermal treatment in air was performed using an annealing temperature of 250, 350, and 500 °C for 3 hours.

The morphology of ZnO / TiO2 core-shell NW heterostructures was studied by top-view and cross-sectional view field-emission scanning electron microscopy (FESEM) imaging using a ZEISS Ultra FEG-SEM. TEM specimens were prepared by scratching the ZnO / TiO2 coreshell NW heterostructures using a diamond tip and put on Holey Carbon copper grid. TEM and HRTEM images were recorded with a JEOL JEM 2010 LaB6 microscope operating at 200 kV with a 0.19 nm point-to-point resolution. EDS mapping was collected by STEM with a JEOL 2100F FEG microscope operating at 200 kV with a 0.2 nm resolution in the scanning mode and equipped with a JEOL SDD Centurio detector with a large solid angle of up to 0.98 steradians.

The local structural properties of the ZnO / TiO2 core-shell NW heterostructures were further investigated by using an automated crystal phase and orientation mapping (ACOM) with precession (ASTAR) system implemented in the JEOL 2100F FEG microscope. The crystal phase and orientation maps were obtained by precession of the primary electron beam around the microscope's optical axis at an angle of 1.16° while collecting the electron diffraction patterns at a rate of 100 frames per second with a step size of 1 nm.

In situ and ex situ Raman scattering measurements were performed using a Jobin Yvon/Horiba Labram spectrometer equipped with a liquid nitrogen cooled charge-coupled device detector. The 488 nm excitation line of an Ar + laser with a power around 2 mW at the sample surface was used. The light was focused to a spot size close to 1 µm 2 using a 50x long working distance objective. Raman spectra were calibrated at room temperature using a silicon reference sample with a theoretical position of 520.7 cm -1 . The annealing temperature was controlled using a commercial Linkam heating stage (THMS600) placed under the Raman microscope, and varied in the range of 20 -600 °C. In situ and ex situ XRD measurements were performed with a Rigaku SmartLab diffractometer and a Bruker D8 Advance diffractometer, respectively, using the CuKα1 radiation according to the Bragg-Brentano configuration. The annealing temperature was controlled using an Anton Paar DHS 1100 chamber, and varied in the range of 20 -600 °C. In situ Raman spectra and XRD patterns were recorded with an acquisition time varying from 5 to 10 min depending on the annealing temperature.

Steady-state and time-resolved photoluminescence (PL) studies were carried out using an Edinburgh Instrument FLS980 spectrometer in front-face configuration. The steady-state PL spectra were measured with a continuous ozone-free xenon arc lamp (450 W) for excitation in a double grating Czerny-Turner monochromator for both excitation part and emission part to reach a total stray light rejection to less than 1:10 -10 . Both excitation / emission slits were opened to a bandwidth of 3 nm for steady state. The emission was detected at 90° from excitation by means of a high-gain Hamamatsu photomultiplier tube (PMT) detector mounted after the second emission monochromator. The time-resolved PL experiments were performed in single photon counting mode using a picosecond 375 nm laser diode as an excitation source (2 MHz) and a microchannel plate PMT (MCP-PMT) Hamamatsu detector cooled at -20°C. This latter was settled after the first emission monochromator to minimize instrumental time resolution down to ca. 80 ps time scale. The slit opening of the excitation monochromator was 10 nm wide to maximize the photon counting. A long pass filter of 410 nm was used to avoid the counting of incident photons directly reflected by the samples without being rejected by the monochromator. The laser energy was ca. 100 nJ/cm 2 . Prior to post-deposition thermal treatment, several well-defined lines occur in the Raman spectrum and are typically attributed to the wurtzite phase of ZnO NWs. Two intense lines at 100 and 438 cm -1 are assigned to the E2 low and E2 high modes, respectively, while the much weaker lines at 333 and 590 cm -1 are due to the E2 high -E2 low and E1(LO) modes, respectively. [START_REF] Cusco | Temperature Dependence of Raman Scattering in ZnO[END_REF] Correlatively, the diffraction peak at 34.4° is mostly ascribed to the (002) plane of the wurtzite phase of ZnO NWs. The diffraction peaks at 31.8, 36.3, 56.8, and 62.9° entirely correspond to the (100), ( 101), (110), and (103) planes of the wurtzite phase of polycrystalline ZnO seed layer. [START_REF] Guillemin | Controlling the Structural Properties of Single Step, Dip Coated ZnO Seed Layers for Growing Perfectly Aligned Nanowire Arrays[END_REF] The Raman spectra and XRD patterns recorded from RT to 260°C are very similar to each other, showing that no drastic change occurs in the amorphous TiOx shell and thus that the crystallization process has not started in that annealing temperature range.

RESULTS AND DISCUSSION

Morphology and structural properties of

In situ analysis of the crystallization process of

At the annealing temperature of 280 °C, an additional line at 149 cm -1 occurs in the Raman spectrum. It is assigned to the low-frequency Eg mode of the anatase phase of the TiO2 shell. [START_REF] Chang | Thermo-Raman Studies on Anatase and Rutile[END_REF] Correlatively, a small shoulder occurs in the XRD pattern with a diffraction peak around 37.9° corresponding to the (004) plane of the anatase phase of the TiO2 shell. The start of the crystallization process of the TiOx shell thus proceeds at 280 °C through the formation of the anatase phase. In the range of 280 to 600 °C, the intensity of the Raman line at 149 cm -1 is much more pronounced and typically shifts towards higher wavenumbers while broadening, as expected from its temperature dependence. [START_REF] Du | Variable-Temperature Raman Scattering Study on Anatase Titanium Dioxide Nanocrystals[END_REF] Also, the additional lines at 198, 398, 513, 519, and 638 cm -1 progressively occur in the Raman spectrum and are assigned to the Eg, B1g, A1g, B1g, and Eg modes, respectively, corresponding to the anatase phase of the TiO2 shell. [START_REF] Chang | Thermo-Raman Studies on Anatase and Rutile[END_REF] Correlatively, the intensity of the diffraction peak around 37.9° slightly increases in the range of 280 to 600 °C. This indicates that the crystallization process of the TiO2 shell is more active and that its crystallinity is improved in that annealing temperature range. After cooling down, all the phonon modes corresponding to the anatase phase of the TiO2 shell are clearly shown in the wavenumber range of 100 -700 cm -1 while the diffraction peak around 37.9° occurs, revealing that the crystallization process has efficiently been proceeded. No sign for the presence of the rutile/rocksalt phases of TiO2 is indicated, showing that the shell has crystallized into the pure anatase phase. An ex situ post-deposition thermal treatment of the amorphous TiOx shell on ZnO NWs was achieved at 250, 350, and 500 °C for 3 hours in air to corroborate the in situ analysis. The corresponding XRD patterns and Raman spectra are presented in Figure S1 and S2. The XRD pattern reveals that the TiOx shell is still amorphous following the post-deposition thermal treatment at 250 °C with no sign for the presence of diffraction peaks. In contrast, the Eg line at 144 cm -1 of the anatase phase already occurs in the Raman spectrum, giving direct evidence of the crystallization onset that is likely localized highly. The longer annealing time as compared to the in situ analysis is thus sufficient to very locally activate the crystallization, showing the importance of kinetic phenomena in that process. After thermal treatments at 350 and 500 °C, both techniques show that the TiO2 shell is well crystallized into the anatase phase, as shown by the occurrence of the diffraction peaks at 25.3, 37.9, 38.6, and 48.1° that are assigned to the (101), (004), ( 112) and (200) planes, as well as of the Raman lines at 144, 198, 398, 513, 519, 638 cm -1 that correspond to the Eg, Eg, B1g, A1g, B1g, Eg modes, respectively. [START_REF] Chang | Thermo-Raman Studies on Anatase and Rutile[END_REF] It should be noted here that the post-deposition thermal treatment in the range of 280 -350 °C is compatible with the use of glass covered with a transparent conductive oxide layer as a typical substrate in the field of photovoltaics. It is not the case for the post-deposition thermal heat treatment above 500 °C. Also, the integration of an amorphous TiO2 shell is not favorable for the charge carrier separation in such nanostructured solar cells. [START_REF] Consonni | Nanowires for Solar Cells: A Comprehensive Review[END_REF] In the following, the interface properties between the ZnO NWs and TiO2 shell are thus investigated in details for the most appropriate condition using the post-deposition thermal treatment at 350 °C. (c) Corresponding SAED pattern revealing the ZnO wurtzite and TiO2 anatase structures of both phases (i.e., scale bar = 5 nm -1 ).

Interface properties between the ZnO nanowires and

Owing to the inherent spatial localization of the HRTEM imaging on the nanometer scale, the structural properties of the TiO2 shell and the related ZnO / TiO2 interface properties were investigated on a larger scale by ASTAR measurements. [START_REF] Rauch | Automated Crystal Orientation and Phase Mapping in Tem[END_REF][START_REF] Midgley | Precession Electron Diffraction -a Topical Review[END_REF] In this technique, the incident electron beam had a few nanometers in size and was precessed to reduce dynamical effects and to enhance the indexing quality. The electron beam was simultaneously scanned over the area of interest to record an electron diffraction pattern at each location. The experimental electron diffraction patterns were basically compared with the complete set of theoretical diffraction patterns, which were computed for every expected crystalline phases and for a large number of orientations. The best match between the experimental and theoretical electron diffraction patterns permitted to identify both crystalline phase and orientation with a high precision. [START_REF] Consonni | Identifying and Mapping the Polytypes and Orientation Relationships in ZnO/CdSe Core-Shell Nanowire Arrays[END_REF] The maps of the crystalline phases and orientations of ZnO / TiO2 core-shell NW heterostructures are presented in Figures 5 and6, respectively. It is clearly shown in Figure 5 that the TiO2 shell on the m-plane sidewalls and c-plane face of ZnO NW exclusively crystallizes into the anatase phase, which is in agreement with the previous Raman scattering and XRD measurements. No sign for the presence of a residual amorphous phase or of rutile/rocksalt phases or of Zn2TiO4 spinel phase is indicated. [START_REF] Yang | Multitwinned Spinel Nanowires by Assembly of Nanobricks Via Oriented Attachment: A Case Study of Zn 2 TiO 4[END_REF] Also, the orientation map in Figure 6 reveals that the TiO2 shell is epitaxially formed on the m-plane sidewalls and c-plane face of the ZnO NW located on the left. The orientation relationship between the TiO2 shell and c-plane face of ZnO NW is the following: the (001) and (0001) planes as well as the (120) and (101 0) planes are parallel to each other with only a very small misorientation. The orientation relationship between the TiO2 shell and m-plane sidewalls of the ZnO NW is identical and as follows: the (120) and (101 0) planes as well as the (001) and (0001) planes are parallel to each other with only a very small misorientation also. It is worth noticing that the lattice mismatch between the TiO2 and ZnO materials lies in the range of 10 % in both orientation configurations, indicating that the epitaxial strain has completely been relieved plastically during the post-deposition thermal treatment. In contrast, the TiO2 shell formed on the m-plane sidewalls and c-plane face of nearby ZnO NW located on the right is slightly more inhomogeneous and typically exhibits a local epitaxy with a large number of orientation relationships. Overall, the present finding shows that the epitaxial deposition of the TiO2 shell can be achieved by ALD followed by a post-deposition thermal treatment according to a double-step process, which is remarkable to get high quality ZnO / TiO2 core-shell NW heterostructures. The orientation relationships are however multiple and the local epitaxy is inevitably relieved totally. It should be noted here that the epitaxial deposition by ALD has only been reported a few times in the literature regarding a couple of dedicated materials systems in a thin film configuration including TiO2 / Al2O3. [START_REF] Vasu | Atomic Layer Deposition of P-Type Epitaxial Thin Films of Undoped and N-Doped Anatase TiO 2[END_REF] The structural quality of epitaxial TiO2 shell is nevertheless dependent to a small extent upon the considered ZnO NW because the deposit of the TiOx shell by ALD is likely affected by the varying free space between the nearby ZnO NWs. Steady-state and time-resolved room temperature PL measurements of bare ZnO NWs and ZnO / TiO2 core-shell NW heterostructures annealed at 350 °C for 3 hours in air are presented in Figures 7 and8. The PL mapping shows that, for both samples, the spectra are composed of a sharp near-band edge (NBE) emission centered at 375 nm (i.e., 3.31 eV) and of a broad and more intense visible emission band lying in the range of 500 -750 nm (i.e., 1.65 -2.48 eV).

The NBE emission is related to radiative bimolecular recombination processes involving neutral donor bound A-excitons, typically through the I4 and I5 lines labeled in the I nomenclature [START_REF] Meyer | Bound Exciton and Donor-Acceptor Pair Recombinations in ZnO[END_REF] that are assigned to substitutional hydrogen on the oxygen lattice site (HO) [START_REF] Lavrov | Identification of Two Hydrogen Donors in ZnO[END_REF] and zinc vacancy -hydrogen (VZn-3H) defect complexes, [START_REF] Heinhold | Hydrogen-Related Excitons and Their Excited-State Transitions in ZnO[END_REF] respectively. The involvement of interstitial hydrogen in bond-centered sites (HBC) is also expected in the NBE emission. [START_REF] Lavrov | Identification of Two Hydrogen Donors in ZnO[END_REF] The present hydrogen-related defects originating from the crystallization process of bare ZnO NWs in aqueous solution act as shallow donors and thus are responsible for their high electrical conductivity. [START_REF] Cossuet | Polarity-Dependent High Electrical Conductivity of ZnO Nanorods and Its Relation to Hydrogen[END_REF] In contrast, the broad visible emission band from the red/orange to yellow/green region stems from radiative processes involving electron-hole recombination mediated by deeper states in the bandgap of ZnO NWs. Its assignment has been a long debate over the last two decades and often attributed to oxygen vacancies for explaining the sensing properties of ZnO NWs. [START_REF] Barbagiovanni | Radiative Mechanism and Surface Modification of Four Visible Deep Level Defect States in ZnO Nanorods[END_REF] The most recent DFT calculations have however established that the emission energies related to oxygen vacancies are smaller than 1 eV and thus cannot account for the present visible emission band obtained by optical spectroscopy. [START_REF] Lyons | First-Principles Characterization of Native-Defect-Related Optical Transitions in ZnO[END_REF] Instead, zinc vacancies as well as (VZn-H) and (VZn-2H) defect complexes [START_REF] Lyons | First-Principles Characterization of Native-Defect-Related Optical Transitions in ZnO[END_REF][START_REF] Frodason | Zn Vacancy-Donor Impurity Complexes in ZnO[END_REF] creating deeper states are involved both in the bulk of ZnO NWs and on their surfaces to account for the red/orange/yellow and green/blue emission bands, respectively. Additionally, zinc dangling bonds have recently been associated with an emission energy around 2.41 eV. [START_REF] Lyons | First-Principles Characterization of Native-Defect-Related Optical Transitions in ZnO[END_REF] They may thus contribute also to the visible emission band here through their location on the surfaces of bare ZnO NWs. Interestingly, these two different radiative processes taking place in ZnO NWs are drastically modified when their surfaces are protected by the epitaxial TiO2 shell grown by ALD and crystallized by postdeposition thermal treatment in air. First, the intensity of the NBE emission is strongly enhanced. This is the result of a reduction of the monomolecular non-radiative recombination processes, which are in this case rather a surface-dominant mechanism than a bulk process owing to the high aspect ratio of ZnO NWs. Second, the broad visible emission band has almost vanished. This result is particularly insightful as it highlights that the hydrogen-related defects in the bulk of ZnO NWs are redistributed following the post-deposition thermal treatment in air while the one on their surfaces very likely get passivated by the epitaxial anatase-TiO2 shell.

These results clarify the origin of the enhancements widely reported in the literature through ZnO/TiO2 core-shell NW heterostructures. In particular, the epitaxial TiO2 shell affords to reduce the energy losses from the excited states of ZnO NWs through the passivation of surface defects, thus enhancing cell photo-voltage and charge carrier collection efficiency in photoelectrochemical devices. The dynamic of these two radiative processes was investigated by time-correlated single photon counting (TCSPC) experiments. The two samples were excited at 375 nm and the PL decays were measured either in the end tail of the sharp NBE emission at 420 nm (Figure 8 a-b) or within the broad emission band in the visible range at 610 nm (Figure 8 c-d). The evolution of the PL intensity I(t) as a function of the delay time t systematically requires three simple exponential components to accurately account for the decays, as follows:

I(t) = f e ( )
where fk is the fraction of the k th process, and τk is the lifetime of the k th process.

Although the origin of these three contributions can be debated, we favored such a mathematical function rather than a stretch bi-exponential for which the origin of lifetime distribution is also questionable. The two aforementioned radiative processes have a thoroughly different dynamic, as one could expect from their distinct origin. The decay from the NBE emission involving shallow donors is significantly faster than through the deeper traps in the visible emission band, as reported in

CONCLUSIONS

  x-ray diffraction (XRD) measurements. The interface properties of the resulting ZnO / TiO2 core-shell NW heterostructures are investigated in details by transmission electron microscopy (TEM)-based experiments as well as steady-state and time-resolved photoluminescence (PL) measurements. It is shown that the TiO2 shell with the anatase phase can epitaxially be formed on ZnO NWs by combining ALD with post-deposition thermal treatment in air, resulting in the fabrication of high quality ZnO / TiO2 core-shell NW heterostructures with improved interface properties.

  ZnO / TiO 2 core-shell nanowire heterostructures. The structural morphology of ZnO / TiO2 core-shell NW heterostructures annealed at 350 °C for 3 hours in air is presented in Figure1. ZnO NWs grown by CBD on a c-axis oriented polycrystalline ZnO seed layer by sol-gel process[START_REF] Guillemin | Controlling the Structural Properties of Single Step, Dip Coated ZnO Seed Layers for Growing Perfectly Aligned Nanowire Arrays[END_REF] exhibit a mean diameter and length of 81 ± 22 nm and 892 ± 102 nm, respectively, using an equimolar mix of zinc nitrate and HMTA.[START_REF] Parize | Effects of Hexamethylenetetramine on the Nucleation and Radial Growth of ZnO Nanowires by Chemical Bath Deposition[END_REF] They retain the typical hexagonal facet on their top face following the ALD of the TiO2 shell. However, the surfaces on their c-plane top face and m-plane sidewalls have a granular morphology indicating the formation of the TiO2 shell. No change in the structural morphology of ZnO / TiO2 core-shell NW heterostructures before and after annealing has been revealed by FESEM imaging.

Figure 1 .

 1 Figure 1. (a) Top-view and (b) cross-sectional view FESEM images of ZnO / TiO2 core-shell NW heterostructures annealed at 350 °C for 3 hours in air. The scale bars denote 100 and 200 nm, respectively.

  TiO 2 shell. In situ Raman scattering and XRD measurements were performed from room temperature (RT) to 600 °C to thoroughly investigate the transformation of the amorphous TiOx shell into the crystallized TiO2 shell during the crystallization process. The corresponding in situ Raman spectra and XRD patterns recorded in air from RT to 600 °C, and after cooling down, are presented in Figures2 and 3, respectively.

Figure 2 .

 2 Figure 2. In situ Raman spectra of ZnO / TiO2 core-shell NW heterostructures annealed from RT to 600 °C in air.

Figure 3 .

 3 Figure 3. In situ XRD patterns of ZnO / TiO2 core-shell NW heterostructures annealed from RT to 600 °C in air.

4 .

 4 TiO 2 shell. TEM and HRTEM images of a ZnO / TiO2 core-shell NW heterostructure annealed at 350 °C for 3 hours in air are presented in Figure The deposition of the TiO2 shell on the m-plane sidewalls of ZnO NW is highly conformal with a constant thickness of 8.5 nm from its top to its bottom, as revealed in the TEM image of Figure 4a. The thickness of the TiO2 shell on the c-plane face of ZnO NW is however slightly larger. The HRTEM image in Figure 4b collected close to the ZnO / TiO2 interface reveals the alignment of the (0002) plane of the ZnO NW with the (004) plane of the TiO2 shell. The related SAED pattern in Figure 4c confirms that the ZnO NW and TiO2 shell crystallize into the wurtzite and anatase phases, respectively. It further shows that the TiO2 shell has an orientation relationship with the ZnO NW, following the post-deposition thermal treatment at 350 °C in air.

Figure 4 .

 4 Figure 4. (a) TEM image of a ZnO / TiO2 core-shell NW heterostructure after annealing at 350 °C for 3 hours in air (i.e., scale bar = 100 nm). (b) Corresponding TEM image of ZnO / TiO2 core-shell NW heterostructure at higher magnification (i.e., scale bar = 25 nm). The inset on the right hand side shows a HRTEM image of the ZnO / TiO2 interface (i.e., scale bar = 5 nm).

Figure 5 .

 5 Figure 5. (a) ASTAR map representing the crystalline phases of ZnO / TiO2 core-shell NW heterostructures annealed at 350 °C for 3 hours in air (i.e., scale bar = 50 nm). The ZnO wurtzite and TiO2 anatase phases are shown in red and green, respectively. (b, c) Corresponding electron diffraction patterns collected at points (1) and (2), respectively. The red and green open circles represent the theoretical electron diffraction patterns of wurtzite ZnO and anatase TiO2, respectively.

Figure 6 .

 6 Figure 6. ASTAR maps representing the crystalline orientations of the wurtzite ZnO and anatase TiO2 phases within the ZnO / TiO2 core-shell NW heterostructure annealed at 350 °C for 3 hours in air, plotted along the X, Y and Z axes. The color scales for the wurtzite ZnO and anatase TiO2 phases are given in the insets.

Figure 7 .

 7 Figure 7. Steady-state room temperature PL measurements of bare ZnO NWs (left) and ZnO / TiO2 core-shell NW heterostructure annealed at 350 °C for 3 hours in air (right) as a function of the excitation wavelength. The black inset corresponds to a 5 times zoom-in in intensity of the portion below 450 nm to better show the weaker NBE emission for sake of clarity.

  representing the predominant contribution to the decay. The presence of the epitaxial TiO2 shell slightly accelerates the PL decay and significantly promotes the fast component with a fraction increasing from 11.4 % to 32.16 %. This suggests the possibility of a photon-induced electron transfer from the ZnO NW core towards the epitaxial TiO2 shell. The PL contribution from the TiO2 shell is however not detected in our case, thus suggesting a process faster than the time resolution offered by TCSPC. By probing the visible emission band at 610 nm, the faster component of ca. 1.4 ns to the decay is found to be much longer. The second and third components have a lifetime of ca. 7.4 ns and ca. 54.2 ns, respectively, this latter being the major contribution to the decay. In this case, the epitaxial TiO2 shell does not influence significantly the excited state dynamic of ZnO NWs. This can be anticipated since the deeper position of the states located in the bandgap of ZnO NWs cannot afford any electron transfer towards the TiO2 shell. Overall, these optical measurements indicate that the epitaxial TiO2 shell grown by ALD leads to an efficient passivating effect of the surfaces of ZnO NWs as well as to a redistribution of hydrogen-related defects following the post-deposition thermal treatment.

Figure 8 .

 8 Figure 8. Time-resolved PL decay for (a,c) bare ZnO NWs and (b,d) ZnO / TiO2 core-shell NW heterostructures annealed at 350 °C for 3 hours in air for an excitation wavelength at 375 nm and emission wavelengths at (a,b) 420 nm or (c,d) 610 nm (laser repetition of 2 MHz, energy of 100 nJ/cm 2 , long pass filter of 410 nm). The blue dots correspond to the experimental data and the red curve to the experimental fits after reconvolution of the instrumental response function. The residual is reported at the bottom of each decay.

Table 1 .

 1 A fast component of ca. 70 ps is deduced for the NBE emission, in addition to a second component at ca. 1 ns and a third component at ca. 8.3 ns, this latter

Excitation at 375 nm / Emission at 420 nm τ 1 (ns) f 1 (%) τ 2 (ns) f 2 (%) τ 3 (ns) f 3 (%) χ 2

	ZnO	0.069	11.40	0.996	25.17	8.32	63.42	1.060
	ZnO/TiO 2	0.067	32.16	0.724	27.85	6.70	39.98	1.149
				Excitation at 375 nm / Emission at 610 nm
		τ 1 (ns)	f 1 (%)	τ 2 (ns)	f 2 (%)	τ 3 (ns)	f 3 (%)	χ 2
	ZnO	1.436	14.6	7.445	29.43	54.154	55.96	1.003
	ZnO/TiO 2	1.552	5.70	8.852	26.03	53.543	68.27	1.026

Table 1 .

 1 Lifetime τk and fraction fk values of the k th processes obtained after reconvolution of the instrumental response function for bare ZnO NWs and ZnO / TiO2 core-shell NW heterostructures annealed at 350 °C for 3 hours in air for an excitation wavelength at 375 nm and emission wavelengths at 420 nm or 610 nm. χ 2 is the chi value of the fit.

ACKNOWLEDGEMENTS

The authors acknowledge the financial support by the French Research National Agency through the project DOSETTE (ANR-17-CE24-0004). Funding by Cross-Disciplinary Program of the IDEX Université Grenoble Alpes (ANR-15-IDEX-02) through the project ECO-SESA is also acknowledged. The authors further acknowledge the facilities, and the scientific and technical assistance of the CMTC characterization platform of Grenoble INP, which is supported by the Centre of Excellence of Multifunctional Architectured Materials (LabEx CEMAM) under the contract ANR-10-LABX-44-01 funded by the "Investments for the Future" Program.

The chemical instability of ZnO NWs over a broad range of conditions in solution and gas phases as well as the high density of traps on their surfaces require the development of protective, passivating shells for improving their technological integration and maximizing the performances of the related devices. We have developed a double-step process using ALD and post-deposition thermal treatment in air to form a conformal, epitaxial TiO2 shell on the ZnO NWs with a range of orientation relationships, as shown by TEM-based experiments. The crystallization process has been found to start around 280 °C in air through the formation of the only anatase phase, using an in situ analysis combining temperature-dependent Raman scattering and XRD measurements. Furthermore, the epitaxial TiO2 shell has been revealed to induce a beneficial passivating effect and to redistribute the hydrogen-related defects in the center of ZnO NWs following the post-deposition thermal treatment at 350 °C in air. The optimal conditions for the double-step process are thus compatible with the use of glass covered with a transparent conductive oxide layer as a typical substrate in the field of photovoltaics.

These findings report a powerful tool to form ZnO / TiO2 core-shell NW heterostructures with high structural and optical quality. They further open the way for developing the double-step process in other nanostructured material systems to master the related surface and interface properties.

ASSOCIATED CONTENT

XRD pattern of as-deposited ZnO / TiOx core-shell NW heterostructures and corresponding XRD patterns obtained after annealing at 250, 350 and 500 °C for 3 hours in air (Figure S1); Raman spectrum of as-deposited ZnO / TiOx core-shell NW heterostructures and corresponding Raman spectra collected after annealing at 250, 350 and 500 °C for 3 hours in air (Figure S2).

AUTHOR INFORMATION Corresponding Author

*E-mail : vincent.consonni@grenoble-inp.fr