Agnes Burel

Marie-Thérèse Lavault

Clément Chevalier

Helmut Gnaegi

Sylvain Prigent

Antonio Mucciolo

Stéphanie Dutertre

Bruno M Humbel

Thierry Guillaudeux

Irina Kolotuev
email: irina.kolotueva@unil.ch

A targeted 3D EM and correlative microscopy method using SEM array tomography

Keywords: correlative light and electron microscopy (CLEM), array tomography, volume reconstruction, model organisms, C. elegans, Drosophila, Development, Accepted AT -Array Tomography CLEM -Correlative Light and Electron Microscopy EM -Electron Microscopy FIB -Focused Ion Beam

Using electron microscopy to localize rare cellular events or structures in complex tissue is challenging. Correlative light and electron microscopy procedures have been developed to link fluorescent protein expression with ultrastructural resolution. Here, we present an optimized scanning electron microscopy (SEM) workflow for volumetric Array Tomography for asymmetric samples and model organisms (Caenorhabditis elegans, Drosophila melanogaster, Danio rerio). We modified a diamond knife to simplify serial section arrays acquisition with minimal artifacts. After array acquisition, we transfer the arrays to a glass coverslip or silicon wafer support. Using light microscopy, we rapidly screen the arrays for initial recognition of global anatomical features (organs or body traits). Then, using SEM, we perform an in-depth study of the cells and/or organs of interest. Our manual and automatic data acquisition strategies make 3D data acquisition and correlation simpler and more precise than do alternative methods. This method can be used to address questions in cell and developmental biology that require the efficient identification of a labeled cell or organelle.

Introduction

Imaging methods aid the analysis and understanding of biological processes. Advanced fluorescent light microscopy (FLM) techniques can answer questions about the localization, dynamics, and interactions of proteins in various organs and cell structures. Genetically encoded fluorescence tools enable the study of live processes. However, many cellular processes cannot be fully visualized, due to the insufficient resolution of fluorescent labeling and the lack of visible reference structures within the cell. Much higher resolution of cellular components can be obtained with electron microscopy, which is crucial for the analysis of cellular ultrastructure. Recently, cellular ultrastructure has been directly correlated with a fluorescence signal using correlative light and electron microscopy (CLEM; [START_REF] Caplan | The power of correlative microscopy: multi-modal, multi-scale, multi-dimensional[END_REF][START_REF] Muller-Reichert | Preface. Correlative light and electron microscopy II[END_REF].

Serial section transmission electron microscopy (TEM), as well as tilt series combined with electron tomography, have been the primary tools for reconstructing ultrastructure within a defined volume. These methods have significantly contributed to our current understanding of the local 3D organization of biological samples (White et al., 1986;[START_REF] Bumbarger | Three-dimensional fine structural reconstruction of the nose sensory structures of Acrobeles complexus compared to Caenorhabditis elegans (Nematoda: Rhabditida)[END_REF][START_REF] Noske | Expedited approaches to whole cell electron tomography and organelle mark-up in situ in high-pressure frozen pancreatic islets[END_REF][START_REF] Anderson | Exploring the retinal connectome[END_REF][START_REF] Saalfeld | Elastic volume reconstruction from series of ultra-thin microscopy sections[END_REF][START_REF] Bock | Network anatomy and in vivo physiology of visual cortical neurons[END_REF][START_REF] Doroquez | A highresolution morphological and ultrastructural map of anterior sensory cilia and glia in Caenorhabditis elegans[END_REF][START_REF] Lee | Anatomy and function of an excitatory network in the visual cortex[END_REF]. Unfortunately, these procedures require a high level of training and are both time-consuming and prone to occasional loss of sections, that is, loss of information. Recently developed alternative methods for serial sectioning such as Serial Block Face -SEM (SBFSEM) and Focused Ion Beam -SEM (FIB-SEM), that use backscattered electrons to image the block surface, have helped to address biological questions that require the analysis of large EM volumes [START_REF] Denk | Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure[END_REF]Heymann et al., 2006;[START_REF] Knott | Positional correlative anatomy of invertebrate model organisms increases efficiency of TEM data production[END_REF]Kizilyaprak et al., 2014;[START_REF] Titze | Volume scanning electron microscopy for imaging biological ultrastructure[END_REF]. During these approaches, the block surface is imaged, and then sections are removed. The new block surface is then imaged again. This cyclical repetition results in the generation of an automatically aligned stack of numerous sections. Although these approaches result in efficient 3D data acquisition, re-imaging the same sample at another region of interest or at a different resolution is impossible, as is on-section immunolabeling or post-staining contrast enhancement with heavy metals. These problems can be overcome, as has been shown in several studies that correlated light and FIB-SEM data on cells in culture, in brain tissue, and developing blood vessels in zebrafish [START_REF] Bushby | Correlative light and volume electron microscopy: using focused ion beam scanning electron microscopy to image transient events in model organisms[END_REF][START_REF] Lucas | Bridging microscopes: 3D correlative light and scanning electron microscopy of complex biological structures[END_REF][START_REF] Maco | Correlative in vivo 2 photon and focused ion beam scanning electron microscopy of cortical neurons[END_REF][START_REF] Lucas | Correlative 3D imaging: CLSM and FIB-SEM tomography using high-pressure frozen, freeze-substituted biological samples[END_REF][START_REF] Bosch | FIB/SEM technology and high-throughput 3D reconstruction of dendritic spines and synapses in GFP-labeled adult-generated neurons[END_REF][START_REF] Blazquez-Llorca | Correlation of two-photon in vivo imaging and FIB/SEM microscopy[END_REF]. Notably, such Development • Accepted manuscript elegant studies require rare and expensive equipment, available only in specialized labs with limited external access.

Array Tomography was developed by Micheva and collaborators to study protein localization in synapses (Micheva and Smith, 2007;[START_REF] Micheva | Single-synapse analysis of a diverse synapse population: proteomic imaging methods and markers[END_REF][START_REF] Collman | Mapping synapses by conjugate light-electron array tomography[END_REF]. The samples are embedded in a hydrophilic resin and sectioned into long ribbons of serial sections (the arrays) using an ultramicrotome. The sections are then transferred to a glass coverslip (instead of a microscope grid) and analyzed by light microscopy followed by SEM. To localize a large number of proteins in a single series of sections, several rounds of fluorescence labeling were performed, using antibodies combined with an antibody elution step before each additional round. The EM-like sample preparation procedure enabled the analysis of the cellular ultrastructure with the localization of fluorescent signal.

Array Tomography (AT) is used in different procedures, depending on the research goal: a. Multiplex FLM is used to detect numerous fluorophores (Table 1, Multiplex labeling). The mild sample treatment and hydrophilic resin embedding usually compromises the quality of the ultrastructure.

b. Serial EM is used for volume acquisition and Region Of Interest (ROI) recognition, when ultrastructural preservation is critical (Table 1, Volume reconstruction). Since the backscattered SEM mode is used, high contrast with multiple heavy metal stains is required. This preparation uses standard or enhanced contrast TEM with epoxy resin based embedding. c. Correlative Fluorescence-EM is used to preserve fluorescence and ultrastructure quality, both en bloc and on section (Table 1, CLEM). Freeze-substitution with acrylic resin embedding can provide efficient fluorescence retention [START_REF] Nixon | A single method for cryofixation and correlative light, electron microscopy and tomography of zebrafish embryos[END_REF][START_REF] Kukulski | Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision[END_REF][START_REF] Watanabe | Protein localization in electron micrographs using fluorescence nanoscopy[END_REF][START_REF] Peddie | Correlative and integrated light and electron microscopy of in-resin GFP fluorescence, used to localise diacylglycerol in mammalian cells[END_REF][START_REF] Mcdonald | Rapid embedding methods into epoxy and LR White resins for morphological and immunological analysis of cryofixed biological specimens[END_REF][START_REF] Burette | Knowing a synapse when you see one[END_REF]Kremer et al., 2015;[START_REF] Paez-Segala | Fixation-resistant photoactivatable fluorescent proteins for CLEM[END_REF]this study). However, the retention of the fluorescence signal with this approach is unpredictable and largely dependent on the specific fluorescent fusion protein used.

Although array tomography is a powerful technique, its use has been predominantly restricted to addressing questions in neurobiology, for which it was developed [START_REF] Oberti | Projection neuron circuits resolved using correlative array tomography[END_REF].

Recent studies have applied AT to other fields [START_REF] Jahn | Shedding light on cell compartmentation in the candidate phylum Poribacteria by high resolution visualisation and transcriptional profiling[END_REF][START_REF] Markert | Filling the gap: adding super-resolution to array tomography for correlated ultrastructural and molecular identification of electrical synapses at the C. elegans connectome[END_REF]. For AT, sections are cut and transferred to a solid support, such as a coverslip, an indium-tin oxide-coated coverslip, a silicon wafer or a Kapton plastic tape (Micheva and Smith, 2007;[START_REF] Pluk | Advantages of indium-tin oxide-coated glass slides in correlative scanning electron microscopy applications of uncoated cultured cells[END_REF][START_REF] Kasthuri | Saturated Reconstruction of a Volume of Neocortex[END_REF] using special slide/wafer retraction mechanisms Development • Accepted manuscript [START_REF] Wacker | Array tomography[END_REF][START_REF] Hayworth | Imaging ATUM ultrathin section libraries with WaferMapper: a multi-scale approach to EM reconstruction of neural circuits[END_REF]. AT-SEM is increasingly used as an alternative to TEM serial section SBF and FIB-SEM imaging methods to analyze large surface areas in a non-destructive manner. It allows the direct 3D correlation of CLEM samples with both fluorescence and electron microscopy data. Unlike other volume sections transferred to a solid support, these samples can be stored, re-imaged multiple times, and even re-contrasted with heavy metals to improve the signal-to-noise ratio [START_REF] Schwarz | Correlative light and electron microscopy using immunolabeled sections[END_REF].

Large volume data analysis is required to answer some biological questions. Other questions can be answered by analyzing limited volumes from numerous independent samples or events. To provide a method to answer these questions that is accessible to the larger scientific community without technical limitations, we have modified and adapted the AT technique. Our method encompasses the entire procedure from sample preparation to data analysis. We transfer serial sections to glass coverslips or silicon wafers (hereafter referred to as 'wafer' for simplicity), label with heavy metals, and use high-resolution volume FLM and EM. We have optimized sample preparation, configured a diamond knife to facilitate the transfer of sections to support, and devised an efficient sectional screening strategy. This method can be used with a variety of model organisms. It does not require additional expensive equipment beyond that of most modern research facilities. To demonstrate our technique, we analyzed both Caenorhabditis elegans and Drosophila melanogaster.

Results

Sample preparation methods and sectioning strategy

When analyzing polarized samples and small model organisms, the relevant cellular structure, or region of interest (ROI), can be located using the known morphology and anatomy of the tissue as landmarks. The principal problem with these complex samples is that only one ROI may be present in each sample. Since imaging only one individual structure would be insufficient to make a statistically significant observation, numerous samples must be analyzed.

Our strategy to efficiently localize the ROI in a single sample includes two-step flat embedding, precise block trimming (Figs 1A, B;Kolotuev, 2014), and serial sectioning. We use light microscopy to rapidly locate the ROI for subsequent EM analysis, precisely orient the sample, and delimit the area for ultrathin sectioning. To optimize the stability of the Development • Accepted manuscript ribbon of consecutive sections, we remove the excess embedding resin surrounding the sample. With homogeneous tissue, excess embedding resin can be easily trimmed away from a sample (we refer to this as "tight trimming"). With non-homogeneous tissue, such as C. elegans or Drosophila, even "tight trimming" cannot entirely remove the excess resin. Thus, we use our trimming diamond tool for "tight trimming" to leave a minimal amount of excess resin around the sample. To acquire long ribbons of consecutive sections (>300 sections), we prefer to shape the surface of the block to a rectangle, with the edge parallel to the knife, such that the block length measures twice the length of the side edges. A larger sectional surface tends to produce more folds, while sections with a smaller surface tend to curl.

To produce horizontally aligned sections on support (glass coverslips or wafers), we used a modified diamond knife that greatly facilitates the efficient generation of long ribbons of consecutive sections (Figs. 1C-H; see detailed description below). Left intact, these long ribbons can complicate subsequent manual data acquisition: Without obvious structural variation, mistakes will often be made during the monotonous analysis of long ribbons of consecutive sections. Thus, for our rapid screening approach, we carefully subdivide the long ribbons initially produced into shorter ribbons of consecutive sections (50-100 sections), and then horizontally align the shorter ribbons alongside each other, side by side, in rows on the support. This way, 10 short ribbons (i.e., over 500 sections) can be easily aligned on a small support, be it a coverslip, or a wafer (Fig. 1H). Then, instead of thoroughly analyzing every section within a long ribbon in a serial "vertical" fashion (from 0 to 100+), we "leap" between centrally-located sections across the short horizontally-aligned ribbons to visually screen for the ROI (see detailed example below). Sequential section analysis using TEM grids is very tedious: Numerous problems can emerge.

If the sectional surface is relatively large, then very few sections will fit on one grid. If multiple sections do fit on one grid, then sample collection is error-prone due to the risk of sectional loss. Data analysis is also very time consuming because each section on the grid must be manually screened to find the ROI and orient the sample. For serial section collection, even a relatively short sequence of sections (e.g. 100) transferred to support (glass coverslips or wafers) presents significant advantages compared to sections on TEM grids (Figs. 1I,1J). On support, 115 serial sections can be analyzed in less than 15 minutes after orienting the trimmed block optimally relative to the AT knife. To analyze 115 sections on TEM grids would require much more time and result in significantly less precise data. For any Development • Accepted manuscript scientific question that needs a resolution compatible with FIB and SBF-SEM, our transfer technique has clear benefits over serial section TEM.

Modified diamond knife

Both the histo and the ultra-Jumbo knives (Diatome, Switzerland) hold a coverslip in their large boat at an optimal angle for collecting semi-thin sections. These knives can be used to generate arrays of sections, that is, long ribbons of consecutive sections. In the published AT procedure, the array of sections is moved to the rear edge of the coverslip where it touches the interface between the glass and the water (Micheva and Smith, 2007). To attach the sections to support, water is drained from the side of the knife boat using a syringe (Micheva and Smith, 2007) or the coverslip is pulled out of the trough using a micromanipulator [START_REF] Horstmann | Serial section scanning electron microscopy (S3EM) on silicon wafers for ultra-structural volume imaging of cells and tissues[END_REF][START_REF] Wacker | Hierarchical imaging: a new concept for targeted imaging of large volumes from cells to tissues[END_REF]. In our hands, this method compromises the control of water during draining. The resulting rapid drying of the sections on support can create wrinkles or folds in the sections and cause ROIs to be lost from view.

We have developed a water-draining system for the histo jumbo diamond knife, which reduces the incidence of sectional folding and wrinkling. Also, we have replaced the blade on the diamond knife and increased the size of the knife boat to better suit AT:

1. We replaced the diamond blade for the histo jumbo diamond knife for semi-thin sectioning (~500 nm) by the diamond blade for the ultra 35º knife for cutting ultrathin sections (~50 nm).

Ultrathin sections have better quality than semi-thin sections: Unlike semi-thin sections, ultrathin AT sections with a thickness of 50-100 nm give better z-resolution and generate fewer folds. Ultrathin sections also improve the quality of subsequent analysis, as they adhere better to the support and provide higher ultrastructural resolution.

2. We increased the size of the knife boat to completely accommodate a coverslip on a flat support on the bottom of the trough at a 0º angle. The support for the coverslip is surrounded with a ditch to allow for user-controlled water draining. A hole in the bottom of the boat facilitates the insertion of a flexible tube attached to a syringe (Figs. 1C,D). Using the syringe, water can be added to or retracted from the boat without creating turbulence, which had presented problems while using previous methods (Fig. 1E). To attach the sections to support, water is drained slowly through the hole in the bottom of the boat. Slow water removal avoids turbulence and allows the sections to dry slowly, without wrinkles and folds.

Development • Accepted manuscript

This technique of attaching the section ribbons to support is the same for all types of support:

After generating the ribbon of the desired length (Fig. 1F), the water level in the boat is gradually lowered, by draining the water slowly through the hole in the bottom of the boat, either actively by aspiration with the syringe or passively, by allowing the water to drip through the tube without a syringe attached to it (Fig. 1G). When the water level reaches the height of the transfer support, the sections are manually aligned and arranged side by side (Figs. 1G,H, Movie 1). In our experience, this method greatly facilitates the ease and accuracy of section transfer for CLEM and volume EM. The same principle can also be used to collect histological sections for light microscopy analysis.

Multiplex immunolabeling

The immunolocalization of antigens is compromised by low Z-resolution for both standard and super-resolution light microscopy. Also, the co-localization of multiple antigens using antibodies generated in the same species is often problematic. The advantage of AT is that sections can be cut as thin as 40 nm, and images of several cycles of immunofluorescence labeling can be acquired individually and then superimposed. Physical ultra-thin sectioning can achieve a Z-resolution of a few tens of nanometers, which is an order of magnitude better than the Z-resolution of currently available super-resolution microscopes.

C. elegans is a transparent worm, which makes it an ideal model organism for light microscopy. Unfortunately, most specific primary antibodies available for C. elegans research are restricted to either mouse monoclonal or rabbit polyclonal. Thus, the co-localization of multiple antibodies on the same sections is often impossible. To demonstrate the power of the AT approach, we labeled serial sections of C. elegans larvae prepared by high pressure freezing (HPF-FS; see the Materials and Methods sample preparation section for details) on coverslip support. We stained/labeled the arrays of 100 nm thick sections with DAPI (nuclei, blue), anti-tubulin (red), and anti-erm-1 (EB1, green) in the first cycle and, after destaining, we stained for DAPI, anti-actin (red), and anti-vha-5 (green) in the second cycle (Figs 2A,B).

Both rounds of immunolabeling gave a strong signal, equally visible on individual sections and on the merge of all 25 sections (Figs 2A, B, Movie 2). Before relabeling and reanalyzing the samples, we extensively washed the sections between the labeling cycles, and verified that the previous labeling had been completely removed. We used the manual acquisition of the images, improved their alignment by developing a new Fiji macro, and superimposed all markers in one data set (Fig. 2C). DAPI nuclear staining served as a useful reference marker Development • Accepted manuscript to facilitate the alignment of the complementary signals provided by the two independent labeling cycles.

To monitor the quality and feasibility of EM acquisition, and to correlate the labeling data on sections after two labeling cycles, we imaged the corresponding ROIs with the SEM using the back-scattered electron (BSE) detector. The fluorescence signal could be identified quickly, and the overall ultrastructure was well preserved (Fig. 2D). To improve the accuracy of superposition, we used DAPI nuclear staining and examined the nuclear ultrastructure and other structural features of the sample (Fig. 2E).

Localization of ROI and volume reconstruction

In complex tissues, drawing conclusions about the ultrastructure from a single section or a small number of sections can be impossible. Volume reconstruction of cells and tissues using the SBF and FIB-SEM methods is an efficient way to investigate the relationship between cells and organelles in their complex 3D cellular context. For both approaches, however, the ability to precisely target the ROI remains challenging and requires endogenous landmarks [START_REF] Karreman | Intravital Correlative Microscopy: Imaging Life at the Nanoscale[END_REF]. To illustrate the advantages of an AT-based solution in targeting the ROI, we imaged the Drosophila ovarian chamber at stage 3. Our previous efforts with the ovarian chamber had already exposed us to the difficulties of localizing the ROI in this tissue, and had actually prompted us to develop our current AT method that enables more direct sampling and screening.

Drosophila oogenesis is often used to model cellular processes, such as cell division, cell migration, and stem cell differentiation [START_REF] Horne-Badovinac | Mass transit: epithelial morphogenesis in the Drosophila egg chamber[END_REF][START_REF] Fuller | Male and female Drosophila germline stem cells: two versions of immortality[END_REF][START_REF] Mclaughlin | Drosophila melanogaster Oogenesis: An Overview[END_REF]. During development of the ovarian chambers, the exterior follicular cells, the internal nurse cells, and the oocyte undergo numerous structural changes. Nurse cells, which supply the necessary nutrients and cell building material to the oocyte, do not accomplish abscission but remain interconnected through the network of ring canals. During development and cell growth, the initially small ring canals expand in diameter by about ten-fold [START_REF] Robinson | Morphogenesis of Drosophila ovarian ring canals[END_REF][START_REF] Hudson | Drosophila Kelch functions with Cullin-3 to organize the ring actin cytoskeleton[END_REF][START_REF] Mclean | Protein equilibration through somatic ring canals in Drosophila[END_REF][START_REF] Hudson | Actin Cytoskeletal Organization in Drosophila Germline Ring Canals Depends on Kelch Function in a Cullin-RING E3 Ligase[END_REF]. Imaging the ring canals (RCs) in their entirety using EM is difficult, mainly due to their large size and random distribution inside the ovarian chamber. On single sections, the RCs frequently appear as two spaced bars or brackets, and not as a circular structure, as in FLM micrographs. Based on our experience, Development • Accepted manuscript localizing the RCs with FIB-SEM or SBF-SEM tomography is problematic [START_REF] Loyer | Drosophila E-cadherin is required for the maintenance of ring canals anchoring to mechanically withstand tissue growth[END_REF].

We prepared the ovarian chambers using chemical fixation and flat embedding, sectioned, and then transferred the ribbons of sections to wafers. Our fast screening strategy rapidly localized the RCs within the arrays of sections at low magnification. Then, we assessed the general orientation of the organism in a flat embedded sample based on the anatomy of the sample (Fig. 3A). After sectioning, we localized the RCs based on known landmarks in the organism.

For this, screening the sections on wafer at low magnification resulted in the rapid identification of regions to analyze at higher resolution (Figs 3B,C). Initially, we collected ribbons of consecutive sections, aligned them side by side, and then tracked cellular features (or landmarks) along these ribbons (In Fig. 3B, each ribbon of sections is colored distinctly).

For faster screening, the sections were not screened in consecutive order but rather "horizontally," by scanning the middle section from each ribbon for RCs (Fig. 3C; the color of the frame corresponds to the color of a given portion of the array, selected in proximity to the white arrow at the panel B). Once a RC was recognized and located (Fig. 3C, orange), we could easily track the entire relevant sequence of sections by sequentially following the sections along the ribbon, using the reference section as an anchor. This simplifies the screening logic: One starts from the beginning of the relevant sample portion and then collects sections for as long as necessary. In this way, the localization of anatomical features is more efficient, rapid and precise, avoiding the acquisition of unnecessary data sets.

To localize the four ring canals that connect the future ovary and the nurse cells, we applied the above-described screening strategy. At low magnification, we identified ring canals in sections and as expected, the canals appeared only partially and did not resemble rings (Fig. 3D). After acquisition, the overlay of the sections allowed the modeling of the rings in their entirety, as well as the identification of the position of the ring on the cell surface (Movie 3).

The same set of sections can be reused to reconstruct the interaction between additional pairs of cells in the same ovariole, or the sections can be reimaged to analyze the structure of the RC in ovarioles of another developmental stage. Since the sections remain intact on the surface of the wafer, a ring of particular interest can be revisited using different acquisition parameters for more detailed study (Fig. 3E, Movie 4). The aligned data set can be further analyzed using different modeling and rendering programs, providing the precise identity of different cells, their spatial distribution, and interactions (Figs 3F, G, Movie 5). The resulting data can be further mined by targeted analysis of relevant 3D data portions (Fig. 3H, Movie Development • Accepted manuscript 5). Once the various stages of ovarian chambers and ring canal maturation have been collected and maintained on wafers, a library can be established. As in the case of ovarian chambers, any sample collected on wafers can be reanalyzed and reimaged when new questions arise. Arrays can be stored indefinitely and, with proper annotation, can be used by several different research groups. In addition to the examples presented here, this strategy can be applied to answer similar questions in other complex tissues (Fig. S1).

Correlative labeling of samples on wafers: fluorescence retention

We next extended our multiplex immunolabeling protocol and 3D screening with a reconstruction strategy, focusing on an organism bearing genetically encoded fluorescent markers. To retain the endogenous fluorescence en-bloc for AT, we tested several preparation protocols. Using high-pressure cryofixation and quick freeze substitution in acetonecontaining uranyl acetate with subsequent embedding in HM20 methacrylate resin gave the most satisfactory results. With this, we could visualize various fluorescent constructs, both enbloc and on section (Figs 4A-D), facilitating the localization of the ROIs in 3D. Moreover, searching for the fluorescence signal in a light microscope helped to localize the ROI faster, than does EM screening for desired morphological features. We processed C. elegans larvae using this protocol. In addition to the endogenous signal, the sections could be labeled with antibodies, providing additional data (Fig. 4C). Here again, the superposition of the fluorescent and the EM images was reliable and straightforward, making the direct CLEM reconstruction and analysis precise and unambiguous (Fig. 4D).

To further facilitate rapid correlation of ROIs and automate the acquisition process, we used the FEI 'MAPS' software. This provided automatic acquisition of both fluorescent and SEM images. We then superimposed these images, allowing the transfer of the same "geographical" parameters from one type of microscope to another [START_REF] Loussert Fonta | Correlative microscopy[END_REF]. Using the FEI CorrSight light microscope and MAPS software, large regions can be acquired automatically at low magnification (x10) with the separate single images stitched together to form one large file. This provides an overview of the sections on the support that is useful to identify the current location within the ribbons of consecutive sections. Imaging is then done using various objectives, imaging modes, and several fluorescence channels to localize the proteins of interest. For correlation, high-magnification (x63, x100), large-scale images of the fluorescent structures on the relevant sections can be acquired by tiling (Fig. 5A). After light microscopy, the project, containing the recorded light micrographs can be transferred to a (FEI) scanning electron microscope (Fig. 5B). Using Development • Accepted manuscript anatomical landmarks, all light micrographs can be aligned and overlaid on the map of the electron micrograph and high-resolution electron micrographs can be recorded [START_REF] Schwarz | Correlative light and electron microscopy using immunolabeled sections[END_REF].

To highlight the versatility of this technique, we analyzed the excretory canal of C. elegans.

This single cell organ is located in the pharyngeal region of the worm and, during development, sprouts four tubular extensions that run the length of the entire body of the animal (Fig. 5C; Kolotuev et al., 2010;[START_REF] Kolotuev | A pathway for unicellular tube extension depending on the lymphatic vessel determinant Prox1 and on osmoregulation[END_REF][START_REF] Sundaram | The Caenorhabditis elegans Excretory System: A Model for Tubulogenesis, Cell Fate Specification, and Plasticity[END_REF].

Fast HPF-FS preparation enabled the preservation of the fluorescent vha-1::GFP signal (Fig. 5D). This greatly facilitated the recognition of the ROI after transfer to SEM, as the image has a similar appearance in both modalities.

The MAPs program can facilitate the automatic acquisition of both fluorescence and SEM images, subsequently superimpose these images, and provide a result analogous to that of the "Google Earth" principle: First, images are acquired using low magnification (Fig 5B ) and then higher magnification is used (Figs 5E,F). Finally, "stitched images" are composed of several single images at a given resolution so that both the overall field of view and the individual images of particular ROIs can be reused for future analysis. Compared to analyzing sections on TEM grids, using arrays permits the analysis of larger sections. Thus, more individual samples can be simultaneously collected (Fig. 5E, n>30, yellow). The fluorescent signal is easily detected on sections, facilitating identification of animals in the desired orientation and/or developmental stage. After the appropriately oriented sample is identified via FLM, SEM using manual correlation, based on either the position of the sections; alternatively, a MAPS-mediated site localization can easily detect it. After the ROI has been found on a reference section, one can select the desired resolution for the EM acquisition (Figs 5G,H). Then the necessary ultrastructural data can be obtained by volume reconstruction (Fig. 5I). Within the tissue, the desired cell type can be ultrastructurally indistinguishable and/or difficult to localize. One example is the cell body of the early larval stage excretory canal, which is located in a densely packed environment and is not evident on routine ultra-thin sections. The fluorescence signal can aid in identifying a particular cell at the ultrastructural level (Fig. 5G). Once the fluorescence signal allows recognition of the cell of interest, EM analysis of the subsequent sections no longer requires correlation with the fluorescent data: The position of the cell on subsequent sections becomes evident (Fig. 5H).

The adult excretory canal extension is 100-1000 m long and 50-100 nm in diameter, making it difficult to image the overall volume of the canal arms using SEM. Only very rarely does Development • Accepted manuscript the fixed animal remain straight after processing, so orienting for longitudinal sectioning is a tedious procedure. Usually, only a small portion of the body can be correctly oriented.

Locating an ROI may require screening and acquisition of hundreds of carefully collected serial sections (Fig. 6A). Thus, it is complicated to precisely orient these extensions for longitudinal sectioning. The specimen often curves after fixation, and the recognition of the defragmented structure without the overall body context is often difficult, as the same structure appears differently and in a different location depending on the sectioning axis (Fig. 6B). In this case, the fluorescence served as a landmark to permit the easy recognition of the cells of interest. For this purpose, we labeled the excretory cell in green, and a random cell in its vicinity in blue. Superficially, these cells appeared identical, and if one had to distinguish them by a 'blind search', it might be impossible.

Here again, the combination of fluorescence-mediated screening with subsequent targeted EM acquisition facilitates precise orientation. First, the appropriate fluorescent image is found on section. It is useful to label the sections with the nuclear marker (DAPI, Draq5) to assess the orientation of the sample and to avoid confusion in repositioning the fluorescent signal on the EM data. Second, fluorescence data rapidly guides the analysis to the relevant ROI, after which the required number of sections or the desired volume of data is collected (Fig. 6C). In our example, the sections are spaced less than 200 nm apart, and the portion of the canal that connects the extension to the cell body is no longer seen from one section to another (Fig. 6D, arrow). Without fluorescence, the detached part would not have been evident for the recognition using only the EM data only (Fig. 6, arrows).

Our results demonstrate that when the endogenous fluorescence signal is preserved throughout the preparation, the random-sectioning approach facilitates identification of randomly-oriented samples. When combined with the MAPS correlation software, the structures of interest can be rapidly identified and imaged at high-resolution by EM, enabling fast-throughput analysis of a statistically significant number of organisms.

Development • Accepted manuscript

Discussion

This study presents an efficient array tomography (AT) procedure to facilitate the analysis of a single region of interest (ROI) in a whole organism on serial sections that is applicable for numerous biological samples. On serial sections, the ROI can be labeled with a fluorescent tag and correlated with the corresponding electron micrograph. The serial sections can then be quickly screened using fluorescence or directly SEM.

Besides the SEM, our method does not require additional expensive equipment for an EM lab or facility. The newly designed diamond knife we introduce can be used with any ultramicrotome, and its application is technically easier than performing classical ultrathin sectioning. In contrast, the approach using serial-block-face with focused-ion-beam scanning microscopy has a very high cost and limited accessibility. An alternative approach using the ATUMtome machine for automatic section collection from neuronal samples was recently demonstrated [START_REF] Kasthuri | Saturated Reconstruction of a Volume of Neocortex[END_REF]. Likewise, an alternative AutoCUTS method was recently used to collect sections of C. elegans larvae [START_REF] Li | Large scale threedimensional reconstruction of an entire Caenorhabditis elegans larva using AutoCUTS-SEM[END_REF]. However, this timeconsuming and more expensive strategy is only worthwhile when there is a requirement for thousands of sections, as is often needed for brain samples. In contrast, our direct AT method requires a modified diamond knife, while the ATUMtome strategy requires an expensive and specialized machine (available at selected facilities only).

For both the ATUMtome and our direct AT method, libraries of serial sections can be stored indefinitely and reanalyzed to address future research questions. For studies involving multiple organisms or organs, for which neither serial section TEM nor volume EM approaches would be efficient, our screening method provides an accessible solution. For questions that can best be answered by analyzing many different samples (e.g., wild-type vs. mutants), libraries of AT sections can be extremely useful. A single 2 x 4 cm wafer can contain all the necessary information to permit the analysis of a small organism quickly. Also, the researcher needs not to immediately acquire the entire sectional area at high resolution, as is done with block-face microscopy. High-resolution collection of only relevant data results in greater precision and shorter acquisition time. Less electronic data are stored, as information that is irrelevant to the initial question are not collected. Thus, statistically significant EM volume data can be acquired in a timely fashion. Further, compared to block-face methods, the sections generated by AT can first be imaged at low magnification for orientation, and Development • Accepted manuscript then the ROI can be imaged at higher resolution for a more detailed analysis. Direct CLEM, with or without automation, can provide an additional level of volume correlation and can simplify the EM identification of similar structures. The fields of cell and developmental biology, model organism analysis, and even clinical research could benefit from this technique to address such mechanisms as cell migration, tissue remodeling, and the fine structure of cell division and abscission (Fig. S1).

Most of the technical problems with the use of the existing SEM imaging fields and modalities relate to the lack of precision of automatic acquisition. Current software systems that support array tomography include Atlas, with Zeiss scanning electron microscopes, and MAPS, with FEI electron microscopes. With both software systems still in development, future improvements could facilitate the automation of robust and efficient serial section acquisition: For example, the acquisition process could be automated, and more solid and user-friendly data alignment algorithms could be provided.

In conclusion, our AT method can be used to resolve long-standing questions in cell and tissue biology that require 3D ultrastructural analysis, and to localize antigens in defined structures. This approach could be useful for both biological research and clinical analysis, as high-quality results can be efficiently achieved using equipment that is, to a large extent, already present in many imaging facilities.

Materials and methods

Sample fixation and processing

The samples were either chemically fixed or processed using the high-pressure freezingquick freeze-substitution method (Table 1).

Chemical fixation: The samples were fixed for 2h at ambient temperature in 2% (w/v) formaldehyde and 2.5% (w/v) glutaraldehyde dissolved in 0.1 M phosphate buffer, pH7.4 (EMS, 19340-72, 11650, 19344-10 respectively). They were then post-fixed in 1% (w/v) osmium tetroxide (EMS, 19152) and 1.5% (w/v) potassium ferricyanide in cacodylate buffer (EMS-26604 1A) for 1h at ambient temperature, followed by 1h incubation in 1% (w/v) tannic acid (Sigma-Aldrich, #403040). After extensive washes in water, the samples were incubated in 2% osmium tetroxide followed by 1h in 1% (w/v) uranyl acetate in water and dehydrated in an ascending series of ethanol followed by an ascending concentration of Development • Accepted manuscript EPON (EMS, 1420) in ethanol mixtures [START_REF] Starborg | Using transmission electron microscopy and 3View to determine collagen fibril size and threedimensional organization[END_REF]Kolotuev, 2014). Finally, the samples were flat embedded using a two-step procedure, and then polymerized at 60°C for 48h as previously described (Fig. 1A, Table 1; Kolotuev, 2014).

Cryo fixation quick freeze-substitution was performed using a modified version of the previously described method [START_REF] Mcdonald | Rapid embedding methods into epoxy and LR White resins for morphological and immunological analysis of cryofixed biological specimens[END_REF]. Carriers containing HPF immobilized samples were transferred to vials containing freeze substitution media (0.1% (w/v) uranyl acetate, 2% (v/v) H2O in acetone) and then placed in the freeze-substitution unit (AFS2; Leica Microsystems, Vienna), cooled to -140°C, and heated to -50°C over 8h. The samples were then infiltrated by ethanol and resin mixtures. After freeze-substitution, the samples were flatembedded, either in EPON embed 812 resin (EMS, 1420 morphology analysis), or in the monostep HM20 hydrophobic methacrylate resin (EMS, ref 14340) for the samples containing fluorescently-tagged proteins, or those to be used for immunofluorescent labeling (Table 1; Kolotuev, 2014).

Block preparation and serial sectioning

Blocks were trimmed to ∼100×200 µm cuboids using a trim 90-diamond blade (Diatome).

Sample block sides parallel to the knife were coated with a mixture of one part Weldwood contact cement (SADER® NÉOPRÈNE LIQUIDE) to three parts xylene. This mixture increased the adhesiveness of the sections to each other to produce a long ribbon of consecutive sections (>300). The resulting long ribbons of consecutive sections were subdivided into shorter ribbons (50-100 sections) and then aligned on support, side by side (Kolotuev, 2014).

To generate planar arrays of sections on support, we used a modified ultra 35º diamond knife with a custom-built boat (Figs 1C, D; Diatome, Switzerland; see Results for details) mounted on a Leica UC7 ultramicrotome (Fig. 1E; Leica Microsystems; Vienna, Austria). Ribbons of consecutive sections were generated with a length that varied according to the eventual goal of the AT procedure: 50-200 sections per ribbon, 50-150 nm thick. Each array of ribbons aimed for multi-labeling was transferred to a glass coverslip (VWR ref 631-1575), or, if it was dedicated for EM reconstructions and CLEM, to a silicon wafer (Ted Pella, No 16015).

Before use, glass coverslips were cleaned and coated with 0.5 % gelatin solution (EMS 16564) added to 0.05 % chromium potassium sulfate (Sigma-Aldrich, ref 243361), following a previously described procedure [START_REF] Micheva | Single-synapse analysis of a diverse synapse population: proteomic imaging methods and markers[END_REF]. We followed the manufacturer's Development • Accepted manuscript instructions to obtain a 1-2 nm carbon layer for the samples collected on the glass slide using CCU-0101 LV machine (Safematic GmbH, Switzerland). A thicker layer can compromise the quality of the SEM images. Image collection on the ITO coated slides should eliminate the need in this step (unpublished observation, IK, AM).

Round wafers were cut into 2x4 cm rectangles using glass breaking pliers 6'' (Ted Pella,ref 7295), cleaned, and glow discharged for 30 seconds using the current plasma HT of 15mA (Cressington 208, Cressington Scientific Instruments Ltd, UK). We did not carbon coat the samples on wafers since we did not detect any charging effect during the image acquisition.

Improving quality of arrays on sections: avoiding folds

Sectioning problems can compromise both the quality of the sample and the reliability of the subsequent analysis. The appearance of wrinkles on the surface of sections can ruin the entire AT data set. The impact of a fold on data analysis is difficult to quantify, mainly because only folds that affect the ROI are deleterious.

We distinguish two types of wrinkles that can appear on the surface of sections: (a) folds, causing global damage to the section, and (b) micro-folds, small wrinkles in limited areas of the sample surface. We could detect both types of wrinkles on sections, but frequently they affected only the excess embedding resin surrounding the sample. Also, on several sections, the wrinkles could be found on the sample surface, without affecting the ROI (Figure S2).

Even in very extreme cases of severe wrinkles, it is difficult to interpret their impact on data analysis. In Figure S3, four independent samples of a section are shown. Even if the samples No. 1, 2, or 3 have large folds or debris, this will not affect the data analysis. However, if the sample No. 4 had a fold in the bottom half, it would only be ruined if we were interested in its bottom half. If we wanted to analyze its upper half, then the wrinkle would not affect our analysis. The situation with micro folds is very similar if they happen to occur within the sample but not within the ROI (Figure S3). Thus, we decided not to quantify the number of folds but rather to characterize the conditions under which they appear and possible ways to prevent them.

Below we list factors that can lead to folds and wrinkles and how these can be minimised.

Development • Accepted manuscript

Block surface size and section homogeneity

The size of the sample surface appears to be essential: Smaller sections result in fewer wrinkles because there is less surface to generate wrinkles. Further, density differences between the sample can influence the quality of sectioning. Epoxy resin will create a copolymer within the biological sample, which has a different density than the empty resin.

Thus, excess embedding resin should be removed. Within homogeneous tissue, all excess resin is removed, leaving only a rectangular block of embedded tissue. For smaller nonhomogeneous samples such as C. elegans or Drosophila, it is difficult or impossible to remove the entire resin around the sample. Thus, we advise maintaining a similar amount of resin on each side of the sample.

For better stability of the ribbon of consecutive sections, it is important to trim the block as close to the sample as possible, shaping its surface into a rectangle, with the edge touching the knife measuring twice the width of the side edges at the side. A larger section surface produces more folds, while sections with a smaller surface tend to curve more. Curving of the ribbon of consecutive sections that is, section curling, is problematic, especially for ribbons that will be aligned side by side on support. With some experience, this problem can be overcome (Movie 1).

Section thickness

This parameter is as important as the block edge size and has a significant effect on the quality of the resulting sections. Ultra-thin (50-80 nm) sections attach better to the support and have fewer micro-folds compared to semi-thin sections (100-250 nm).

The speed of water retraction during section drying

We found the rate of water retraction to be critical for successful transfer of the ribbons of consecutive sections to the slide support. The support surface should be hydrophilic.

Excessive hydrophobicity of the support can cause folds in the sections. Thus, supports dedicated for SEM analysis (wafers, glass slides, or Kapton tape) should be carefully pretreated using a standard glow discharge procedure. Even with a very disorganized collection of sections, if the process of drying was optimal, there is a lower chance of the appearance of folds (Figure S4, can be zoomed in) and the development of microfolds in nonhomogeneous samples is much less likely to occur.

Occasional debris on the section surface can be difficult to eliminate. To reduce the occurrence of debris, the knife and the basin must be meticulously rinsed with distilled water Development • Accepted manuscript filtered with a 0.22 µm syringe filter. Coated glass slides must be carefully prepared, and wafers, thoroughly cleaned. Water filtering and knife cleaning before the generation of the sections can help to minimize the accumulation of dust and debris on the surface of the sections.

Sample treatment for FLM, EM, and CLEM

Multiplex fluorescent labeling: Labeling the arrays with a subset of different antibodies was carried out using a previously described procedure [START_REF] Micheva | Single-synapse analysis of a diverse synapse population: proteomic imaging methods and markers[END_REF]. Briefly, ribbons of consecutive sections on a glass coverslip were incubated in a humidity chamber with 50 mM glycine solution in TBS (Sigma-Aldrich, T5030) for 5 min., then with blocking solution containing 0.1 % BSAc (Aurion, 900-099) in TBS for 5 min. Specific primary antibodies mouse anti-actin (Cedarlane CLT 9001), mouse anti-tubulin (Invitrogen 322500), rabbit anti VHA5 (courtesy of Dr. Labouesse) and rabbit anti EB1 (Santa Cruz SC 15347) were diluted to 1:100 v/v in TBS and incubated overnight at 4°C [START_REF] Nicolle | Adaptation of Cryo-Sectioning for IEM Labeling of Asymmetric Samples: A Study Using Caenorhabditis elegans[END_REF]. Secondary antibodies Cy2 anti-mouse, (Abcam Ab-6944) and Cy3 anti-rabbit, (Abcam Ab-6939) were diluted to 1:100 v/v in TBS and incubated at ambient temperature for 2h. Samples were mounted using the VECTASHIELD ® Antifade Mounting Medium (Vector Laboratories, H1200) and imaged the same day. DE staining and relabeling of the arrays was done according to the experimental requirements (Micheva and Smith, 2007). For removing the bound antibody complex, a mixture of 0.2 M NaOH with 0.02 % SDS was applied to the samples for 20 min. followed by extensive washing cycles. Before relabeling using the same method as the initial labeling, the samples were dried in an oven at 60ºC for 20 min.

After completion of fluorescent image acquisition, the samples were extensively washed in double distilled water, dried and contrasted for 15 min. in a 4% uranyl acetate solution followed by a 10 min. incubation in Reynolds lead citrate solution.

For SEM acquisition, samples prepared using the high-contrast procedure were directly observed without further manipulation. For all other acquisitions, the sample contrast was increased using the same parameters as for CLEM experiments.

Development • Accepted manuscript

Fluorescence on sections

Multiple fluorophores and fluorescence preservation procedures for CLEM have been developed [START_REF] Nixon | A single method for cryofixation and correlative light, electron microscopy and tomography of zebrafish embryos[END_REF][START_REF] Kukulski | Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision[END_REF][START_REF] Watanabe | Protein localization in electron micrographs using fluorescence nanoscopy[END_REF][START_REF] Peddie | Correlative and integrated light and electron microscopy of in-resin GFP fluorescence, used to localise diacylglycerol in mammalian cells[END_REF][START_REF] Mcdonald | Rapid embedding methods into epoxy and LR White resins for morphological and immunological analysis of cryofixed biological specimens[END_REF][START_REF] Burette | Knowing a synapse when you see one[END_REF][START_REF] Johnson | Correlative in-resin super-resolution and electron microscopy using standard fluorescent proteins[END_REF][START_REF] Kopek | Diverse protocols for correlative super-resolution fluorescence imaging and electron microscopy of chemically fixed samples[END_REF].

Though efficient for many types of proteins, the level of fluorescence of genetically encoded markers on sections varies from construct to construct. Although we did our best, we could not predict under which preparation conditions it would be preserved at all, and to what extent. For some constructs, fluorescence remains visible inside the block and on sections even without using an anti-bleaching solution. For other samples, even if we were not able to detect signal inside the block the fluorescence was often visible in the sections. For these samples, we could preserve the fluorescence signal for at least one year after the preparation.

For some samples, however, although the fluorescence signal was initially present, we were not able to preserve the fluorescence in blocks or on section. A systematic analysis of fluorescence preservation as a function of a protein type (transmembrane, cytosolic, secretory, etc.) could further facilitate our understanding of the problem of fluorescence preservation. In the future, we hope to combine our observations with those of other groups that have experience with fluorescence retention using other species and fluorescent constructs to understand the factors influencing the fluorescence retention and to develop a robust preparation protocol.

Intuitively, thicker sections should provide a better fluorescence signal when genetically encoded markers are used. However, there is a thickness-to-stickiness relation, in addition to the loss of EM details in thicker sections: Thinner sections had better adherence to the surface. Also, photo-bleaching was even more troubling. Thus, to find a reliable way to quantify the "signal emission" from sections of different thickness might require complicated math/physics calculations, which would likely not be applicable for all types of fluorescent constructs. However, the overall preservation of sectional fluorescence is a concern because, after fixation, the signal is not linear and varies with the type of construct.

Development • Accepted manuscript

Image acquisition

Fluorescent microscopy acquisition and image treatment

Wide-field fluorescent image acquisition was made using a Leica DMIRB inverted microscope or a Leica DMRXA upright microscope equipped with a 20x/0.4 NA plan, a 63x/0.7 NA PL Fluotar or 100x/0.7 air and 1.4 NA PL APO oil objectives, and a Coolsnap HQ2 CCD camera (Photometrics, Tucson, USA).

Confocal fluorescent images were taken using a Leica TCS SP8-SMD confocal microscope equipped with both 63x/1.4 NA Oil HC PL APO and 40x/1.3 NA Oil HC PL APO objectives (Leica Microsystems, Mannheim, Germany). Fluorescence signals were collected sequentially through Leica photon-counting hybrid detectors to increase the signal-to-noise ratio.

For light microscopy tile acquisitions, the CorrSight light microscope (FEI Company Munich, former Till Photonics) running the MAPS software (Thermo Fischer, former FEI Company, Eindhoven) was used [START_REF] Loussert Fonta | Correlative microscopy[END_REF]. The microscope is equipped with Zeiss objectives (Zeiss, Munich): 5x NA 0.15, 20x NA 0.8, 40x NA 0.9 and 40x NA 1.3 oil immersion; and DAPI, 488 nm, and mCherry filters. The light micrographs, bright field, and fluorescence were recorded with MAPS and stored as an aligned package in a project.

For SEM acquisition, we used a JEM 7800F with retractable backscattered electron detector (JEOL, Japan), with the following parameters: Accelerating voltage: 8 kV, Probe current: 1.6 nA, WD: 5mm, dwell time: 10 µs/pix with 4096 x 4096 frame size. For MAPS-related experiments, FEI Quanta FEG 250 SEM (Eindhoven, Holland) was used with the following parameters: Accelerating voltage: 10 kV, Probe current: 0.8 nA, WD: 7mm, dwell time: 30 µs/pix with 4096 x 4096 frame size. The MAPS projects were transferred to the Helios 650 FIB-SEM (Thermo Fischer, former FEI Company, Eindhoven, Holland). A reference image was taken with SEM. Light micrographs were then aligned to this image and used to find the region of interest to be analyzed at high resolution using SEM [START_REF] Loussert Fonta | Correlative microscopy[END_REF].

Development • Accepted manuscript

Alignment and reconstruction

To align the fluorescent stacks of sections, we developed the computational volumetric analysis protocol, based on Fiji and ICY programs (Schindelin et al., 2012;[START_REF] De Chaumont | Icy: an open bioimage informatics platform for extended reproducible research[END_REF]. The co-registration procedure will depend on the data set. Using Fiji software, we selected C. elegans embryos in each stack using an automatic threshold, and signals located outside of the chosen area were deleted. The first two steps can be done automatically using a rigid registration. Indeed, as images come from the same array with a microscope always using the same settings, the displacements between two images are rotation and translation. In the image processing community, numerous algorithms aim to solve rigid registration task. We used the "Register Virtual Stack Slices" available on Fiji (http://imagej.net/Register_Virtual_Stack_Slices). This method uses the SIFT feature to find corresponding regions in images pairs. To facilitate the use of this registration algorithm for array data, we developed a Fiji macro that runs in three steps:

The co-registration procedure will depend on the data set.

(1) "Split Stack": this step splits the array hyperstack into various image sequences, one sequence for each staining of the FLM data. The sequences are stored in output subfolders C1, C2 … The operator need only to specify the number of channels (i.e., staining) in the stack.

(2) "Register Reference": this step calculates the registration transformation matrices using the reference staining. Usually, the DAPI staining is used as a reference for FLM. The operator needs only to provide the reference channel directory (C1 or C2 or …), and the output directory where the transformation matrices will be stored. This directory is already created by the macro, the operator can simply select it. The algorithm settings are (a) reference channel: channel used to estimate the transformation. (b) Registered image size: size of the output image canvas. It should be larger than the original image for all of the images that have been transformed to fit into the canvas.

(3) "Apply transformation" This step applies the transformations calculated in (2), to all of the staining series (C1, C2, …) Depending on the image content, the registration algorithm may fail to locate any corresponding regions between the separate array images. This can be resolved using pre-filtering of the reference staining before registration (i.e., between Step (1) and Step (2)). The operator can then apply a filter to enhance the brightness, the contrast, or the gradient of the array. This optional step had not been implemented in the macro since it is application dependent. The use Development • Accepted manuscript of the macro is similar to registering LM and EM array, but for the EM alignment, a single channel C1 is used. EM / FLM registration: This registration cannot be done automatically since the resolution, and the content of images are different. Thus a supervised registration method is required. For this, landmarks that match the reference regions on FLM and EM micrographs are used with the ICY plugin ec-CLEM [START_REF] Paul-Gilloteaux | eC-CLEM: flexible multidimensional registration software for correlative microscopies[END_REF]. We co-registered arrays obtained with the macro described above and registered the first image of the C1 FLM array and the first image of the EM array with ec-CLEM. A detailed tutorial of ec-CLEM is available at http://icy.bioimageanalysis.org/plugin/ec-CLEM. ec-CLEM generates a file containing the registration transformation matrix. The operator can then apply this transformation to all of the FLM arrays with ec-CLEM.

For the SEM data alignment and 3D modeling, we used IMOD, Fiji, and Photoshop software (Schindelin et al., 2012;Kremer et al., 1996). To achieve a TEM-like appearance, the SEM micrographs were contrast-inverted using IMOD or Adobe Photoshop. HPF-QFS: high-pressure freezing-quick freeze substitution. OTO: enhanced contrast procedure. After fixation, the embedding medium differs: hydrophilic acrylic resin is preferred when there is a need to preserve antigenicity and fluorescence signals; hydrophobic epoxy resins are used when the ultrastructure is the main goal. The microscope used depends on the imaging goal. Note that the cells surrounding the labeled cells appear identical, and in the absence of the superposition, it could be impossible to recognize the cell of interest. Scale bar, 5 µm.

Development • Accepted manuscript

FiguresFigure 1 ,Figure 4 .F:

 14 Figures

Figure 6 .

 6 Figure 6. AT of nonlinear sample

Table 1

 1

			Volume
	Steps	Multiplex labeling	reconstruction	CLEM
	Fixation	HPF-QFS	OTO	HPF-QFS
	Embedding resin	Acrylic	Epon	Acrylic
	Sectioning and
	transfer	Coverslip	Silicon wafer	Coverslip or silicon wafer
				Direct observation or 1° and
	Stain	1° and 2° Abs	Direct observation	2° Abs
	Microscope type	widefield /confocal	FEG-SEM	widefield /confocal FEG-SEM
		0.2 M NaOH, 0.02
	Destaining	% SDS	NA	NA
	Restain	1° and 2° Abs		UAc/Pb
	Reconstruction		Fiji/3Dmod

Table 1 .

 1 A simplified summary of AT procedures with the changes in the flow necessary for three preparation strategies.

Acknowledgements

We thank the following colleagues for their help: Kristina Micheva, Gareth Griffiths, Lucy O'Brien, Till Matzat, Paola Moreno-Roman, and Karen Lunde for critical reading of the manuscript and fruitful discussions. We thank Loic Joanny (SCANMAT, Université de Rennes 1) for the help with SEM, Jean Daraspe for sharing the 3Dmod script, Willy Blanchard for the macro imaging and Tom Garber for the help in 3D image rendering. The project was supported by the UMS Biosit University of Rennes 1/CNRS/Inserm "Risky projects" grant.

Development • Accepted manuscript

C.

A drawing of a modified AT-boat diamond knife enlarged and flattened to facilitate sectional collection on glass or wafer support. As part of the modifications, we carved water evacuation ditches (black arrows) and drilled a hole (white arrow) in the bottom of the boat.

D.

A syringe needle is connected to the hole and covered with double-sided sticky tape. A catheter tube connects it to a syringe. The internal diameter of the needle determines the speed of water retraction. Thus, water can be retracted, or drained, from below the water surface, either actively, using the syringe, or passively, by allowing water to drip at a steady pace through the catheter, leaving both hands of the operator free to align sections on the support.

E.

A complete description of tasks necessary for array generation: the modified AT diamond knife is connected to a syringe and installed on the ultramicrotome. Before sectioning, the support (a coverslip or wafer) is entirely submerged in water and lies parallel to the water surface.

F. A ribbon of consecutive sections floating inside the modified boat while sectioning (yellow arrow). A rectangular wafer lies on the bottom of the boat. Scale bar, 1 cm. G. After sectioning, the ribbons of consecutive sections are aligned side by side, and water is gradually drained from the boat. After either assisting with a syringe or allowing the water to drain away completely, the support remains at the bottom of the boat until any remaining moisture has evaporated completely from its surface (Movie 1).

H. Aligned ribbons

B.

Immunolabeling the same sections after eluting the initial labeling, washing, and relabeling with a second set of markers (actinred, VHA-5green, DAPIblue). DAPI nuclear staining served as an alignments reference to combine the first and second datasets.

Development • Accepted manuscript

C. Merged image of both labeling rounds, labeling all five markers (tubulinred, EB1green, actinred, VHA-5green, DAPIblue).

D. An inverted contrast SEM image of one section from the middle of the array that corresponds to the labeled region (vulva and embryos). The mother cells that initiate such clusters by undergoing multiple rounds of cell division are randomly scattered throughout the spermatheca. Our AT approach with "blind" sectioning greatly facilitates locating the mother cells compared with standard sectional analysis. Black box encircles a potential ROI. Scale bar, 10 µm.

C. Close-up of two pairs of precursor cells highlighted in blue and in yellow (Box in Panel G).

After a single section containing the ROI has been identified by "horizontal" screening (Figure 4C), the sections sequentially upstream and downstream of the initially identified section (i.e., "vertical") within the same ribbon of consecutive sections can be analyzed. Scale bar, 2 µm.

D.

A sequence of serial images obtained from the blue area (Panel G). None of the sections was affected either by the folds or the microfolds. Scale bar 2 µm.

Development • Supplementary information

Supplementary Movies

1. Sectioning. The time required for sectioning depends on the desired number of sections and the speed of sectioning. We used a rate of 0.8-1.0 µm/sec.

2. Alignment of ribbons of consecutive sections. During the first step, one or several ribbons can be generated. Multiple ribbons can be arranged side by side using gentle movements of an eyelash attached to a thin stick.

3. Water draining. Water retraction can be achieved by active aspiration through the syringe and by passive dripping through the plastic catheter. The entire procedure takes a couple of minutes at the most. 4. Readjustment of ribbons. While draining the water from the basin, the aligned ribbons often become misaligned. While water remains on the surface of the wafer, we can readjust the ribbons until the surface starts to dry. 5. Gradual evaporation of water from the surface of the wafer. We consider this step to be critical to avoid folds in the sections. The visual zoom-in on a drying portion of the ribbon illustrates this concept well. The length of time for drying will largely depend on the size of the surface area and the surrounding environmental conditions. In practice, drying takes 10 to 30 minutes for a wafer of 2x4 cm. // split the channels to individual files for(c =1 ; c <= channelNumber ; c++){ File.makeDirectory(outputDir + "C" + c); } splitStackToImages(channelNumber); } else if(macroChoice == "Register Reference"){ // get parameters Dialog.create("Register tomo fluo"); Dialog.addChoice("Reference channel", newArray("C1", "C2", "C3", "C4", "C5", "C6")); Dialog.addNumber("Registered Iage Size", 1024); Dialog.show(); var referenceChannel = Dialog.getChoice; var imageMaxSize = Dialog.getNumber; var outputDir = getDirectory("Select the Directory containing the Cx input images folder"); // register reference stack File.makeDirectory(outputDir + "reg_C1"); File.makeDirectory(outputDir + "reg_C2"); File.makeDirectory(outputDir + "reg_C3"); File.makeDirectory(outputDir + "reg_C4"); File.makeDirectory(outputDir + "reg_C5");


Issam El Gmati

Pierre Francois Calmon

Ali Boukabache

Patrick Pons

Hatem Boussetta

Mouhamed Adel Kallala

Kamel Besbes

RF MEMS continuous reversible variable inductor based on a microfluidic network

In this work, RF MEMS continuous reversible variable inductor has been fabricated by using microelec-tronic technology and lamination process. We review, evaluate and compare this variable inductor with other work. The proposed inductor is a dual circular coil and has an inductance of few nH. The fundamental idea is to place a liquid droplet between the metal turns of a coil in order to modify the capacitive/resistive coupling between metal tracks and hence to change the stored magnetic energy. The SU-8 resin was used to realize the microfluidic channels and Au as metallic tracks.

To prove the reversibility of the inductor, two cases were studied: filling and emptying of channels. The tuning range of the inductance is approxi-mately 107 % at 1.6 GHz, making these devices very suitable as building blocks in many RF applications.

Introduction

Tunable inductors are important building blocks in many radio-frequency (RF) applications such as voltage-con-trolled-oscillators, tunable filters and antennas, and adaptative impedance matching circuits [START_REF] Yoshihara | Wide tuning range LC-VCO using variable inductor for reconfigurable RF circuit[END_REF]Ito et al. 2005;[START_REF] Lucyszun | High performance MMIC Narrow band filter using tunable active inductors IEEE Microwave and Millimeter-wave monolithic circuits Symposium[END_REF]. Most of these applications required a high quality factor and wide tuning range. Many techniques were developed to tune the inductance value, most of which rely on the variation of the distance between two inductors to change the mutual coupling, as in [START_REF] Lubecke | Self-assembling MEMS variable and fixed RF inductors[END_REF][START_REF] Tassetti | New tunable RF MEMS microinductors design[END_REF][START_REF] Zine-El-Abidine | Tunable radio frequency MEMS inductors with thermal bimorph actuators[END_REF]. A bus of switches was used in [START_REF] Zhou | A monolithic variable inductor network using microrelays with combined thermal and electrostatic actuation[END_REF] to select a certain number of turns of a spiral inductor resulting in a set of selectable discrete values of inductance. A magnetic core sliding inside a solenoid inductor was used in [START_REF] Sarkar | Microassembled tunable MEMS inductor[END_REF]. In this paper we present an RF MEMS continuous reversible inductor with a large tuning range. The variability of the inductance varies according to the liquid position, which can be increased from zero to the total spiral length (l). RF the MEMS continuous reversible inductor is compared with each other in relation to some parameters such as tuning range, quality factor, self resonance frequency, excitation method and tuning method.

Principe of variable inductor

The proposed inductor, represented in Fig. 1, is a dual circular coil designed to have an expected inductance value of a few nH. The metallic (Au) lines of the inductor have 20 lm widths, spaced by 10 lm, while the inside and outside coil diameters are around 600 and 1,200 lm respectively. The dual spires must be fabricated inside the 70 lm width channel. When liquid moves along this channel, it changes the capacitive coupling between inter-spires leading to reduction of the stored magnetic energy, and hence scales down the inductance. The inductance value varies according to the liquid position, which can be increased from zero to the total spiral length (l). The principal technological steps used to fabricate our devices is represented in Fig. 2 and show the sequential operations allowing to obtain thick gold spiral inductors deposited on a glass substrate. The technological process requires six masks level.

Gold spiral inductors

A Ti/Au (500/500A˚ ) seed layer was homogeneously evaporated on the top side of a 500 lm thick glass substrate (Fig. 2a). A positive photoresist (AZ 4,562) was subsequently patterned in order to create the electroplating mould (thickness &5.5 lm) (Fig. 2b). Then, 2-lm-thick gold lines, was electroplated into the resist mould and the latter was finally removed and the seed layer was chemically etched (Fig. 2c).

Microfluidic channel

In order to obtain channels with subsequent high aspect ratio, we have used the photosensitive resin SU-8 as a structural material. A 10 lm thick film was firstly deposited by spin coating 4 ml of SU-8 3,005 at 900 rpm with 900 rpm s-1 acceleration for 30 s. The resist was then soft baked during 1 min at 65 C, ramped at a rate of 10 C min-1 up to 95 C, held at 95 C for 7 min and finally cooled down to room temperature. The resist was then exposed to UV light (90 mJ cm-2), and post-baked during 1 min at 65 C and 3 min at 95 C to cross-link the photoresist. Finally, the resist was developed in PGMEA developer (Propylene Glycol Methyl Ether Acetate) during 9 min (Fig. 2d). A photosensitive SU-8 dry film, 10 lm in thickness, was then laminated on the glass wafer. A lamination pressure of two bars and a temperature of 65 C were used to ensure perfect adhesion between SU-8 films. This new glass/SU-8 wafer is used as a classical wafer and the laminated SU-8 layer can be patterned using classical UV equipment and appropriate mask. Access holes are then opened into SU-8 layer in order to reach embedded channels (Fig. 2e). The alignment between the different layers are obtained classically thank to gold alignment marks fabricated during the first steps. This process steps are described in detail in [START_REF] Fulcrand | Development of a flexible microfluidic system integrating magnetic micro-actuators for trapping biological species[END_REF]; [START_REF] Abgrall | A novel fabrication method of flexible and monolithic 3D microfluidic structures using lamination of SU-8 films[END_REF].

Microfluidic access

Microfluidic access channel was then performed by the same lamination technique. A second photosensitive SU-8 dry film (10 lm in thickness) was laminated and patterned on top of the previously structure in order to realize the access channel walls (Fig. 2f). Finally, a 10 lm thick SU-8 dry film was laminated and patterned to close the access channel and create access holes for the fluid (Fig. 2g).

Fluidic connexion

In order to perform fluidic connection between micro-pipe and fluidic channels, polydimethylsiloxane (PDMS) disks were sealed at the microchip inlet and outlet using photo curable glue (Fig. 2h-i). Figure 3 shows the successful integration of circular inductance by combining standard electroplating technique and lamination process. A general view of a gold circular micro-inductor is given in Fig. 3a). The ''inductance channel'' in SU-8 presents an excellent alignment with micro-inductance (Fig. 3b). Access holes are opened in the SU-8 layer (Fig. 3c), a second photosensitive SU-8 dry film is laminated to realize the access channel walls (Fig. 3d), PDMS disks were sealed at the microchip (Fig. 3e) and the complete microsystem is represented in Fig. 3f).

Experiments results

The S-parameters were measured from 50 to 20 GHz with an Agilent 8510 network analyzer and Microtech GSG probes Channels were filled with salt water using a syringe and molded PDMS blocks achieve the fluidic connection. The set-up is shown in Fig. 4. The inductance L and the quality factor Q were calculated by using the following equations. During the RF measurement, fluid was manually moved by connecting a syringe to one of the external tube. Figure 5 specifies the different liquid positions of salt water inside the channel used for the RF characterization. To prove the reversibility of the inductor, two cases were studied: filling and emptying of channels. As illustrated in Fig. 6a, the maximum quality factor is close to 12 and is obtained at 1.6 GHz without liquid in the channel. At this frequency the minimum quality factor is around five when the entire channel is filled with salt water. The measured inductance at 1.6 GHz can be tuned from 5.4 down to 2.6 nH (Fig. 6b). Once the channels are filled with salt water, it makes the handling of emptying channel through the same positions of fluid positions. Figures 7 show respectively the variations of the measured results (I) versus saturated salt water positions (filling channels) at a frequency of 1.6 GHz. Most of the variation occurs for different liquid positions as was expected from the measured results 2 (emptying channels). It can be seen in Table 1, that at 1.6 GHz the tuning range of the inductor is above 107 % and the Q-factor is between 5 and 12. The measured results (II) differ slightly from the measured results two. For the inductance (L) and the quality factor (Q) value, the differences are lower than 2 and 3 % respectively and show in Fig. 8. The effects of the droplet location in the microfluidic channel can cause this slight difference; in fact the droplet may either be in direct contact with the solid metal or it could form a capacitive contact through a thin layer. When the droplet or slug is removed from the RF signal path, the inductance presents very low loss that is dominated by the transmission line loss itself. These results demonstrate reversibility of the inductance.

Comparison with other work

A few variable inductors are already documented in literature in order to obtain tuned inductors. In [START_REF] Zhou | A monolithic variable inductor network using microrelays with combined thermal and electrostatic actuation[END_REF], inductance variability was accomplished through the use of micro relays. While large inductance changes were possible, the values were discrete and the maximum quality factor was 3.3. A continuous variation of the inductance can be achieved by altering the mutual inductance of closely coupled coils by changing the distance between them [START_REF] Lubecke | Self-assembling MEMS variable and fixed RF inductors[END_REF]; by this way, a tuning range of 18 % was achieved with a quality factor of 12. In Zine-El-Abidine et al. (2003), two bimorph, inductors were connected in parallel with the outer inductor fixed. The tuning range was 8 % and the quality factor is not exceeding 9 at 5 GHz. Electrostatic actuation was also used to change the gap between two inductors [START_REF] Tassetti | New tunable RF MEMS microinductors design[END_REF]) the tuning range was closed to 50 %, but the quality factor was as low as three. Another technique consists to use a spiral inductor with a metal shield above it [START_REF] Sugawara | Widerange RF variable inductor on Si CMOS chip with MEMS actuator[END_REF])it results with 38 % of inductance variation when the separation between the inductors is changed. A spiral inductor fabricated with a flexible metal film [START_REF] Yokoyama | On-chip variable inductor using microelectromechanical systems technology Japan[END_REF]) can be used to change the spiral height, resulting in an inductance variation with a 10 % maximum tuning range, and a 7.2 for the quality factor. In [START_REF] Chang | A tunable RF MEMS inductor on silicon incorporating an amorphous silicon bimorph in a lowtemperature process[END_REF], tunable inductor was based on the bi-morph effect by using amorphous silicon deposited on aluminum layers: a 32 % tuning range with high inductance (5.6-8.2 nH) is achieved by the application of an electric potential. In [START_REF] Kim | Tunable MEMS spiral inductors with optimized RF performance and integrated large-displacement electrothermal actuators[END_REF], the inductance of a spiral inductor is tuned by controlling the relative position of a magnetically coupled short circuited loop. The tunable inductor shows a measured *2:1 inductance tuning ratio over 25 GHz with a maximum quality factor that varies between 10 and 26. The main drawbacks of these tunable inductors are generally the low tuning range and quality factor. Table 2 presents the various studies cited in literature. They can be characterized by the fre-quency of work, the quality factor and the tuning range. In this work, we present a continuous reversible variable RF inductor. The tuned inductor is obtained through the salt water flowing inside the SU-8 micro-channel. Experiments give very interesting results compared to those obtained by the different approaches experienced up to now (Table 2): we have obtained inductance variation from 5.4 to 2.6 nH with a tuning range of 107 % at 1.6 GHz frequency by using liquid circulation in 3D microfluidic RF MEMS developed in our laboratory. This approach gives the proof of potentialities offered by liquids to modify the RF behavior of inductors. We have fabricated and evaluated RF MEMS continuous reversible variable inductor using photosensitive lamination process and MEMS technology. Experiments give very interesting results compared to those obtained by the different approaches experienced up to now. The tuning range of the inductance is approximately 107 % and Q-factor is approximately 12. To prove the reversibility of the inductor, two cases were studied: filling and emptying of channels. Variable inductor can be exploited in the design of components, subsystems and/or systems in mobile communication systems to achieve new compatibility and improved electrical performance over a wide range and frequency, smaller size, lower power consumption and less weight.

Fig. 1

 1 Fig. 1 Top view of the 3D model structure of the proposed variable inductor

Y

  ind is the two-port admittance matrix of the de-embedded inductor. De-embedding was performed by using the formula where Y meas and Y open are the admittance matrices of the measured device and the open pattern, respectively. The scattering parameters (S-parameters) are measured at different input powers and the interconnect lines with open standard.

Fig. 3

 3 Fig. 3 Photographs of the studied devices during the fabrication process

Fig. 6

 6 Fig. 6 Measured results 1 (filling channels) a quality factors and b inductor for different salt water position

Fig. 8

 8 Fig. 8 Comparison between measured results 1 (filling channels) and measured results 2 (emptying channels) characteristics of the variable inductance at 1.6 GHz for different salt water positions

Table 1

 1 Comparative measured resultsTable2Comparative result of various works presented in literature

	6 Conclusion