Marek Cyrklaff

Michael Lanzer
email: michael_lanzer@med.uni-heidelberg.de

Nicole Kilian

Philipp Henrich

Kilian

using different electron tomographic approaches

Keywords: Cryo-electron tomography, Maurer's cleft, protein trafficking Cryo-electron tomography, Maurer's cleft, protein trafficking Abbreviations CEMOVIS -Vitrified sections, Cryo-ET -cryo-electron tomography, MC -Maurer's cleft, PM -Plasma membrane (of erythrocyte), PV -Parasitophorous vacuole, PVM -Parasitophorous vacuole membrane, RBC

3D analysis of the Plasmodium falciparum Maurer's clefts using different electron tomographic approaches.

Introduction

Malaria remains a major infectious disease causing over 500 million clinical cases and over one million deaths annually [START_REF] Snow | The global distribution of clinical episodes of Plasmodium falciparum malaria[END_REF]. The high morbidity and mortality of malaria relates to the intra-erythrocytic stages of P. falciparum and their ability to sequester in the deep vascular bed of inner organs -resulting in a broad range of pathologies including localized hypoxia, inflammatory reactions and the syndromes associated with maternal and cerebral malaria [START_REF] Marti | Signal mediated export of proteins from the malaria parasite to the host erythrocyte[END_REF].

By propagating within human erythrocytes, the parasite exploits a unique ecological niche. Human erythrocytes are highly differentiated cells, designed for the key purpose of distributing oxygen within the body. Accordingly, they lack many cellular functions. For example, they are anucleated and transcriptionally-and translationally-inactive; they lack a secretory apparatus; they possess only a limited repertoire of solute and ion transporters and they are readily removed from the circulation when damaged. To survive within such a hostile environment, the parasite has to remodel its adopted host cell by exporting several hundreds proteins into the infected erythrocyte [START_REF] Hiller | A host-targeting signal in virulence proteins reveals a secretome in malarial infection[END_REF]. Some of these proteins alter the biomechanical properties of the host erythrocyte. Other exported proteins create, within the erythrocyte plasma membrane, new permeation pathways for solute uptake and ion regulation [START_REF] Kirk | Membrane transport in the malaria-infected erythrocyte[END_REF][START_REF] Ginsburg | The permeability properties of the parasite cell membrane[END_REF]. A third group of proteins renders the infected erythrocyte cyto-adhesive or contributes through other means to immune evasion and modulate the severity of the infection [START_REF] Hiller | A host-targeting signal in virulence proteins reveals a secretome in malarial infection[END_REF].

To deliver proteins to different destinations within the host erythrocyte, such as the cytoplasm and the plasma membrane, the parasite has to generate a protein export machinery within the host cell -outside the boundaries of its own plasma membrane. A central structural component of this 'extracellular' protein export system are Maurer's clefts [6]. Maurer's clefts were discovered more than a century ago as stipplings within the cytoplasm of Romanowsky-stained infected erythrocytes [START_REF] Maurer | Die tupfelung der wirtszelle des tetrianaparasiten[END_REF]. Electron microscopy identified the Maurer's clefts as stacked unilamellar membrane profiles that appeared electron translucent, and which were frequently accompanied by vesicle-like structures [START_REF] Aikawa | Plasmodium: the fine structure of malaria parasites[END_REF]. Other membrane profiles seen on single ultra-thin sections include whorls, loops and long tubules extending from the parasitophorous vacuolar membrane, which separates the parasite from the cytoplasm of the host cell. These early micrographs provided only a two-dimensional and thus limited view of the Maurer's clefts, leaving it up to speculation as to whether the different membrane profiles observed in single ultra-thin sections are independent compartments or whether they are connected with each other. Recent studies have attempted to address these issues. Wickert et al. [START_REF] Wickert | Maurer's cleft organization in the cytoplasm of Plasmodium falciparum-infected erythrocytes: new insights from threedimensional reconstruction of serial ultrathin sections[END_REF] proposed a spatial model of Maurer's clefts on the basis of 3-D reconstructions of consecutive serial sections. According to their model, Maurer's clefts are much more elaborate than is evident from single ultra-thin sections, spanning much of the distance between the parasite and the erythrocyte plasma membrane. Moreover, structures that on single sections appeared separated from each other merged into a continuous membrane network of intertwined tubular and disk-like profiles in the 3D-reconstructions. Electron tomograms confirmed the complex morphology of the Maurer's clefts [START_REF] Hanssen | Electron tomography of the Maurer's cleft organelles of Plasmodium falciparum-infected erythrocytes reveals novel structural features[END_REF][START_REF] Tilley | The Twists and Turns of Maurer's Cleft Trafficking in P. falciparum-Infected Erythrocytes[END_REF], depicting a single cleft as a flattened, highly buckled sack-like bladder, decorated with nodules and having surface crevices and convoluted edges. Electron-dense tether-like structures seem to connect the Maurer's clefts to the erythrocyte membrane-skeleton [START_REF] Tilley | The Twists and Turns of Maurer's Cleft Trafficking in P. falciparum-Infected Erythrocytes[END_REF], consistent with biochemical findings showing that proteins of the Maurer's clefts interact with components of the erythrocyte cytoskeleton [START_REF] Blisnick | Pfsbp1, a Maurer's cleft Plasmodium falciparum protein, is associated with the erythrocyte skeleton[END_REF].

Even though these studies provided novel insights into the fine structure of the Maurer's clefts (reviewed in [START_REF] Frischknecht | The Plasmodium falciparum Maurer's clefts in 3D[END_REF]), a concern remains that these employed exclusively chemical fixation to preserve the specimen. As shown in other systems, chemical fixation can alter the morphology of, or can even destroy, sub-cellular structures [START_REF] Al-Amoudi | Nanostructure of the epidermal extracellular[END_REF]. In an effort to overcome these limitations, we have applied four techniques that preserve the sample by rapid freezing and evaluated the structures by electron tomography. Rapid freezing assures preservation of a sample within milliseconds, and water molecules around and within the sample are immobilized in the original, unordered state, so called vitreous water, without forming crystalline ice [15]. First, we used a whole cells approach, whereby the cells were grown or deposited on EM grids [START_REF] Lucić | Structural studies by electron tomography: from cells to molecules[END_REF][START_REF] Garvalov | Luminal particles within cellular microtubules[END_REF], plunge frozen and examined at cryo-conditions using an electron microscope. The thinner parts of intact cells could be imaged without necessity of sectioning. Second, we prepared fully hydrated sections, where the material was frozen at high pressure, then sectioned and examined in the EM at cryo-conditions [START_REF] Al-Amoudi | Cryo-electron microscopy of vitreous sections of native biological cells and tissues[END_REF]. Third, we generated freeze-substituted sections, where the sample is high-pressure frozen, after which it undergoes a process similar to classical EM preparations including fixation, dehydration, plastic embedding, but under cryo-conditions. The sections were produced at room temperature and examined by EM [START_REF] Studer | High pressure freezing comes of age[END_REF]. Finally we prepared 'Tokuyasu' sections, where chemically fixed material was incubated in high concentrations of sucrose and frozen for sectioning. The sections were then thawed and immunolabelled with specific antibodies coupled to electron dense markers and imaged by EM at room temperature [START_REF] Griffiths | Fine structure immunocytochemistry[END_REF]. The Tokuyasu method preserves well the membranes profiles and is particularly suitable for immuno-histological studies. We called the first two approaches a bona-fide cryo-EM as these do not involve chemical fixation and also that the samples were in a fully-hydrated vitrified state when examined in the electron microscope at cryo-conditions. The cryo-EM showed novel morphological details of the Maurer's clefts, such as the density in the lumen that is comparable to the exterior, the narrow connections between flat lamellae, as well as that with parasitophorous vacuole, and notably the absence of tethers thought to supposedly interconnect the Maurer's clefts with the plasma membrane of infected cells.

Materials and Methods

The P. falciparum strains HB3 and Dd2 were cultured as previously described [START_REF] Trager | Human malaria parasites in continuous culture[END_REF] with some modifications. Briefly, we incubated the parasites in A+ human erythrocytes in Petri dishes of 10 and 25 cm diameter containing HEPES-buffered RPMI medium 10% A+ heat-inactivated human serum and antibiotics. The cultures were incubated in an atmosphere of 3% CO 2 , 5% O 2 and 92% N 2 , at 37°C and 95% humidity. The cultures were harvested at min of 5% of parasitemia. Preparation for EM: 1. Cryo-electron tomography (Cryo-ET) of perforated, wholly-mounted cells: The parasitized cells (mid trophozoites /schizonts, approx 5% parasitemia) were purified on a magnetic column and perforated using streptolysin O (SLO/Sigma Chemical Co. -St. Louis, MO, USA) (modified after [22], without chemical fixation) to deplete the cytosol. Briefly, we incubated 10 8 cells with 25 units of post-activated SLO for 6 min at RT. Digestion was stopped by putting the sample on ice, and SLO was washed away by 2X centrifuging at 1000g for 2 min and washing with cold PBS, and put in medium for approx. 20 min at RT for cells to recover. 3-4 µl of cell suspension was transferred on holey carbon grid with the fiducial gold markers, the excess of liquid was blotted from the opposite side of the grid and the grids were plunge frozen in liquid ethane at the temperature of -180°C [23]. 2. Cryo-hydrated sections (CEMOVIS): Infected erythrocytes (not permeabilised) were high-pressure frozen using EM PACT2 (Leica Microsystems, Vienna, Austria) at 2000 bars after brief infiltration (2-4 min before freezing) with 20% of 40kD dextran [START_REF] Al-Amoudi | Cryo-electron microscopy of vitreous sections of native biological cells and tissues[END_REF]. Thin sections of 80 nm were cut on a Leica microtome at -160°C. 3.

Freeze substitution: High pressure freezing -as for CEMOVIS but without dextran. The perfusion of the vitreous samples and slow exchange of bound water with organic solvents (anhydrous acetone) containing 0.1% glutaraldehyde, 0.25% uranyl acetate, and 0.01% osmium tetroxide. Substitution with Lowicryl resins was initiated at -90°C and the Deleted: The P. falciparum strains HB3 and Dd2 were cultured as previously described [START_REF] Trager | Human malaria parasites in continuous culture[END_REF]. [START_REF] Studer | High pressure freezing comes of age[END_REF]. Sections of 200 nm were cut at room temperature. 4. Tokuyasu sections: The cells were fixed in 4% formaldehyde, embedded in gelatine, perfused in 2.3 M sucrose and frozen in liquid nitrogen. Frozen blocks were sectioned in a cryo-microtome (Leica EM UC6) at -110°C, thawed on methyl cellulosesucrose and labelled with STEVOR antibodies followed by 10 nm PA-gold [START_REF] Griffiths | Fine structure immunocytochemistry[END_REF].

For EM tomography the samples were examined and tilt series recorded in an FEI Polara electron microscope (Max-Planck-Institute for Biophysics, Frankfurt, Germany), as well as FEI CM300 FEG (Max-Planck-Institute for Biochemistry, Martinsried, Germany), with the acceleration voltage of 300kV. The tomograms were calculated by back-projection of 50-60 images (recorded in the tilt series covering approx ±60°, with 2-3° intervals, and objective lens defocus of 8-14 µm) aligned with fiducial markers (EM processing package [START_REF] Hegerl | The EM Program Package: A Platform for Image Processing in Biological Electron Microscopy[END_REF]). The tomograms for CEMOVIS sections were aligned using cross correlation alignment (ImageJ / plugin: TomoJ) and reconstructed with SIRT (TomoJ). The thickness of Maurer's cleft cisternae was measured between centres of membrane masses in 50 nm intervals along the cisternae. Visualization and surface rendering was done on AMIRA (Visage Imaging GmbH, Berlin).

Results and Discussion

We first undertook a whole-cell-approach by preparing tomograms of cryo-preserved entire P. falciparum-infected erythrocytes. As the electrons transmit through and form an interpretable image only of samples thinner than 0.5-1 µm, it became necessary to partially deplete haemoglobin by perforating the RBC plasma membrane with bacterial toxins. This was achieved with a modified procedure after Jackson et al [22], but without chemically fixing the cells. The resulting tomographic maps of what appeared to be the intact architecture of the infected RBCs covered approx 1-2 cubic microns of cell cytoplasm. Various membrane systems were clearly visible in single sections and in the tomograms. This included the parasite plasma membrane, the parasitophorous vacuolar membrane, the Maurer's clefts and the erythrocyte plasma membrane (Fig. 1A). The knobs (insert in Fig. 1A) appeared as protrusions of the erythrocyte plasma membrane, with a layer of electron dense material underneath. A careful inspection of the tomograms revealed known as well as new features of the Maurer's clefts (Fig. 1B, C and Fig. 2).

Stacks of flat cisternae were observed within the host cell cytoplasm, indicative of Maurer's clefts (Fig. 1B). The lengths of the different cisternae typically varied between 100 and 500 nm and the neighbor lamellae were separated by 30-100 nm of cytoplasm. The lumen of the cisternae was typically 35 nm wide, but could vary between 20 and 200 nm (Fig. 1D).

We did not distinguish different classes of Maurer's clefts based on the width of the lamellae.

Occasionally some cisternae flattened towards the ends. Apart of the flat lamellae we also observed other membrane morphologies such as whorls that in some tomograms were intermingled with the flat stacks, whereas in some other appeared isolated. Notably, the whorls had a similar distribution of the luminal thickness as the flattened disks (not shown).

Moreover, we noted a thickening of the membranes at the main body of the cisternae by F o r P e e r R e v i e w 9 approx. 3 nm, as compared to the ends, due to additional material, possibly of protein origin, attached to the cytoplasmic side of the membrane (Fig. 2A). The cytoplasmic surface of the cisternae had a smooth appearance and the transition between thinner and thicker profiles was smooth, with no detectable abruptions or gaps. Such features were described in classical preparations and referred to as 'electron dense coat' that appears as strongly corrugated material that massively accumulates heavy metal stains [START_REF] Atkinson | Ultrastructure of malaria-infected erythrocytes[END_REF]. Thick membrane profiles were ubiquitous in the unstained cryo-hydrated preparations that are free of an uncontrolled precipitation of material in random volumes. We regard these densities as a characteristic trait for the Maurer's cleft cisternae. The most typical width of 35 nm was measured predominantly in the regions with thick membrane profiles, which raises the question whether these additional densities endow the characteristic disk shape of the cisternae. We made these measurements on the preparations of whole cells that were perforated before freezing. As this treatment might induce changes in osmolarity [START_REF] Lew | Excess hemoglobin digestion and the osmotic stability of Plasmodium falciparum-infected red blood cells[END_REF], which then might affect the volume of cisternae, we made additional measurements on the vitreous sections (CEMOVIS) (along the direction of sectioning), which confirmed the accuracy of the measurements (not shown).

One of the unsolved issues of the Maurer's cleft architecture is whether the cisternae are interconnected with each other, and furthermore, whether they are connected to other membrane compartments of the infected cells [START_REF] Wickert | Maurer's cleft organization in the cytoplasm of Plasmodium falciparum-infected erythrocytes: new insights from threedimensional reconstruction of serial ultrathin sections[END_REF][START_REF] Hanssen | Electron tomography of the Maurer's cleft organelles of Plasmodium falciparum-infected erythrocytes reveals novel structural features[END_REF]. Solving this issue would strongly advance the studies on parasitic protein export [6]. The 3D EM attempts carried out so far on classically prepared samples provided a diverse picture. Wickert et al. [START_REF] Wickert | Maurer's cleft organization in the cytoplasm of Plasmodium falciparum-infected erythrocytes: new insights from threedimensional reconstruction of serial ultrathin sections[END_REF] described highly interconnected networks of the export machinery, and found connectors to both the parasitophorous vacuole and the plasma membrane of the infected cell. On a contrary, the tomograms of plastic sections showed non-interconnected cisternae, spread in the RBC cytoplasm [START_REF] Tilley | The Twists and Turns of Maurer's Cleft Trafficking in P. falciparum-Infected Erythrocytes[END_REF]. The latter study further provided evidence for tethers, potentially proteinaceous connectors between clefts and the plasma membrane of infected RBC. Our tomograms of cryo-hydrated preparations revealed tubular structures that seemed to interconnect lumina of adjacent cisternae (Fig. 2B). These tubular connectors were shaped like two apposing funnels that merged at the narrowest sites (Fig. 2B). Such interconnections could not be detected between all of the apposing cisternae. The funnel-like connectors were composed of single membranes that lacked the additional protein depositions at the cytoplasmic side, similar to membranous extensions observed at the edges of individual cisternae.

Further, we occasionally observed tubular connections between the Parasitophorous Vacuolar Membrane (PVM) and cisternae (Fig. 2B). The insert in Fig 1A shows a typical distance between the PVM and the plasma membrane of the parasite that is also considered as the minimum distance. Notably, the PVM in cryo-hydrated preparations often appeared at larger distances in respect to the parasite plasma membrane, unlike to that seen in the classical TEM preparations. These differences could be due the osmotic changes [START_REF] Lew | Excess hemoglobin digestion and the osmotic stability of Plasmodium falciparum-infected red blood cells[END_REF] after depletion of the excess of hemoglobin for the 'whole cell' preparation. In none of the 16 tomograms examined so far, we could detect membranous connections between the cisternae and the erythrocyte plasma membrane. However, this does not rule out their existence. The cisternae consistently ended well below the inner leaflet of the erythrocyte plasma membrane. Moreover, in one case we detected a seemingly proteinaceous density that was attached at one end to a cisterna, but without connecting it to any other structure (not shown). We often observed proteinaceous material scattered in the cytoplasm between the membrane stacks that typically appeared as diffuse clouds (Fig. 2C) rather than the compact tethers that are frequently seen in the classical EM preparations [START_REF] Tilley | The Twists and Turns of Maurer's Cleft Trafficking in P. falciparum-Infected Erythrocytes[END_REF]. The discrepancy between different preparations is likely due to the precipitation of material during fixation and dehydration or the selective accumulations of the stain. Nevertheless, such material observed in cryo-preparation could also be remnants of the extracted cytoplasm after cell perforation. We next investigated the morphology of the Maurer's clefts using cryo-hydrated CEMOVIS sections [START_REF] Al-Amoudi | Cryo-electron microscopy of vitreous sections of native biological cells and tissues[END_REF]. CEMOVIS sections are more challenging to produce and image in the cryo-EM, but notably, they allow thicker parts of the cells to be imaged. The resulting tomographic maps represent a true distribution of densities within the sample, as there is no dilution of the haemoglobin prior to freezing. The tomograms recorded from CEMOVIS sections confirmed the overall architecture of the Maurer's clefts as depicted in cryo-preserved whole cells.

Again stacks of flattened cisternae were observed (3A and B). Furthermore, the cisternae also flattened at the ends and the membranes were thicker in the main body of the cisternae than at the ends (Fig. 3C).

On the tomograms of the CEMOVIS sections we measured the electron density of different subcellular compartments and determined that both the lumen of the clefts and the surrounding erythrocyte cytosol exhibited comparable densities (Fig. 3A). Our finding contrasts with previous reports, using classical EM techniques, where the lumen of the cisternae appeared electron lucent [START_REF] Tilley | The Twists and Turns of Maurer's Cleft Trafficking in P. falciparum-Infected Erythrocytes[END_REF]. This finding suggests that the Maurer's clefts are filled with more material than previously appreciated. Questions remain concerning the composition of the luminal material, its origin, and the possible role in maintaining the shape of the flattened disks.

The two remaining methods of specimen preservation used in this study involve freezing, but at subsequent steps of the preparation procedures the material is dehydrated and imaged in the EM at room temperature. We next examined the morphology of Maurer's clefts in samples prepared by freeze-substitution. To this end, infected erythrocytes were high pressure frozen, chemically fixed, stained with heavy metal salts and then embedded, at cryo-temperatures, in a plastic material that replaced vitreous water. Although the overall morphology of the Maurer's clefts was preserved in the tomograms of freeze-substituted sections (Fig. 4A and First, the clefts appeared consistently less electron dense than the surrounding erythrocyte cytosol. Second, due to unequal deposition of the stain, the characteristic thinning of membranes towards the ends of cisternae was not distinguished. Third, the cytoplasmic surface of the cisternae had a rough and bumpy texture (Fig. 4C). Occasionally, we observed connections between individual cisternae (Fig. 4C) that resembled tethers seen in classical EM preparations [START_REF] Wickert | Maurer's cleft organization in the cytoplasm of Plasmodium falciparum-infected erythrocytes: new insights from threedimensional reconstruction of serial ultrathin sections[END_REF][START_REF] Tilley | The Twists and Turns of Maurer's Cleft Trafficking in P. falciparum-Infected Erythrocytes[END_REF]; however, we were unable to discern whether or not the lumina were continuous. The advantage of freeze-substitution is that it offers the possibility of immunolabelling as well as the tomography of serial sections [START_REF] Höög | Organization of interphase microtubules in fission yeast analyzed by electron tomography[END_REF] to examine larger volumes of the extended membranous networks of the parasite export system.

Finally we examined specimens prepared by the so-called Tokuyasu technique where chemical fixation is followed by freezing. The tomograms of the sections depicted Maurer's clefts as a complex membrane system (Fig. 5A andB). However, little structural detail could be gleaned from these preparations. The texture of the membranes appeared rough and bumpy and the variations in membrane thickness between ends and middle sections of cisternae was not observed. The lumen of the cisternae appeared electron dense.

As shown in other systems, Tokuyasu sections and freeze-substituted sections are better suited for immuno-EM studies [START_REF] Webster | Preparation of cells and tissues for immuno EM[END_REF]. Using parasites expressing a STEVOR-GFP fusion protein, we were able to localize the protein to the Maurer's clefts in Tokuyasu sections (Fig. 5A andB). However, owing to the size of an antibody and the gold label the resolution is approximately 10-15 nm. Thus, for the Maurer's clefts stacks, it would be difficult to determine whether the antigenic sites are located on the membranes or on connectors between the membranes, without statistical analysis [START_REF] Vogel | Dynamic subcompartmentalization of the mitochondrial inner membrane[END_REF]. A co-localization of the detected antigenic

Conclusions:

In all preparations the general features of the Maurer's cleft were discernible, and the detailed analysis was remarkably advanced in the three-dimensional maps achieved by electron tomography in the frozen-hydrated EM preparations over that of chemically fixed samples.

The membranes of the Maurer's clefts viewed in cryo preparations appeared more continuous than anticipated [START_REF] Wickert | Maurer's cleft organization in the cytoplasm of Plasmodium falciparum-infected erythrocytes: new insights from threedimensional reconstruction of serial ultrathin sections[END_REF][START_REF] Tilley | The Twists and Turns of Maurer's Cleft Trafficking in P. falciparum-Infected Erythrocytes[END_REF]. This also applied to the connections with the other membranous formations in the infected cell, such as the PVM. The lumen of the lamellae had the same electron density as the surrounding cytoplasm, and the proteineous tethers that supposedly connect the lamellae with the plasma membrane of the infected erythrocyte were not detected. Thus, when exploited in a more systematic manner the methods presented here should be capable of addressing critical questions concerning the mechanisms underpinning the export of adhesive factors to the surface of infected cells. This knowledge may facilitate the development of strategies to inhibit protein export and prevent severe malaria-associated complications, such as cerebral and placental malaria.

4

 4

  by 10°C per hour

  there were clear distinctions when compared with bona fide cryo-hydrated preparations.

  structural details can only be answered by correlation with the bona-fide cryopreparations.

14 the

 14 European Commission Network of Excellence "Biology and Pathology of Malaria Parasite (BIOMALPAR)", and the DFG Priority Programme 1399 "Host-Parasite Coevolution". by cryo-electron microscopy of vitreous sections of human skin.

Fig 1 .

 1 Figures captions

Fig 2 .

 2 Fig 2. Details of Maurer's cleft morphology: a whole cell cryo-EM approach as in Fig 1. A. Section through a reconstruction and surface rendered view showing the continuity between thicker (black arrowheads) and thinner (white arrowheads) membranes of the Maurer's cleft. B. Tubular connections between parasitophorous vacuole membrane PVM and Maurer's cleft (black arrowhead), and between cisternae of the Maurer's cleft (white arrowhead). Pfparasite. The inserts below views the same volume at different angle, and a surface rendered view. C. Diffused material (presumably protein) filling gaps between the stacks of Maurer's clefts (indicated with arrows). Thickness of cell in A-C: 300-450 nm (Scale bars: 100 nm).

Fig 3 . 19 (

 319 Fig 3. Tomogram of a cryo-hydrated section through the Maurer's cleft. A. Section through the tomogram. The lumen of MC (white asterix) has similar density as cytoplasm of RBC

Fig 4 .

 4 Fig 4. High pressure frozen -freeze substituted cells. A. Slice through a tomogram showing lamellae of Maurer's cleft B. Surface rendered view of the structures in A. (light blue -Maurer's cleft lamellae, dark blue -erythrocyte plasma membrane, green -parasite). C. Connectors (violet) between lamellae (light blue). Thickness of the section: 200 nm. (Scale bars: 200 nm).

Fig 5 .

 5 Fig 5. 'Tokuyasu' section of infected erythrocyte, immunolabelled against STEVOR. A. Section through a tomogram showing morphology of MC and STEVOR labelling. B. surface rendered view of the area in A. (blue -Maurer's cleft, dark blue -outline of erythrocyte plasma membrane, green -outline of parasitophorous vacuole, red -positions of gold markers). Thickness of the section: 200 nm. (Scale bar: 200 nm).

Wiley-VCH Biotechnology Journal

Acknowledgements:

We thank Stefan Prior for his excellent assistance in sample preparation, electron microscopy and image processing; we also acknowledge Wolfgang Baumeister and Jürgen Plitzko for using the cryo-electron microscopy facility at the MPI for Biochemistry in Martinsried, Germany; Werner Kuehlbrandt and Derryck Mills for using the cryo-electron microscopy facility at the MPI of Biophysics in Frankfurt. We thank Friedrich Frischknecht and Gareth Griffiths for careful reading the manuscript and useful comments. This work was funded by


Ulrike Engel
email: ulrike.engel@bioquant.uni-heidelberg.de

Imaging centers spread across the globe -partnerships between cooperation and academia ((biotech highlight))

Keywords: Light microscopy, Live imaging, Imaging facility NIC, Nikon Imaging Center

Imaging centers spread across the globe -partnerships between cooperation and academia ((biotech highlight))

The Nikon Imaging Centers as collaboration between Nikon and Academia

There are currently 8 Nikon Imaging Centers world wide, three in the United States, two in Asia and three in Europe. The first Nikon Imaging Center (NIC) was opened in 2001 at Harvard Medical School (Boston, MA, USA). It was the idea of Marc Kirschner, who was head of the Cell Biology Department at that time. He and his colleagues in the department had already pioneered live microscopy for the quantitative understanding of cell and developmental questions. So the idea was not to bring microscopes to needy scientists, who have never seen a microscope equipped with a digital camera, but to use the synergy of scientific excellence and cutting edge equipment. This collaboration was a big success with the scientific community at Harvard Medical School and has encouraged the formation of more NICs. In the meantime, 7 more NICs were created all over the world based on this working formula. The NIC at the University of Heidelberg, Germany, opened in September In all cases, these centers are set up as a partnership between a University Institute and the Nikon Cooperation. The directors and staff of the facilities are hired by the University, and the centers are embedded in the scientific community door to door with research labs.

The NICs are built on local initiative and serve the needs of the scientific community they operate in. The NIC@HD was initiated by Thomas Holstein at the Institute of Zoology and Jörg Kukulies from Nikon, Germany. After one and a half years in the Zoology Institute it is now located at the new Bioquant building. Like other light microscopy facilities, the NICs main mission is to make equipment accessible to a large number of scientists, and to provide guidance in using it. Currently, the NIC@HD is serving 240 active users from more then 90 research groups. The type and number of equipment is negotiated between the academic partner and the local Nikon branch. We started with 5 systems and then rapidly agreed to add more as the demands grew. After its first 3 years the NIC@HD now offers 9 imaging setups (see Table 1) and we have considerably diversified the range of possible applications.

Visitors to the NICs are often impressed by the abundance of equipment and cannot help to wonder whether there is a catch somewhere as the equipment is loaned to the University free of charge. Are these centers just "outlets" of Nikon, where the University provides the sales location? Is the staff trying to talk users into buying equipment? This is not how the Nikon Imaging Centers operate. Nevertheless, it is clear that the local Nikon branches and Nikon Japan have endorsed the NICs in their global marketing strategy and it is important to them, that the NICs are showcases for Nikon's imaging products. They are set up prominently in research hot spots, where a large number of postdocs and future group leaders become familiar with Nikon systems. However, the "displayed" products are put to the acid test as users work on them more than 10 hours a day. The NICs operate within an environment where users have the choice: Heidelberg has an astounding number of advanced microscope The exchange of experience between facility users and the NIC staff also involves the product specialist of Nikon. This way, Nikon learns about new applications and imaging demands.

The good relation with the Nikon specialists helps the facility to operate and maintain the highly sophisticated equipment. The relation between academic staff and Nikon employees is generally relaxed and frank. A main reason for this might be the agreement between University and Nikon that the NICs will not purchase microscopes from Nikon's main competitors. This way the Nikon specialists are not in the position where they have to "advertise" their products and thus they share a lot more information with the NIC staff. In turn, the NIC staff is trying to give critical feedback to improve setups. At the NIC@HD we have given a lot of input directly to programmers during the early development of the NIS-Elements software.

In addition to Nikon, other companies also contribute equipment to the NICs. These are often local alliances and differ from NIC to NIC. In the case of the NIC@HD, Hamamatsu Photonics is sponsoring cameras, the German company AHF Analysentechnik provides filters, the Dutch company Scientific Volume Imaging is bringing its worldwide renowned deconvolution package to the NIC@HD. Perkin Elmer also operates as a major contributor, as one of the two spinning disk systems on a Nikon inverted microscope is sponsored by them.

And the second one? It was bought through a German Research program grant, as well as the LaVision Biotec 2-photon system on the Nikon fix stage microscope (Table 1). It is not uncommon, that sponsored and bought equipment is hosted in the NICs, as the foremost interest of the facility is to provide a comprehensive choice of equipment. For the grant Technically, the equipment is loaned and does not become property of the University. This works as a big advantage for the academic partner. Equipment is replaced when advanced models become available. In the last 3 years we have exchanged all the inverted microscopes from TE2000 to the new Ti series with automated xy-stages and hardware focus. The TIRF setup was completely motorized, and we added 3 entirely new systems. As the systems are loaned, planning is much more flexible. Often, new innovations make existing instruments completely unpopular to users and upgrading the instrument is sometimes not possible or extremely costly. NICs have the opportunity to test whether an instruments suits the needs of the scientific community and can otherwise negotiate for a replacement. Of course, there is a down side to the exchange of instrumentation as it contradicts the fundamental principle to "never change a running system". Installing a new system usually means a lot of trouble shooting and extra work for the staff and the new setup is sometimes met by initial skepticism on the user side. Even changing just the software on a single microscope station can mean that 50 users have to be retrained.

The equipment

The dream for both users and staff would be to have one type of microscope that covers all applications, or at least have every setup driven by a single software package. Unfortunately, doing microscopy is more like traveling: you have to change the means of transport all the time. Let us assume you want to look at actin dynamics in a moving cell. The obvious choice for a 3D time lapse study is a spinning disk confocal microscope (Fig. 1 and Fig. 2A-D), yet even finer details of the actin cortex can be imaged with total internal reflection fluorescence microscopy (TIRFM; Fig. 2E). Then again, fluorescence recovery after photobleaching Deleted: . Deleted: . Deleted: . 1) are confocal microscopes or systems that also generate optical sections like the TIRFM and the 2 photon setup. In cases where specimens are flat, wide field fluorescence microscopes with sensitive cameras provide a valuable alternative as they offer an excellent signal to noise ratio for weak fluorescence signals.

Imaging of live cells or entire organisms is a big challenge. To keep an organism alive on stage while preventing it from moving out of view is just one problem. Being able to visualize the signal over an extended time period without phototoxicity or photobleaching is another.

There is no general solution, given that each experiment has different priorities (speed, resolution …) and problems (poor signal, thick specimen...). Live imaging and in-vivo imaging in particular requires much more sophisticated instrumentation [START_REF] Misgeld | In vivo imaging of the diseased nervous system[END_REF] and more optimization by the researcher. Ironically, the resulting images are poorer in resolution and blurry because of noise and/ or autofluorescence in the tissue. Yet compared to the crisp images of fixed specimen, their beauty lays in the temporal information and the physiological relevance. For this reason, the NIC@HD puts a lot of effort in optimizing live experiments together with users. Most microscopes can be equipped with heated chambers and CO 2 incubation.

The software that drives the microscopes

Using different equipment also means that users will encounter different types of software.

Even though we have endorsed the Nikon NIS-Elements software at the NIC@HD and work with Nikon microscopes only, we are currently using 3 additional software packages for image acquisition (Volocity from Improvision, ImSpector Software from LaVision Biotec, Nikon Biostation software). The quality of the software is key to the overall performance of the system. Nowadays it is possible to perform microscopy without oculars (e.g. on the Nikon Biostation, see Table 1), but not without using software to drive the different hardware components and sensors. The automation of microscope components in combination with a smart software allow to store microscope settings and recall them with one mouse click. The facility staff can prefabricate imaging protocols and pass them on to users. This way, even researchers without profound knowledge of microscopy can perform complicated imaging routines, which were previously the privilege of the experts.

In addition to the microscope setups, the NICs provide additional extra computers for data evaluation, storage of data on servers, specialized software for data rendering and deconvolution. While the NIC@HD relies on commercial software in the facility, we also use and teach ImageJ for image analysis [2], simply because it is a non-proprietary software which is available to users in their labs, at home, and in their future career.

Service to the scientific community

In many respects the NICs run like other light microscopy facilities. They provide equipment, training of researchers, maintenance of microscopes and the logistics that make it possible to share instrumentation among many people [START_REF] Demaggio | Running and setting up a confocal microscope core facility[END_REF][START_REF] Anderson | Design and Function of a Light-Microscopy Facility[END_REF]. Different from other core facilities, light microscopy facilities have quickly moved away from "full service" where staff would acquire images for researchers. Instead providing a working environment that allows researchers to perform cutting edge microscopy has been proven to be much more successful. This way, researchers become "users" empowered to generate and improve imaging results. The by far most time consuming task for the NIC staff is to train and advise users. In their very resourceful article on light microscopy facilities, the staff of the light microscopy facility at the Max Planck Institute for Cell Biology and Genetics, Dresden, devote a whole paragraph to users [START_REF] Anderson | Design and Function of a Light-Microscopy Facility[END_REF]. They state: "Users are what it is all about; they can be a blessing and a curse! Good F o r P e e r R e v i e w 8 users push staff for assistance with cutting-edge applications, provide valuable feedback about the state of equipment, and are key to recognizing new trends…Bad users are selfish monsters who expect the equipment to be in top working condition when they want to use it, but give no thought to the condition in which they leave it". Little needs to be added to this angled but apt characterization, though I should mention that all shades between good and bad exist. Also, in quantitative terms, the ratio between good and bad is something like 100:1. As a consequence, the staff gets used to good users very quickly and is then annoyed at the occasional black sheep.

In all the NICs, the researchers are trained individually, on their own samples. This is demanding for the staff, as they do not show (or show off with) beautiful samples they already know, but try to optimize settings together with the researchers. For this reason, most staff members are PhD qualified and often have a post-doctoral or other professional research experience. In order to optimize the imaging experiment, the staff must know the microscope, but also get a quick understanding of the sample. It is ridiculous to painfully enhance the sensitivity of the sensor, when it would have been very easy to increase the expression level of a GFP construct, or to improve the staining protocol. Experienced staff can save the users a lot of trouble not related to the microscope directly. They help in setting up live experiments, getting expression levels right, discriminating autofluorescence from real signal. This does not mean that for all imaging experiments, solutions are straightforward. There are live specimen, which are very dim and nevertheless very interesting. Also, an application may demand extremely quick acquisition and requires that the staff optimize the instrument for sensitivity, speed and resolution.

The projects at the NIC@HD range from simple morphological analysis to experiments where imaging is used as a read-out of physiological activity in the cell (e.g. Calcium Imaging, FRETsensor) and protein interaction (e.g. FRAP and fluorescent resonance energy transfer). To setup a successful experiment detailed knowledge about all imaging components is required [START_REF] Waters | Live-cell fluorescence imaging[END_REF] and as for Deleted: e.g.

Deleted: e.g. 9 every scientific experiment appropriate controls need to be incorporated. Otherwise, serious misinterpretation might occur, for example crosstalk between channels might be taken as colocalization of two markers. To see researchers jump to more demanding imaging techniques without an understanding of the basics has a disheartening effect on the staff of the imaging facilities [START_REF] Pearson | The good, the bad and the ugly[END_REF]. It is their job to make researchers aware of pitfalls, but ultimately the interpretation of the results falls into the responsibility of the researchers. To promote and deepen the understanding of principles in microscopy and discuss advanced applications the NICs also provide courses on a regular basis, which are extremely popular with PhD students.

Outlook

The research conducted at the NIC@HD over the last three and a half years has contributed to around 50 peer-reviewed research papers. Given the delay between data acquisition and publication this is likely to rise steeply during the next years. The projects are very diverse and it is only possible to list a few examples (Table 1). Looking back on the last three years, we were able to exchange and improve setups, making new applications possible (fast FRAP experiments, simultaneous photoactivation and acquisition). Other experiments have become much more productive (e.g. timelapse acquistion is now done with hardware focus and multi-point acquisition). To take part in the fast progress of light microscopy-based research, the NICs depend on the dedication of both Nikon and the University to continuously invest into the NICs. They relay on Nikon's sustained commitment to provide hardware and new innovations. As we are increasingly combining high content with high throughput imaging, we need high resolution microscopes which can perform fast and parallel acquisition without compromising on sensitivity. To evaluate the resulting experimental data, smart ways to handle and analyze the large data sets need to be developed by University IT specialists. It is the task of the NIC staff to integrate knowledge and pass it on to users. Each of the NICs worldwide has its own focus of expertise, but systematic exchange of information amongst the NICs and other imaging centers is a largely untapped resource, and will help to develop solutions to common Comparative immunocytochemistry, such as analysis of knockout mice phenotypes [START_REF] Moers | Gα12/Gaα13 deficiency causes localized overmigration of neurons in the developing cerebral and cerebellar cortices[END_REF]. FRAP experiments [START_REF] Renz | Invasive breast cancer cells exhibit increased mobility of the actin-binding protein CapG[END_REF]. Photactivation in hippocampal neurons.

FRET [START_REF] Jarve | Surveillance of siRNA integrity by FRET imaging[END_REF] and FRETbased sensors [START_REF] Gutscher | Real-time imaging of the intracellular glutathione redox potential[END_REF] and other ratio imaging.

In-vivo cell migration in cancer models.

  2005. Next came the Nikon Imaging Center at Hokkaido University (2005), Oxford University (2006), the University of California San Francisco (2006), the Singapore Bioimaging Consortium (2007), the Institut Curie in Paris (2007), and Northwestern University in Chicago (2008). I have witnessed the early NIC at Harvard Medical School (NIC@HMS) as a post-doctoral fellow and I have planned and directed the NIC at the University of Heidelberg (NIC@HD) 3 years. Based on my experience as both a user and staff of the NIC, I will try to outline the philosophy of the NICs and what they bring to the scientific community.

  the University and other research centers. An enormous advantage for the researchers at NICs is the support that is added to the instrumentation. The instruments are kept in top working condition and there is always somebody who knows how to operate their advanced functions. Frequently, this is not the case when instruments are acquired by individual groups.

  attractive to put their instruments within an existing facility. The choice of what type of sponsored equipment should be part or the Imaging Center is made by the directors and steering committee of the NIC together with the companies who join the effort.

6 (

 6 FRAP) experiments have to be done on a laser scanning confocal microscope, unless the spinning disk has a photo bleaching unit. If the cellular dynamics need to be monitored in tissue, this might require the use of a 2-photon microscope, which allows to generate fluorescence signals in deeper cell layers. Most of the systems at the NIC@HD (see Table

Figure 1 .

 1 Figures and Tables

Figure 2 .

 2 Figure 2. T-cell line expressing GFP live-act (green) to highlight actin dynamics. The cells settle on a substrate covered with modified HIV virus particles (red). (A-D) Live imaging on a spinning disk confocal microscope (21 z-slices, 0.15 µm spacing, time as indicated). (A) 3D view of the cell in a side projection (B) orthogonal maximum projection. (C) Optical xy-slice and reconstructed xz-and yz-slices allow to determine the exact position of a virus particle that has bound to the cell (white arrow head). (D) Insert detail (3 µm length) is magnified to

Table 1 .

 1 photoactivation while acquiring images. Fast zacquisition with a piezo-based z-stepper. The C1 is equipped with controlled-light exposure microscopy, (CLEM [7]), which reduces phototoxicity. 7 Laser lines cover the range of 405-640nm Plan Apo objectives with chromatic correction from UV

Formatted

  Photokinesis unit allows forFRAP and Photoactivation experiments in region of interests.

Wiley-VCH Biotechnology Journal


C Bambra
email: clare.bambra@durham.ac.uk

Real world reviews: A beginner's guide to undertaking systematic reviews of public health policy interventions

Background:

The systematic review is becoming an increasingly popular and established research method in public health. Obtaining systematic review skills are therefore becoming a common requirement for most public health researchers and practitioners. However, most researchers still remain apprehensive about conducting their first systematic review. This is often because an "ideal" type of systematic review is promoted in the methods literature.

Methods: This brief guide is intended to help dispel these concerns by providing an accessible overview of a "real" approach to conducting systematic reviews. The guide draws upon an extensive practical experience of conducting various types of systematic reviews of complex social interventions.

Results:

The paper discusses what a systematic review is and how definitions vary. It describes the stages of a review in simple terms. It then draws on case study reviews to reflect on five key practical aspects of the conduct of the method, outlining debates and potential ways to make the method shorter and smarter -enhancing the speed of production of systematic reviews and reducing labour intensity whilst still maintaining high methodological standards.

Conclusion:

There are clear advantages in conducting the high quality pragmatic reviews which this guide has described: (a) time and labour resources are saved; (b) it enables reviewers to inform or respond to developments in policy and practice in a timelier manner; and (c) it encourages researchers to conduct systematic reviews before embarking on primary research. Well conducted systematic reviews remain a valuable part of the public health methodological tool box.

BACKGROUND

The systematic review (SR), which has been advanced for many years now in the evidence based medicine literature, is becoming an increasingly popular and established research method in public health as well as some of the social sciences. [START_REF] Petticrew | Systematic Reviews in the Social Sciences: A Practical Guide[END_REF] For example, a quick search of the Web of Science database obtains 707 hits for the subject "systematic review" in the social science literature for the period 1945 to 1999 as compared to over 4596 hits for 2000 to 2008. [2] This includes an increase from 85 hits to 764 hits for the sub-category public, environmental and occupational health. [2] In part, this rapid expansion in the use of the method has been fuelled by the promotional efforts of the Cochrane Collaboration (which now has a public health sub-group, as well as a health equity subgroup) and its social science equivalent, the Campbell Collaboration (which has a welfare sub-group). [START_REF][END_REF][START_REF][END_REF] The other great driving force has been the increased emphasis in public health policy and practice for decisions and interventions to be more "evidence based", the so called "ascendancy of evidence". [5 -8] The SR is now widely considered within policy and practice circles to be a good way of making the sometimes conflicting and complicated results of many different types of study accessible and more useable. For example, a recent UK government commissioned review of promoting public health highlighted the value of SRs in providing robust and reliable evidence on the effectiveness of interventions. [START_REF] Wanless | Securing Good Health for the Whole Population[END_REF] Similarly, they are also a key factor in the formulation of binding recommendations made for the UK National Health Service by the National Institute for Health and Clinical Excellence (NICE). [10] Obtaining, often on the job, skills in SR methodology (both conducting them and interpreting them) are therefore becoming a common requirement for most public health researchers and practitioners. This is reflected in the number of guide books available. [1, 11 -13] However, most researchers still remain apprehensive about conducting their first SR and the voluminous guidebooks and handbooks (most are hundreds of pages long) do little to quell such feelings. The fact that most of the commentaries on conducting SRs still come from the health care or evidence based medicine literatures also makes the method inaccessible to public health policy researchers. SRs of public health interventions draw on an evidence base dominated by observational and qualitative studies in which the measurement of relevant outcomes is often heterogeneous. This means that the advice on SR from the health care and medicine literature, concerned as it is with the quality of experimental (particularly RCTs) studies and the meta-analysis of results, [START_REF] Egger | Systematic Reviews in Health Care: Metaanalysis in Context[END_REF] is difficult for novices to transfer to public health questions. [START_REF] Petticrew | Systematic Reviews in the Social Sciences: A Practical Guide[END_REF] Looking to social science is not particularly helpful either as a lot of the SR literature in this field still highlights the difficulties, as opposed to the advantages, of applying the method in social evaluation. From the outside looking in then, the SR approach can initially seem time consuming, tedious, and overwhelming. Added to this, there are various debates in the SR literature about what a SR is, how it should be conducted, and by whom. This brief guide is intended to help dispel these concerns by providing an accessible, more tailored overview for novices of the SR method and its place in public health policy research. The guide draws upon an extensive practical experience of conducting various types of SRs of complex social interventions in the field of public health policy (Box 1) and in terms of training researchers. [14 -22] Firstly it discusses what a SR is and describes the stages of a review in simple terms. It then reflects on five key practical aspects of the conduct of the method, outlining debates and potential ways to make the SR method shorter and smarter. It concludes by highlighting the ongoing value of the method.

What is a systematic review?

The first issue that any researcher is faced with is establishing what counts as a SR. In simple terms the SR is a method of locating, appraising, and synthesising evidence. However, in practice there are disagreements about what does and what does not constitute a SR. Conventional public health and social science definitions of the SR tended to concentrate on distinguishing it from traditional (nonsystematic) literature reviews. So for example, Oakley and Fullerton defined the SR as "a review of a clearly formulated question that uses systematic and explicit methods to identify, select and critically appraise relevant research, and to collect and analyse data from the studies that are included in the review". [START_REF] Oakley | The lamp-post of research: support or illumination?[END_REF] Similarly, a SR is systematic because it: attempts systematically to locate research, both published and unpublished, and critically evaluate it on grounds of relevancy and predetermined methodological criteria. Only research that is judged to be both relevant to the review question, and which fulfils the methodological inclusion criteria, is combined into the final review analysis. The SR combines the results of these studies and thus provides a summary of the 'best available evidence' on a given question. [START_REF] Higgins | Cochrane Handbook for Systematic Reviews of Interventions[END_REF][START_REF]Systematic Reviews: CRD's Guidance for Undertaking Reviews in Healthcare[END_REF] Following the Database of Abstracts of Reviews of Effects (DARE) guidelines on the minimum requirements of a SR, [START_REF]Database of Abstracts of Reviews of Effectiveness (DARE)[END_REF] recent umbrella reviews (systematic reviews of reviews) have tended to define studies as SRs if they addressed a clearly defined question, and an effort had been made to identify all relevant literature by searching at least one named database combined with either checking references, hand-searching, citation searching, or contacting authors in the field. [25 -27]. Although this is clearly only a minimal definition of a SR and not a definition of good practice, it does illustrate that SRs do not therefore need to search everywhere and synthesise all studies on a given question. In these diverse definitions, the clear distinguishing features of the SR are therefore its formality, transparency and replicability: its "systematic-ness".

Stages of a systematic review

A good quality SR follows a formal procedure which starts with the formulation of a precise question that includes a definition of the participants, the intervention to be assessed and the outcomes to be measured. [START_REF] Higgins | Cochrane Handbook for Systematic Reviews of Interventions[END_REF][START_REF]Systematic Reviews: CRD's Guidance for Undertaking Reviews in Healthcare[END_REF] This is followed by the development of a protocol which a priori outlines the inclusion and exclusion criteria, the searches to be undertaken and the sources to be consulted. A rigorous literature search, which combines electronic database searches, searching the references of identified studies, hand searching relevant journals, and contacting experts in the field, is then conducted. [START_REF] Higgins | Cochrane Handbook for Systematic Reviews of Interventions[END_REF][START_REF]Systematic Reviews: CRD's Guidance for Undertaking Reviews in Healthcare[END_REF] Studies are then selected in terms of whether they meet the strict inclusion criteria (which usually includes criteria to define relevance to the review question and methodological criteria).

Included studies are then data extracted using standardised forms and subjected to a methodological critical appraisal (in which the quality of the studies is assessed). The studies are then synthesised using meta-analysis (a statistical strategy for pooling the results of several studies into a single effect estimate) or narrative synthesis (data synthesis and exploration of heterogeneity using description) taking into account study designs and the results of the critical appraisal. The results of the synthesis then need to be interpreted and reflected upon in terms of the research, policy and practice implications. The final stage of the review is writing up and publication and it is essential that the methods of the SR are clearly described and the limitations of the included studies (and the SR itself) are acknowledged.

CONDUCTING A REVIEW

These suggestions on how to conduct a SR are based on experience of ten reviews (Box 1) and are related to different stages and aspects of the review process.

Asking the right question

It is essential that the review question is right as it dictates the remit of the review. Conventionally, SR guidance has recommended that the review question be kept tight. It should define the intervention of interest, the population receiving the intervention, the outcome(s) of interested, and the study designs deemed worthy of inclusion. [START_REF] Higgins | Cochrane Handbook for Systematic Reviews of Interventions[END_REF] However, the use of SRs in evaluating public health and social interventions has highlighted problems with this approach as policy or practice questions may well be broader and the answer may cover more than one intervention. [START_REF] Bambra | Reviewing the evidence: reflections from experience[END_REF] For example in the welfare to work review (Box 1) the policy (or intervention) under review was "welfare to work", however this covers a multitude of different types of intervention (such as anti-discrimination legislation, vocational rehabilitation, return to work credits etc) which may operate on the outcome in different ways. [START_REF] Bambra | Does "welfare to work" work? A systematic review of the effectiveness of the UK's welfare to work programmes for people with a chronic illness or disability[END_REF] This was also the case in the reviews which examined the health effects of changing the organisation of the work environment in which task restructuring interventions ranged from the introduction of team working to increased task variety. [START_REF] Bambra | The psychosocial and health effects of workplace reorganisation 2: A systematic review of task restructuring interventions[END_REF] Broader review questions can therefore enable the mapping of different interventions related to the overall review question. [START_REF] Ogilvie | Systematic reviews of health effects of social interventions: 2. Best available evidence: how low should you go?[END_REF] The use of a broad question may therefore be essential and advantageous in certain circumstances. However, the use of a broader review question is not without problems as it increases the breadth and size of the review, and potentially makes it harder to complete. It is therefore probably advantageous to break a broad review question down into smaller ones. A pilot scoping study which maps out the potential interventions of interest (and also pilots the search strategy) will also be a useful way of controlling and containing the review. The development of a review protocol is essential in this regard as it sets out the parameters, is an on-going source of reference, and is therefore an essential element in managing the review process. [START_REF] Higgins | Cochrane Handbook for Systematic Reviews of Interventions[END_REF][START_REF]Systematic Reviews: CRD's Guidance for Undertaking Reviews in Healthcare[END_REF] The value of team work One of the most daunting aspects of conducting a review is perhaps the isolation that it can engender.

Attempting to conduct a review as a sole researcher or with very little preparation, help or support is not sensible. The SR methodology literature is full of comments about the importance of using a second reviewer to independently select and critically appraise studies, as well as check data extraction. [START_REF] Higgins | Cochrane Handbook for Systematic Reviews of Interventions[END_REF][START_REF] Petticrew | Systematic reviews from astronomy to zoology: myths and misconceptions[END_REF] Experience suggests that this is not just beneficial in terms of methodological rigour and preventing bias, but also in terms of sharing the work load and ensuring that there is support throughout the review process. Ideally, an SR should be conducted by a team which includes an experienced reviewer, a subject specialist and a librarian or search expert. If this is not possible then the Cochrane model of a review advisory group should be considered. SRs which use the skills of specialists are much easier to conduct and complete. The use of a search specialist has particular advantages in social science research and in terms of ensuring a specific and sensitive search strategy is developed. [START_REF] Grayson | A difficult business: Finding the evidence for social science reviews[END_REF] This can save time later in the process particularly in terms of sifting titles and abstracts. A subject specialist will also be able to assist in study location (e.g. by pointing out grey literature sources or specialist databases) however, the real benefit will come in terms of the interpretation and synthesis of the results. For example, the SR of partnership working (Box 1) used the combined skills of three researchers, a librarian, an experienced systematic reviewer and a subject expert. [START_REF] Casiday | Volunteering and Health: What Impact Does It Really Have? Report to Volunteering England[END_REF]

Searches: Breadth versus depth

There is an implicit debate in the SR literature about the breadth of searches required with some sources (purists) suggesting that only reviews which include a comprehensive search of all available evidence (via extensive searches of multiple databases, hand searches etc), can be called a SR, whilst others (the pragmatists) are prepared to acknowledge that it is possible to conduct a SR of some of the evidence base (e.g. it is possible to conduct a SR of only one database -albeit accompanied by supplemental searches, see below) as long as this is done systematically and with transparency over the methods used and acknowledgement of the SR's limitations. "Pragmatist" SRs therefore focus on a handful of "first line" health and social science databases, [START_REF] Rivara | Prevention of falls in the construction industry: Evidence for program effectiveness[END_REF][START_REF] Crowther | Helping people with severe mental illness to obtain work: systematic review[END_REF] or supplement this with the use of a subject specialist one, [START_REF] Bunn | Traffic calming for the prevention of road traffic injuries: systematic review and meta-analysis[END_REF][START_REF] Ogilvie | Promoting walking and cycling as an alternative to using cars: a systematic review[END_REF] whilst "purist" SRs have tended to search every available and potentially relevant electronic database. [START_REF] Bambra | Shifting schedules: the health effects of reorganising shift work[END_REF][START_REF] Bambra | A hard day's night? The effects of Compressed Work Week interventions on the health and wellbeing of shift workers: a systematic review[END_REF][START_REF] Egan | New roads and human health: a systematic review[END_REF] Each approach has associated costs and benefits. So for example, SRs which look only at a few prominent databases save time (both at the searching stage and the sifting of titles and abstracts stage) but risk missing potentially relevant studies. Conversely, searching every database known has a high time cost, although additional studies may be located (such studies may however be lower down the evidence hierarchy -see 4 below). There is evidence to suggest that combining first line searches with a more specialist database might be the most fruitful approach as a paper reflecting on the search strategies used in a SR, of the effectiveness of interventions in promoting a population shift from using cars towards walking and cycling, found that the majority of relevant studies were located not in a "first line" database such as Medline or Web of Science but in a specialist transport database. [START_REF] Ogilvie | Promoting walking and cycling as an alternative to using cars: a systematic review[END_REF][START_REF] Ogilvie | Systematic reviews of health effects of social interventions: 1. Finding the evidence: how far should you go?[END_REF] Supplemental searches (hand search, websites, citation follow up, or expert contact) are essential and may in some cases even further negate the need for a broad "purist" search of the electronic databases especially in SRs of social interventions in which the grey literature is likely to be a prominent source of studies. [START_REF] Bambra | Reviewing the evidence: reflections from experience[END_REF] For example, in the two SRs of shift work interventions, although 27 different databases were searched, 40 of the 66 included studies were found in either Medline or Embase, and half (13) of the others were found via citation follow up (Box 1). [START_REF] Bambra | Shifting schedules: the health effects of reorganising shift work[END_REF][START_REF] Bambra | A hard day's night? The effects of Compressed Work Week interventions on the health and wellbeing of shift workers: a systematic review[END_REF] Similarly, in the SR of welfare to work interventions, only three were located via the electronic searches with the rest found via specialist website searches and citation follow up. [START_REF] Bambra | Does "welfare to work" work? A systematic review of the effectiveness of the UK's welfare to work programmes for people with a chronic illness or disability[END_REF][START_REF] Bambra | Reviewing the evidence: reflections from experience[END_REF] Evidence from other reviews also support the value of web site searches and citation follow up in locating new studies. [START_REF] Ogilvie | Systematic reviews of health effects of social interventions: 1. Finding the evidence: how far should you go?[END_REF] Similarly, some reviewers insist on not limiting the searches by time, place or language. However, this not only extensively broadens the remit of the review, but for the evaluation of public health policy interventions, it may result in the inclusion of studies which have little contextual relevance. For example, the SR of shift work interventions included a USA study from the 1930s. [START_REF] Bambra | Shifting schedules: the health effects of reorganising shift work[END_REF] However, the applicability of the findings of this study in the very changed labour market context of, say, the UK 2009 is very limited. Similarly, SRs may often benefit from spatial restrictions as country or cultural context may matter immensely in terms of the implementation and transferability of an intervention. [START_REF] Egan | Reviewing evidence on complex social interventions: appraising implementation in systematic reviews of the health effects of organisational-level workplace interventions[END_REF] Limitation by language may be a more pragmatic decision determined by the skills of the review team and the budget for translation.

The choice of search strategy is therefore a careful balancing act and one which requires awareness of the subject and an informed judgement of the likely results alongside an assessment of resource allocation. A search strategy would therefore often benefit from an initial pilot search exercise which would produce an initial impression of where studies are located. It is not however a necessity -or indeed always that productive -for a SR to search everywhere.

The best available evidence

The transference of the SR methodology from evidence based medicine was accompanied by a discussion about its limitations in respect to the very different and diverse public health and social science evidence base. Much of this focused on the applicability of the hierarchy of evidence with its emphasis on the RCT and experimental designs. Unadulterated attempts to transfer this part of the SR method often led to reviews with no or uncertain conclusions. [START_REF] Petticrew | Why certain systematic reviews reach uncertain conclusions[END_REF] Public health SRs of interventions are much more likely to be evaluated using observational and other study designs. The hierarchy of evidence, it was suggested, is therefore not useful, different approaches (such as typologies) [START_REF] Petticrew | Evidence, hierarchies, and typologies: Horses for courses[END_REF] should be utilised and public health research should look for the best available evidence wherever this may be found. This perspective is not without merit, particularly in terms of the importance of observational studies and qualitative research in evaluating public health and social science interventions. However, in practice, it is has led to the implicit abandonment of any limitation in terms of study design resulting in very broad inclusion criteria.

Broad study design inclusion criteria have merit in terms of mapping interventions. For example an examination of the study designs of included studies in a SR of the effectiveness of interventions in promoting a population shift from using cars towards walking and cycling, found that if only the better quality study designs were included some types of intervention would not have been identified or would not of had any evidence attached to them. [START_REF] Ogilvie | Systematic reviews of health effects of social interventions: 2. Best available evidence: how low should you go?[END_REF] On the other hand, inclusive searching may mean that for well evaluated interventions types, too many lower level studies are located and they add very little additional information to the evidence base. Often when such reviews are written up it is the better quality studies which are focused on in the findings and the results from lower level studies are marginalised. For example, the review of the compressed working week included 40 studies, yet the write up focused only on the five prospective controlled studies. [START_REF] Bambra | A hard day's night? The effects of Compressed Work Week interventions on the health and wellbeing of shift workers: a systematic review[END_REF] The broad study inclusion criteria on this occasion therefore increased the breadth of the review and increased the associated time and other costs (data extraction and critical appraisal are very time consuming even for experienced researchers) but with very little by way of additional information.

There is not an easy solution but experience suggests that a trade off is therefore required. SRs are interested in locating and synthesising the "best available evidence" (not all available evidence) this means that the hierarchy of evidence does need to be applied -albeit in a pragmatic way. The best course therefore might be to initially search for all study designs so that interventions are mapped against the evidence base, [START_REF] Ogilvie | Systematic reviews of health effects of social interventions: 2. Best available evidence: how low should you go?[END_REF] but then when the parameters are known, it is only the better quality studies for each type which are subjected to the lengthy data extraction and critical appraisal process. This will therefore produce an account of the "best available evidence" for each intervention type in the SR. For example, in the case of the compressed working week intervention this would mean controlled prospective cohorts only, [START_REF] Bambra | A hard day's night? The effects of Compressed Work Week interventions on the health and wellbeing of shift workers: a systematic review[END_REF] whilst for the health walk intervention in the SR of transport shifts, [START_REF] Ogilvie | Systematic reviews of health effects of social interventions: 2. Best available evidence: how low should you go?[END_REF] it would mean potentially going as low in the hierarchy as uncontrolled retrospective cohort level evidence. The best available evidence may well vary by intervention and this is part of the mapping. However, it is important not to spend unnecessary time on the data extraction and critical appraisal of studies which do not constitute the best available evidence for any given intervention.

Tools for Reviewers

SRs are very labour intensive and so it is important to avoid unnecessary replication of effort. The popularity of SRs not only in evidence based medicine but also increasingly in public health and social science means that there is the opportunity to use pre-existing and validated data extraction and critical appraisal tools. Designing, developing and piloting a new data extraction and/or critical appraisal tool is time consuming and it may also be challenged by peer reviewers when the review is completed and submitted for publication. [START_REF] Egan | Reviewing evidence on complex social interventions: appraising implementation in systematic reviews of the health effects of organisational-level workplace interventions[END_REF] There are however now numerous data extraction and critical appraisal tools that have already been used successfully by reviewers. These should be examined and where possible adapted for use in any new SR. For example, the Cochrane Reviewers Handbook or the NHS CRD handbook both contain examples of data extraction forms. [START_REF] Higgins | Cochrane Handbook for Systematic Reviews of Interventions[END_REF][START_REF]Systematic Reviews: CRD's Guidance for Undertaking Reviews in Healthcare[END_REF] In terms of critical appraisal, there are various tools which can be used, the validity of which have been extensively examined. For example, the Newcastle-Ottawa scale for the assessment of observational studies has attracted a lot of support and there are also now fairly well used ways of appraising qualitative evidence. [START_REF] Deeks | Evaluating non-randomised intervention studies[END_REF][START_REF] Popay | Rationale and standards for the systematic review of qualitative literature in health services research[END_REF] Another approach would be to examine the tools used by existing SRs in a similar subject area and adapt them. For example, the same data extraction and critical appraisal tools were used in all four of the SRs on the work environment and they were also adapted for use in later reviews. [START_REF] Egan | The psychosocial and health effects of workplace reorganisation 1: a systematic review of interventions that aim to increase employee participation or control[END_REF][START_REF] Bambra | The psychosocial and health effects of workplace reorganisation 2: A systematic review of task restructuring interventions[END_REF][START_REF] Bambra | Shifting schedules: the health effects of reorganising shift work[END_REF][START_REF] Bambra | A hard day's night? The effects of Compressed Work Week interventions on the health and wellbeing of shift workers: a systematic review[END_REF][START_REF] Smith | Partners in health? A systematic review of the effects on health and health inequalities of partnership working[END_REF][START_REF] Casiday | Volunteering and Health: What Impact Does It Really Have? Report to Volunteering England[END_REF] The SR process is thereby considerably simplified and streamlined with valuable time saved for use in the synthesis and analysis stages.

Conclusion

This guide has demonstrated ways in which the "real" world practice of conducting SRs can be made shorter and smarter, enhancing the speed of production of SRs and reducing labour intensity whilst still maintaining high methodological standards. It is not recommending an "anything goes" approachwhat is pragmatic and feasible needs to be weighed up along side considerations of what is robust and appropriate to help ensure that SR findings are useful and not misleading. There are clear advantages in conducting the high quality pragmatic reviews which this guide has described: (a) time and labour resources are saved; (b) it enables reviewers to inform or respond to developments in policy and practice in a more timely manner; and (c) it encourages researchers to conduct SRs before embarking on primary research, thereby reducing replication and helping to ensure that any subsequent primary research undertaken is well informed. Hopefully this guide has quelled some of the concerns of novices, elaborated on debates, and opened up the SR to new audiences so that it will be used more often, by more researchers, policy makers and practioners and that it will continue to be a central part of the public health researchers' tool box. [START_REF] Mulrow | Systematic reviews: rationale for systematic reviews[END_REF] What is known on this subject: Systematic reviews are increasingly popular in terms of evaluating public health policies. However, inexperienced researchers are often apprehensive about conducting a systematic review This is because existing methodological guidance on the conduct of systematic reviews is too prescriptive and promotes an "ideal type" view of what a systematic review should be.

What this study adds:

This study draws on extensive practical experience of the conduct of various "real" systematic reviews across different aspects of public health policy It highlights pragmatic ways in which systematic reviews of public health policy interventions can be undertaken without compromising their methodological integrity It thereby dispels concerns about the systematic review and opens the method up to newcomers.

Box 1 -Case study systematic reviews

Completed Systematic Reviews

Effectiveness of the welfare to work programmes for people with a chronic illness or disability (2005). [START_REF] Bambra | Does "welfare to work" work? A systematic review of the effectiveness of the UK's welfare to work programmes for people with a chronic illness or disability[END_REF] Quantitative and qualitative studies of the employment effects of UK welfare to work programmes directed at people with a disability or a chronic illness were identified using 17 electronic databases, hand searches of the relevant literature, searches of the world wide web, citation follow-up, and contacts with authors. 16 qualitative and quantitative observational studies were included and critically appraised.

Interventions that aim to increase employee participation or control.(2007). [START_REF] Egan | The psychosocial and health effects of workplace reorganisation 1: a systematic review of interventions that aim to increase employee participation or control[END_REF] Experimental and quasiexperimental studies reporting health and psychosocial effects of interventions which increased employee participation or workplace control were examined. 17 electronic databases (medical, social science and economic), bibliographies, and expert contacts were searched. 18 observational studies were included and critically appraised.

Workplace task restructuring interventions (2007). [START_REF] Bambra | The psychosocial and health effects of workplace reorganisation 2: A systematic review of task restructuring interventions[END_REF] Experimental and quasi-experimental studies reporting health and psychosocial effects of changes to the work environment brought about by task structure work reorganisation were examined. 17 electronic databases (medical, social science and economic), bibliographies, and expert contacts were searched. 19 observational studies were reviewed.

The health effects of reorganising shift work (2008). [START_REF] Bambra | Shifting schedules: the health effects of reorganising shift work[END_REF] Systematic review of experimental and quasiexperimental studies that evaluated the effects on health and work-life balance of organisational level interventions that redesign shift work schedules. 27 electronic databases (medical, social science, economic) were searched. 26 observational studies were synthesised.

The effects of Compressed Work Week interventions on the health and wellbeing of shift workers (2008). [START_REF] Bambra | A hard day's night? The effects of Compressed Work Week interventions on the health and wellbeing of shift workers: a systematic review[END_REF] Studies of the effects of the Compressed Working Week on the health and work-life balance of shift workers were systematically reviewed. 27 electronic databases were searched as well as websites, bibliographies, and expert contacts. 40 observational studies were included.

The health effects of volunteering (2008). [START_REF] Smith | Partners in health? A systematic review of the effects on health and health inequalities of partnership working[END_REF] Systematic review of the health effects of volunteering on individual volunteers and on health service users. 11 electronic databases were searched for qualitative and quantitative studies (both intervention studies and comparative studies). Expert contacts used. 87 qualitative and observational quantitative papers were included in the review.

The effects on health and health inequalities of partnership working (2009). [START_REF] Casiday | Volunteering and Health: What Impact Does It Really Have? Report to Volunteering England[END_REF] Systematic review of quantitative (longitudinal before and after) and qualitative studies (1997)(1998)(1999)(2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008) reporting on the health (and health inequalities) effects of public health partnerships in England. 18 electronic databases (medical, social science and economic) were searched as well as websites, bibliographies, and expert contacts. 15 studies were reviewed.

Ongoing Systematic Reviews

Return to work interventions for people with a chronic illness. Update of 2005 review of the effectiveness of welfare to work interventions. [START_REF] Bambra | Does "welfare to work" work? A systematic review of the effectiveness of the UK's welfare to work programmes for people with a chronic illness or disability[END_REF] 16 electronic databases have been searched and 40 studies are currently being synthesised.

Psychosocial work environment and lower back pain. Systematic review of prospective cohort studies of the association between the psychosocial work environment and the development of lower back pain. 4 electronic databases as well as bibliographies have been searched.

Flexible working conditions and their effects on employee health and well being. A Cochrane collaboration systematic review of the effects of flexible working conditions on the health of workers. 6 electronic databases have been searched as well as bibliographies, websites and expert contacts.

Acknowledgements

This guide was originally written to assist novice systematic reviewers in the Department of Social Medicine at the Karolinska Institute, Stockholm whom I visited in February 2009.