Lebensm.-Wiss. u.-Technol.

, 34, 11 } 17 (2001)

Visualization of Food Structure by Confocal Laser Scanning Microscopy (CLSM)

Markus B. Du K rrenberger, Stephan Handschin, Be H atrice Conde-Petit*, Felix Escher

M. B. Du K rrenberger: Biocenter of the University of Basel, Interdepartmental Electron Microscopy, Klingelbergstrasse 70, CH-4056 Basel (Switzerland) S. Handschin, B. Conde-Petit, F. Escher: Swiss Federal Institute of Technology (ETH) Zurich, Institute of Food Science, CH-8092 Zurich (Switzerland) (Received September 19, 2000; accepted December 14, 2000)

Confocal Laser Scanning Microscopy is a rather new technique for structural analysis of biological and food material. In contrast to conventional light microscopy the light source is replaced by laser, a scanning unit and a pinhole in the back focal plane, which improves the limited depth of focus. The potential and limitations of CLSM are discussed based on experimental work. Yam parenchyma and wheat products (wheat dough, bread and pasta) were imaged by CLSM using Acid Fuchsin and Safranin O as yuorescent dyes. CLSM proved to be a useful tool for obtaining three-dimensional information on the cellular structure of yam parenchyma and on the properties of protein and starch networks in wheat products. Furthermore, CLSM allows the study of bread after baking in situ and the analysis of surface properties of pasta in the reyection mode.

2001 Academic Press Keywords:

Introduction Confocal Laser Scanning Microscopy (CLSM) is a relatively new optical tool which is increasingly being applied in the food area (Blonk & van Aalst, 1993; Vodovotz et al., 1996). The development of CLSM goes back to an invention in the nineteenth century. In 1884, Paul Nipkow designed a rotating disk with holes arranged in a spiral o!ering the possibility to scan images and transmit them by telegraphic wire. This principle led to technical developments in television technology, but the Nipkow disk itself fell into oblivion as television technology advanced. Around 1980 the Czech scientist M. Petran adapted the principle of the Nipkov disk to develop a confocal microscope. This progress eliminated the blurring caused by non focal information from images of thick objects. After a visit by Allan Boyde, a U.S. scientist, Petran was invited to continue his research in the labs of Allan Boyde. The result was the "rst commercially available confocal microscope (from NORAN Instruments) which has since found wide application in biological and medical sciences (Boyde, 1994; Pawley, 1990; White et al., 1987). The present publication aims at

giving an overview of the application of CLSM for structural characterization of complex food systems. In the "rst part, the principle but also the potential and limitations of CLSM are described. In the second part, applications of CLSM are presented based on experimental results. Yam (Dioscorea cayenensis rotundata) parenchyma and cereal foods such as bread and pasta have been investigated by CLSM to illustrate the potential of this technique.

The principle The primary value of the CLSM to research is its ability to produce optical sections through a three-dimensional (3-D) specimen, for example a piece of tissue or other thick objects ( Fig. 1). An optical section contains information from one focal plane only. Therefore, by moving the focal plane of the instrument by steps of de"ned distance ( m-range) through the depth of the specimen, a stack of optical sections can be recorded (Lichtman, 1994). This property of the CLSM is fundamental for solving 3-D problems where information from regions distant from the plane of focus can blur the image of such objects. For imaging in the CLSM, either the epi-#uorescence or the epi-re#ection mode is generally used. As

* Corresponding author.

0023-6438/01/020011 # 07 $35.00/0 2001 Academic Press

doi:10.1006/ fstl.2000.0739 All articles available online at http://www.idealibrary.com on

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Fig. 1 Stack of optical sections from focal planes at di!erent depths through a specimen which provides a three-dimensional image of the object

a valuable by-product, the computer-controlled CLSM produces digital images which are amenable to image analysis and processing, and can also be used to compute surface- or volume-rendered 3-D reconstructions of the specimen. Optical sections in a CLSM are composed from a repeated point experiment to get a scanned image (Pawley, 1990; Wilson & Sheppard 1984). To image a specimen point by point (scanned), a collimated polarized laser beam is de#ected stepwise in the x- and y-direction by a scanning unit before it is re#ected by a dichroic mirror (beam splitter) so as to pass through the objective lens of the microscope, and focused onto the specimen. The emitted longer-wavelength #uorescent light (or re#ected light in re#ection mode) from the components of interest stained with #uorescent dyes is collected by the objective lens, passes through the dichroic mirror (transparent for the longer wavelength, or semi-transparent for re#ection mode) and is focused into a small pinhole (the confocal aperture) to eliminate out-of-focus light. Therefore, the CLSM not only provides excellent resolution within the plane of the section (50.25 m in x- and y-direction), but also yields good resolution between section planes (50.25 m in z-direction). A light-sensitive detector, as presented in Fig. 2, which is positioned behind the confocal aperture, records the in-focus information of each specimen point, and the analogue output signal is digitized and fed into a computer to generate an image on a monitor. A stack of serial optical sections, each consisting of a pixel-matrix in digital form through the specimen, o!ers the possibility to compute either a composite projection, or a volume-rendered 3-D representation of the specimen. The confocal part of a CLSM is comparable with an elaborate, highly folded optical bench on which the laser, all optical elements and the detector are mounted. When working in the epi-#uorescence mode, the laser beam is "ltered to select distinct monochromatic wavelengths from one or several lasers (360 nm, 458 nm, 488 nm, 543 nm, 568 nm, 633 nm, 647 nm). Furthermore, a multi dichroic mirror that re#ects excitation and transmits emission wavelengths is used to generate #uorochrome speci"c signals on up to four detectors. For the epire#ection mode no wavelength "lters are needed. Instead, a semi-transparent mirror re#ects 50% of the incident

Fig. 2 Principle of confocal imaging: The numbered elements are: (1) laser, (2) dichroic mirror, (3) objective lens, (4) thick specimen, (5) confocal pinhole, (6) photomultiplier

laser beam through the objective lens to reach the specimen. The mirror transmits 50% of the light re#ected by the specimen and collected by the objective lens, to the detector.

Potential and Limitations The CLSM detects in-focus regions only, the out-of-focus parts appearing black. Therefore, the application is not limited to thin samples. For instance, rather thick food samples can be analyzed by CLSM to obtain structural information. A precise view of the spatial arrangements of structural elements may be obtained by collecting a 3-D data set of the sample. Visualization of the sample occurs at ambient conditions which allows the observation of the sample in the hydrated state. For instance, the microstructure of aqueous phase separated protein-polysaccharide mixtures can be assessed without changing the solvent conditions (Bourriot et al., 1999). A further advantage of CLSM is the possibility to follow in situ the dynamics of processes such as phase separation, coalescence, aggregation, coagulation, solubilization, etc. Specially designed stages, which allow heating, cooling or mixing of the sample, give the possibility to simulate food processing under the microscope (Thorvaldsson et al., 1998). In some cases, only a few preparatory steps are necessary for viewing a specimen by CLSM. This often applies to

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native plant tissue where the naturally occurring #uorescence (auto#uorescence) may be su$cient to generate a contrast. Further examples are biopolymer emulsions containing gelatin, where the auto#uorescence of gelatin allows visualization of the microstructure of the emulsion in situ (Foster et al., 1997). Alternatively, the component of interest in the sample may be labelled. Fluorescent dyes are labelling agents which contain excitable structures that emit #uorescence after illumination by light of a speci"c wavelength. The photons emitted by the #uorescent dyes are visible even below the resolution limit of the microscope as these structures appear as spots with a diameter of the resolution limit of the microscope optics. CSLM may be combined with #uorescence intensity to quantify the labelled component. In aqueous biopolymer mixtures, for instance, this technique allows the quanti"cation of biopolymer concentration and phase volume (Blonk et al., 1995). Furthermore, pH gradients in a sample may be detected by using a #uorescent compound that is sensitive to pH (Hassan et al., 1995). CLSM o!ers the possibility to analyze the topography and surface of samples in the epi-re#ection mode. In this mode the laser light which is re#ected from the sample is collected as signal. To increase the signal intensity the sample can be covered with a thin metal "lm by sputtercoating as for the preparation for scanning electron microscopy. Conventional transmission light imaging is limited when applied to thick sections, because the images can be blurred by out-of-focus information. Recently, with increasing power of image processing computers, an image reconstruction tool called deconvolution was developed to treat focal series of transmitted light images. The results are deblurred 3-D representations similar to confocal stacks of images. Drastic alterations in the design of CLSM's could lead to the possibility of recording transmitted light confocal images in the future. Such methods would imply fewer preparation steps, (e.g. the omission of staining), to be ready for confocal 3-D imaging. A major limitation of CLSM is the fact that most samples require some treatment to be visible. All steps such as staining or quenching of auto#uorescence, which have to be performed in liquids at room temperature can result in artefacts like swelling and solubilization of components. Many #uorochromes are sensitive to laser-illumination and can bleach within the time necessary for searching and acquiring an image. Objective lenses with a high numerical aperture (e.g. 63;NA 1.4, 40;NA 1.0) are normally used to provide good resolving power in x-, yand z-direction. By physical laws their working distance, measured between the front lens and the top of the sample (cover glass), is restricted to about 100 m. The use of objective lenses with longer working distances results in lower resolution of the image. Furthermore, the penetration of the laser beam into samples is restricted and is in a maximum range of 100 to 150 m in zdirection. Short-pulsed double photon illumination, where the energy of two photons comes to addition in the focus plain and can be absorbed by a #uorochrome, will cause emission of higher energy (shorter wavelength)

photons. This can result in extended penetration of the beam into the sample and in reduced bleaching of #uorochromes, but the lasers required are extremely expensive and must be tuned before use. In addition only one #uorescence channel is available, since the principle of double photon shooting does not allow multiple wavelength illumination. The resolving power according to the rule of thumb (half the wavelength of illumination) is not restricting because a shorter wavelength of illumination results. This can be done by interference of a double pulse of the longer wavelength.

Experimental Sample preparation Yam. Fresh parenchyma tissue of yam (Dioscorea cayenensis rotundata cultivar Lopka from the Ivory Coast) was cut into sections of 150 m thickness with a manual microtome (Leica microsystems, CH-Glattbrugg) equipped with a knife-holder for conventional razor-blades. The sections were stained by immersion into aqueous Safranin O solution 0.04% (Sigma-Aldrich, CH-Buchs) for 30 min followed by rinsing in deionized water for 30 min.

Wheat dough and bread. Two procedures were developed for the preparation of wheat dough and bread samples. In one case, wheat dough and bread were prepared and cryo-sectioned (50 m thickness) as described by Hug-Iten et al. (1999). Prior to sectioning the auto#uorescence of dough and bread was removed by soaking small pieces of samples in 10 mL aqueous solution of Heparin (500 i.U. per mL Liquemin, Roche, CH-Basel) for 30 min, followed by rinsing in deionized water for 30 min. Alternatively, a wheat dough without yeast was prepared from low extraction wheat #our, which had previously been bleached with Heparin. For this purpose, 200 g wheat #our was treated with 500 mL aqueous Heparin 500 i.U. for 30 min, followed by rinsing with deionized water (30 min). The dough samples were heated directly under the microscope in a specially designed stage. Baking was simulated by heating from ambient temperature to 80 3C within 35 min. Wheat dough and bread were stained either with Acid Fuchsin (Sigma-Aldrich, CH-Buchs) or Safranin O. Staining with Safranin O was carried out as described for yam. Staining with Acid Fuchsin was carried out as described by Fardet et al. (1998). The cryosections where immersed in Acid Fuchsin solution (0.01 g Acid Fuchsin in 1% acetic acid) for 10 min followed by rinsing in deionized water for 60 min.

Spaghetti. Spaghetti were prepared and dried at 55 3C as described by Zweifel et al. (2000), then cooked to the optimal cooking point and subsequently cooled by immersion in water at room temperature for 1 min. Auto#uorescence was removed as described for bread.

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Spaghetti samples were cryosectioned (50 m) as described by Cunin et al. (1995). Staining with Acid Fuchsin was performed as described for bread. For recording of images of the surface in the re#ection mode the spaghetti samples were sputtered with a 30 nm gold layer in a sputtering device (Baltec, FL-Balzers).

Microscopy Images were recorded with a Leica TCS SP (spectrometer type) CLSM mounted on an upright DM RXE #uorescence light microscope (research type, Leica Lasertechnik GmbH, D-Heidelberg). The samples were mounted on glass slides and covered with deionized water and a cover glass. The samples stained with Acid Fuchsin were illuminated with the krypton laser at 568 nm. Samples with Safranin O with the argon-ion laser at 488 nm. Initially, the maximum of the emission peak was determined with the spectrometer of the microscope. The emission maxima were 540 and 620 for samples stained with Safranin O and Acid Fuchsin, respectively. Therefore, the bandwidth for recording the #uorescence images was set from 530 to 550 nm for Safranin O and from 600 to 620 for Acid Fuchsin. Samples were observed in the epi-#uorescence mode except spaghetti, where re#ection mode was also used. Approximately 40 images were recorded per stack covering a depth of about 40 m in the sample. Surface projections were carried out using Imaris software (Bitplane AG, CH-Zu K rich). For conversion from stack to projection pictures were processed with the shareware-program NIH Image (National Institutes of Health, Bethesda, Maryland, U.S.A.).

Results and Discussion Preliminary experiments with fresh yam tissue showed that yam does not exhibit auto#uorescence at the wavelengths used. Therefore staining was necessary to generate contrast. Fig. 3 presents the microstructure of fresh yam parenchyma tissue after staining with Safranin O. The cellular structure of yam is clearly visible, as well as the native starch granules within the cells. The triangular form of the starch granules, which is typical for Dioscorea cayenensis rotundata, can be recognized. Note that Safranin O stains starch, but also the polysaccharides in the cell wall. Similar studies on the structure of apples (Lapsley et al., 1992), grapes (Gray et al., 1999), and strawberries (Suutarinen et al., 2000) show that structural features of plants in particular cell size and shape, cell adhesion and internal air spaces can be visualized by CSLM. Regarding the starch fraction, details of their morphology have been revealed by this technique such as the pores on the surface of maize and sorghum starch (Huber & BeMiller, 2000). In contrast to yam, wheat products exhibited auto#uorescence. Control experiments con"rmed that bleaching with Heparin completely removes auto#uorescence (micrographs not shown). Fig. 4a and 4b show micro-

graphs of wheat dough and bread stained with Acid Fuchsin. The latter dye is a well known staining agent for protein. The bright areas on the micrograph of dough (Fig. 4a) correspond to the coarse network of wheat protein, also termed gluten. The starch granules are also clearly recognizable. It is likely that proteins associated with the starch surface (Seguchi & Yoshino, 1999) contribute to the visualization of starch. In bread, a "ne stranded gluten network is recognizable (Fig. 4b). The bright spots distributed throughout the micrograph can be attributed to yeast labelled with Acid Fuchsin. The dark areas correspond to swollen (gelatinized) starch. The morphology of starch in bread is less recognizable than in dough, most probably due to the swelling of the starch granules. The micrographs of both dough and bread show that protein and starch are not evenly distributed. In dough, regions are found where starch granules are segregated from protein. Likewise, in dough and bread the protein network is interrupted by accumulations of starch. Spaghetti is another wheat based product where CLSM can be used to assess the continuity of the protein phase. Fig. 5 shows a cross section of cooked spaghetti stained with Acid Fuchsin. The micrograph reveals that protein forms a dense continuous network towards the centre of the spaghetti strand. In contrast, in the outer layer of cooked spaghetti the continuity of the protein network is almost lost. This result con"rms earlier studies on the microstructure of spaghetti using light microscopy, where it was shown that the centre of cooked pasta is dominated by the protein fraction, whereas in the outer layer the strand swelling of starch contributes to a disruption of the protein network (Cunin et al., 1995; Fardet et al., 1998). CSLM was also successfully applied to visualize other protein networks such as the gelation of milk proteins as induced by milk acidi"cation (Hassan et al., 1995). Contrary to protein, visualization of starch by CLSM is more di$cult, since there is little information on speci"c #uorescent dyes for starch in the literature except for methods based on Concanavalin A (Gibson et al., 1997). In the present investigation the suitability of Safranin O for staining starch has been studied. Fig. 6a and 6b show bread directly after baking and after 7 d of ageing. In fresh bread swollen and mostly elongated starch granules, which are not intensively stained, are recognizable. The bright zones within the starch granules and in the intergranular space most probably correspond to phase separated amylose (Fig. 6a). Phase separation of the two starch polymers, amylose and amylopectin, is the result of polymer incompatibility. Phase separation of starch in bread was previously shown by Hug-Iten et al. (1999) using light microscopy. In aged bread, an intensively stained intergranular phase is still visible, but not throughout the sample. Furthermore, the bright areas within the starch granules are no longer visible. This observation suggests that the increase in molecular order of starch, which is known to occur during ageing of bread, has an in#uence on the staining intensity of Safranin O. It is conceivable that densely packed starch, for instance crystallites, are not as easily penetrated by the dye as the plasticized amorphous zones of starch. It

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Fig. 3 CLSM of freshly cut yam (Dioscorea cayenensis rotundata, var. Lokpa), stained with Safranin O. The micrograph is shadowed by image-processing to provide a three-dimensional view of the cells Fig. 4 (a) CLSM of a cryosection of wheat dough stained with Acid Fuchsin for protein (b) CLSM of a cryosection of fresh bread stained with Acid Fuchsin for protein Fig. 5 CLSM of a cryosection of cooked spaghetti stained with Acid Fuchsin for protein

should be added, that Safranin O does not show #uorescence in the absence of starch. The nature of the starchSafranin O interaction is not fully understood and needs further investigation. In order to follow the structural changes of starch during baking of bread, dough without yeast stained with Safranin O was heated to 80 3C under the microscope.

A micrograph of the resulting &bread' is presented in Fig. 7. The microstructure of bread baked under the microscope is very similar to regular bread (Fig. 6a) with the di!erence, that no pores can be seen. The gelatinized starch granules are less intensively stained since dough was stained before heating when starch was in the native state. It is, therefore, concluded that the baking process

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Fig. 6 (a) CLSM of a cryosection of fresh bread stained with Safranin O for starch. Arrow indicates a pore limited by the pore wall. (b) CLSM of a cryosection of bread stored for 7 days at 20 3C, stained with Safranin O Fig. 7 CLSM of bread baked on the microscope stage stained with Safranin O Fig. 8 Topographic image of dried spaghetti, generated by CLSM in the epi-re#ection mode. Arrows indicate starch granules hold in the protein phase

can be followed in situ by CLSM. Detailed information on the transformation of starch, but also of other changes such as the coagulation of proteins could be collected. Finally, the surface of dried spaghetti was investigated by CLSM in the epi-re#ection mode. Fig. 8 shows the surface of dried spaghetti sputtered with a thin gold-layer. The micrographs reveal that the starch granules on the

pasta surface are partly embedded in a protein network. Compared to SEM images, the 3-D dataset of CLSM also contains information on the z-dimension. Therefore, surface properties such as roughness can be calculated based on CLSM results. It should be noted that this sample preparation for CLSM in the re#ection mode is similar to the metal coating for Scanning Electron

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Microscopy (SEM) in that the thickness of the gold layer is below the resolving power of light microscopy and therefore does not in#uence the imaging of structural details.

Conclusions Based on the above presented examples it can be concluded that CLSM broadens the application of conventional light microscopy. It gives the possibility to examine the internal structure of rather thick samples in three dimensions. Samples can be viewed in the hydrated state and, therefore, food transformations during processing may be followed in situ using specially designed stages. The power of CLSM will further increase as more speci"c labelling agents for food components become available. Conventional labelling with #uorescent dyes as well as more sophisticated techniques such as immunolabelling need further investigation. Finally the simultaneous labelling of two or more components of foods with probes which are speci"c for each component is necessary for an even more detailed analysis of food structure. The possibility to combine CLSM with rheological measurements, light scattering and other physical analytical techniques in the same experiments with specially designed stages o!ers the possibility to obtain detailed structural information of complex food systems.

References
BLONK, J. C. G. AND VAN AALST, H. Confocal scanning light microcopy in food research. Food Research International, 26, 297}311 (1993) BLONK, J. C. G., VAN EENDENBURG, J., KONING, M. M. G., WEISENBORN, P. C. M. AND WINKEL, C. A new CSLM-based method for determination of the phase behaviour of aqueous mixtures of biopolymers, Carbohydrate Polymers, 28, 287}295 (1995) BOURRIOT, S., GARNIER, C. AND DOUBLIER, J. L. Micellar casein-kappa-carrageenan mixtures. I. Phase separation and ultrastructure. Carbohydrate Polymers, 40, 145}157 (1999) BOYDE, A. Bibliography on confocal microscopy and its applications. Scanning, 16, 33}56 (1994) CUNIN, C., HANDSCHIN, S., WALTHER, P. AND ESCHER, F. Structural changes of starch during cooking of durum wheat pasta. ebensmittel-=issenschaft und-echnologie, 28, 323}328 (1995) FARDET, A., BALDWIN, P. M., BERTRAND, D., BOUCHET, B., GALLANT, D. J. AND BARRY, J.-L. Textural images analysis of

pasta protein networks to determine in#uence of technological processes. Cereal Chemistry 75, 699 }704 (1998) FOSTER, T., UNDERDOWN, J., BROWN, T., FERDINANDO, D. P. AND NORLON, I. T. Emulsion behaviour of nongelled biG HL opolymer mixtures. In: E. DICKINSON, B. BERGENSTA (Eds). Food colloids, proteins, lipids and polysaccharides. Cambridge: The Royal Society of Chemistry (1997) GIBSON, T. S., SOLAH, V. A. AND MCCLEARY, B. V. A procedure to measure amylose in cereal starches and #ours with concanavalin A. Journal of Cereal Science, 25, 111}119 (1997) GRAY, J. D., KOLESIK, P., HOJ, P. B. AND COOMBE, B. G. Confocal measurements of the three-dimensional size and shape of plant parenchyma cells in a developing fruit tissue. Plant Journal, 19, 229 }236 (1999) HASSAN, A. N., FRANK, J. F., FARMER, M. A., SCHMIDT, K. A. AND SHALABI, S. I. Formation of yoghurt microstructure and three-dimensional visualization as determined by confocal scanning lasser microscopy. Journal of Dairy Science, 78, 2629 }2636 (1995) HUG-ITEN, S., HANDSCHIN, S., CONDE-PETIT, B. AND ESCHER, F. Changes in starch microstructure on baking and staling of wheat bread. ebensmittel-=issenschaft und-echnologie, 32, 255}260 (1999) LAPSLEY, K. G., ESCHER, F. AND HOEHN, E. The cellular structure of selected apple varieties. Food Structure, 11, 339 }349 (1992) LICHTMAN, J. W. Confocal microscopy. Scienti,c American, 271, 40 } 45 (1994) PAWLEY, J. B. he handbook of biological confocal microscopy. Plenum, New York (1990) SEGUCHI, M. AND YOSHINO, Y. Microdetermination of surface proteins of wheat starch granules by dye binding. Cereal Chemistry, 76, 410 } 412 (1999) SUUTARINEN, J., HEISKA, K., MOSS, P. AND AUTIO, K. The e!ects of calcium chloride and sucrose prefreezing treatments on the structure of strawberry tissues. ebensmittel-=issenschaft und echnologie, 33, 89 }102 (2000) THORVALDSSON, K., STADING, M., NILSSON, K., KIDMAN, S. AND LANGTON, M. Rheology and structure of heat-treated pasta dough: In#uence of water content and heating rate. ebensmittel-=issenschaft und-echnologie, 26, 297}311 (1998) VODOVOTZ, Y., VITTANDINI, E., COUPLAND, J., MCCLEMENTS, D. J. AND CHINACHOTI, P. Bridging the gap: use of confocal microscopy in food research. Food echnology, 50, 74, 76-82 (1996) WHITE, J. G., AMOS, W. B. AND FORDHAM, M. An evaluation of confocal versus conventional imaging of biological structures by #uorescence light microscopy. Journal of Cell Biology, 105, 41} 48 (1987) WILSON, T. AND SHEPPARD, C. heory and practice of scanning optical microscopy, London: Academic Press (1984) ZWEIFEL, C., CONDE-PETIT, B. AND ESCHER, F. Thermal modi"cations of starch during high-temperature drying of pasta. Cereal Chemistry, 77, 645}651 (2000)

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