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A Novel Technique for Ice CrystalVisualization in Frozen Solids UsingX-Ray Micro-Computed TomographyRRRRREZEZEZEZEZAAAAA M M M M MOUSAOUSAOUSAOUSAOUSAVIVIVIVIVI, , , , , TTTTTAAAAAGHIGHIGHIGHIGHI M M M M MIRIIRIIRIIRIIRI, P, P, P, P, P.W.W.W.W.W. C. C. C. C. COOOOOXXXXX, , , , , ANDANDANDANDAND P P P P PETERETERETERETERETER J. F J. F J. F J. F J. FRRRRRYERYERYERYERYER

ABSTRAABSTRAABSTRAABSTRAABSTRACTCTCTCTCT: A no: A no: A no: A no: A novvvvvel techniqueel techniqueel techniqueel techniqueel technique, using an X-r, using an X-r, using an X-r, using an X-r, using an X-ray micray micray micray micray micro-computed tomogro-computed tomogro-computed tomogro-computed tomogro-computed tomography system (X-raphy system (X-raphy system (X-raphy system (X-raphy system (X-ray micray micray micray micray micro-CT system)o-CT system)o-CT system)o-CT system)o-CT system)has been developed for visualization of the two- (2-D) and three-dimensional (3-D) ice crystal structures formedhas been developed for visualization of the two- (2-D) and three-dimensional (3-D) ice crystal structures formedhas been developed for visualization of the two- (2-D) and three-dimensional (3-D) ice crystal structures formedhas been developed for visualization of the two- (2-D) and three-dimensional (3-D) ice crystal structures formedhas been developed for visualization of the two- (2-D) and three-dimensional (3-D) ice crystal structures formedduring freezing. The system reconstructs all 3-D images based on a set of 2-D images obtained by multipleduring freezing. The system reconstructs all 3-D images based on a set of 2-D images obtained by multipleduring freezing. The system reconstructs all 3-D images based on a set of 2-D images obtained by multipleduring freezing. The system reconstructs all 3-D images based on a set of 2-D images obtained by multipleduring freezing. The system reconstructs all 3-D images based on a set of 2-D images obtained by multipleslicing of an X-ray shadow image. This study demonstrates the capability of the technique to characterize the iceslicing of an X-ray shadow image. This study demonstrates the capability of the technique to characterize the iceslicing of an X-ray shadow image. This study demonstrates the capability of the technique to characterize the iceslicing of an X-ray shadow image. This study demonstrates the capability of the technique to characterize the iceslicing of an X-ray shadow image. This study demonstrates the capability of the technique to characterize the icecrystal microstructure of mycoprotein products after freezing. Results are presented for the 2-D ice crystalscrystal microstructure of mycoprotein products after freezing. Results are presented for the 2-D ice crystalscrystal microstructure of mycoprotein products after freezing. Results are presented for the 2-D ice crystalscrystal microstructure of mycoprotein products after freezing. Results are presented for the 2-D ice crystalscrystal microstructure of mycoprotein products after freezing. Results are presented for the 2-D ice crystalsformed within mycoprotein frozen at different rates. The method requires freeze-drying of the sample to removeformed within mycoprotein frozen at different rates. The method requires freeze-drying of the sample to removeformed within mycoprotein frozen at different rates. The method requires freeze-drying of the sample to removeformed within mycoprotein frozen at different rates. The method requires freeze-drying of the sample to removeformed within mycoprotein frozen at different rates. The method requires freeze-drying of the sample to removefrozen water before scanning to indicate ice crystal and internal structure of the material at a depth of 1 cm. Thefrozen water before scanning to indicate ice crystal and internal structure of the material at a depth of 1 cm. Thefrozen water before scanning to indicate ice crystal and internal structure of the material at a depth of 1 cm. Thefrozen water before scanning to indicate ice crystal and internal structure of the material at a depth of 1 cm. Thefrozen water before scanning to indicate ice crystal and internal structure of the material at a depth of 1 cm. Thedendrite spacing of ice crystals has been related to the freezing conditions of the material.dendrite spacing of ice crystals has been related to the freezing conditions of the material.dendrite spacing of ice crystals has been related to the freezing conditions of the material.dendrite spacing of ice crystals has been related to the freezing conditions of the material.dendrite spacing of ice crystals has been related to the freezing conditions of the material.

KKKKKeyworeyworeyworeyworeywords: myds: myds: myds: myds: mycoprcoprcoprcoprcoprotein, ice crotein, ice crotein, ice crotein, ice crotein, ice crystal, X-rystal, X-rystal, X-rystal, X-rystal, X-ray micray micray micray micray micro-tomogro-tomogro-tomogro-tomogro-tomographyaphyaphyaphyaphy, scanning electr, scanning electr, scanning electr, scanning electr, scanning electron micron micron micron micron microscoposcoposcoposcoposcopyyyyy, fr, fr, fr, fr, freeeeeeeeeezingzingzingzingzing

Introduction

The use of freezing as a preservation technique is well estab-lished for many commodities as well as processed foods. Freez-

ing often results in substantial textural damage caused by thegrowth of ice crystals within the delicate structure either presentnaturally or created during processing. However, freezing can alsogenerate a textured product from an amorphous protein paste orslurry (Lawrence and others 1986). In myco-protein, composed ofFusarium venenatum mycelium set with egg albumin, freezing isconsidered necessary to produce the required fibrous texture. Theprocess is possibly analogous to the “freeze texturization” used forsome food protein materials, such as kori-tofu (Lawrence and others1986). Freeze texturization relies on the compression of the mate-rial between ice crystals, which result in the polymerization of pro-teins into fibers. Understanding the relationship between thefreezing conditions and the size of ice crystals formed is critical incontrolling product quality and texture.

Observation of the ice crystal size may be direct or indirect. Di-rect observation methods include cryo-scanning electron micros-copy (Russell and others 1999), cold microscopy (Donhowe andothers 1991), and confocal laser scanning microscopy (Evans andothers 1996). Indirect methods such as freeze substitution (Bev-ilacqua and others 1979; Martino and Zarizky (1998), freeze fixation(Miyawaki and others 1992), and freeze-drying techniques (Woinetand others 1998a, 1998b; Fayadi and others 2001) followed by sec-tioning have been used. Indirect methods assume that the origi-nal morphology is maintained during the sectioning into thinenough layers to allow microscopic methods to be used. These tech-niques, thus, have the disadvantages that the microstructuremight change during cutting. They are also limited to observationof a thin layer of material. In addition, conventional optical or elec-

tron microscopy allows visualization of only two-dimensional (2-D)images or thin slices. In most cases, conclusions about the originalthree-dimensional (3-D) ice structures cannot be made on the baseof 2-D information unless a suitable stereological method is ap-plied to relate the 3-D structure to the measured 2-D structure(Underwood 1970; Xu and Pitot 2003).

Three-dimensional information on ice crystal structures can beachieved either by first obtaining the 2-D information and then us-ing image reconstruction techniques or directly from 2-D informa-tion by appropriate software. Ueno and others (2004) and Do andothers (2004) applied a Micro-Slicer Image Processing System(MSIPS) to observe 3-D ice crystal structure in frozen dilute solu-tion and beef. They used algorithms to reconstruct the 3-D imagebased on 2-D cross-sections resulted from multiple-slicing of a fro-zen sample with the minimum thickness of 1 �m. The method iscumbersome and possibly unreliable, as the ice structure itself canbe altered by the preparation technique and the distance betweenthe slices may be too coarse to avoid loss of 3-D information.

The X-ray micro-computed tomography system (X-ray micro-CTsystem) allows visualization and measurement of complete three-dimensional object structures without sample preparation or chem-ical fixation. It uses a combination of X-ray microscopy and tomo-graphical algorithms, based on the contrast in X-ray imagesgenerated by differences in X-ray attenuation (absorption andscattering) arising from differences in density of material within thespecimen. X-ray pass through a specimen that is rotated in manydifferent directions and yield an image that displays differences indensity at thousands of points in the 2-D slices through the spec-imen. Many contiguous slices, each of a certain finite thickness ofaround 18 �m, are generated in this way and stacked up to recon-struct a 3-D distribution of material density within the object. Thetechnique has so far been successfully applied to a wide range ofmaterials such as rock, bone, ceramic, metal, and granules (Farberand others 2003; Salvo and others 2003). The aim of this study is todemonstrate the potential of X-ray micro-CT system as a nonde-structive technique for the study of the internal microstructure offoods, using mycoprotein as a model material.

MS 20050143 Submitted 3/3/05, Revised 4/20/05, Accepted 5/18/05. AuthorMousavi is with Faculty of Agriculture, Shahid Bahonar Univ. of Kerman, 22Bahman Blvd, P.O. Box 76169-133, Kerman, Iran. Authors Mousavi, Miri,Cox, and Fryer are with Centre for Formulation Engineering, Dept. of Chemi-cal Engineering, The Univ. of Birmingham, Edgbaston, Birmingham, U.K.Direct inquiries to author Mousavi (E-mail: rezamousavi_49@yahoo.co.uk).

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E: Food Engineering & Physical Properties

Ice crystal visualization by x-ray . . .

Material and Methods

Three mycoprotein products of paste, dough, and steameddough were taken from the production line of Marlow Foods

Ltd (Stokesley, U.K.). The paste was the final dewatered product ofthe fermentation process. The paste is then mixed with other ingre-dients, such as egg albumin, to form dough. This dough is then giv-en a “steam” treatment to set the egg albumin: this is referred to as“steamed dough.”

Freezing methodFreezing methodFreezing methodFreezing methodFreezing methodFigure 1 shows the experimental setup to provide unidirectional

freezing in a test situation. The freezing cylinder was made from 2Perspex tubes with the same length (6.5 cm), interior tube diameter(2.2 cm), and exterior tube diameter (3 cm). Samples were loadedinside the bottom of the inner tube without leaving any gap be-tween the sample and tube wall to minimize heat transfer from thesides. The middle tube was then located in an exterior tube, sur-rounded by an insulated layer to minimize heat loss. Both tubeswere then clamped between 2 flat aluminum caps at base and topto ensure heat transfer easier in that direction. The freezing cell wasthen placed either (1) on a controlled temperature plate freezer fora slow freezing method or (2) into a liquid nitrogen flask for fastfreezing. Thermocouples, attached to a computer, recorded thetemperature as a function of time during freezing of the material.In other experiments, the temperature at different axial positionswithin the sample was recorded. Figure 2 shows typical tempera-ture profiles for the paste at the lowest (Figure 2a): temperature ofthe base plate set to –5 °C) and highest freezing rate (Figure 2b):liquid nitrogen at –196 °C). These measurements allowed the exactoperating conditions within the specimen to be determined, andalso enable to link ice crystal size information to the freezing rates.

Freeze-dryingFreeze-dryingFreeze-dryingFreeze-dryingFreeze-dryingAfter freezing, both slow and fast frozen cylinders were placed in

a vacuum chamber (3 torr or 4 mbar). The aim of this was to removemoisture from the frozen samples without damaging their struc-ture. A combination of a vacuum pump and its ice trap held at –50 °C was used to recover the condensed water vapor. The devicewas also used to promote ice sublimation. At the beginning, the tem-perature was just below the vitreous transition temperature of thesample to avoid ice melting and collapse effects.

X-ray micro-computed tomographyX-ray micro-computed tomographyX-ray micro-computed tomographyX-ray micro-computed tomographyX-ray micro-computed tomographyMore information about this technique can be found in Maire

and others (2001), Sasov and Van Dyke (1998), Salvo and others

(2003), and Lim and Barigou (2004). Samples were scanned usinga high resolution desktop X-ray micro-CT system (Skyscan 1072,Belgium), which consists of a micro-focus sealed X-ray tube with aspot size of 5 �m operating at a voltage of 100 kV and current of 96�A. For micro-tomographical reconstruction, X-ray images wereacquired from up to 400 views through 180 degrees of rotation. Thescanning process is controlled by Sky scan internal software, whichalso allows micro-tomographical reconstruction. The reconstructionalgorithm is based on the filtered back projection procedure for fan-beam geometry with specific noise reduction corrections (Skyscan2003). The subsequent reconstruction of 1 slice of 1024 × 1024 pix-els from 200 projections takes approximately 20 s.

Before scanning, freeze-dried samples of mycoprotein were cutvertically into 4 equal sections parallel to the heat flux. Each slicewas then irradiated at right angles to the heat flux. Thus a collectedimage was representative of the whole microstructure of voids fromthe base to the top with high resolution. A typical scan took around25 to 30 min. Samples were scanned in their native environmentconditions without any special preparation. The temperature in-side the X-ray chamber was only a few degrees higher than theroom temperature, but to minimize potential evaporation, freshand non–freeze-dried samples were covered by a plastic film andwere sealed completely before the scanning process. Shadow imag-es obtained in such a manner were then converted to 2-D and 3-Dimages by the reconstruction software.

Figure 1—Schematic diagram of unidirectional freezingapparatus

Figure 2—(a) Typical temperature transients formycoprotein paste using 4 thermocouples at different dis-tances from the base plate held at –5 °C. (b) Typical tem-perature transients for mycoprotein paste using 4 thermo-couples at different depths into the specimen from baseplate held at –196 °C (liquid nitrogen).

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Ice crystal visualization by x-ray . . .

Scanning electron microscopy (SEM)Scanning electron microscopy (SEM)Scanning electron microscopy (SEM)Scanning electron microscopy (SEM)Scanning electron microscopy (SEM)An environmental scanning electron microscopy technique

(ESEM) was used to image samples of fresh mycoprotein in wetmode to allow comparison with the X-ray micro-computed tech-nique. Micrographs of these samples were obtained using a scan-ning electron microscope (Philips FAI XL30 ESEM FEG). The sametechnique was also used for a dry mode observation of frozen sam-ples after freeze drying (SEM), but the sample holder for SEM wastransferred to coating equipment and covered by gold beforetransferring them to the SEM chamber.

Image analysisImage analysisImage analysisImage analysisImage analysisA Leica image analysis system (Quantimet 600) was used for ice

crystal width measurement. The scale bar of SEM image was used tocalibrate the image analyser while calibration for X-ray image wascarried out according to specified ruler unit left around the imageafter 2-D reconstruction process. An interactive feature measure-ment was used to draw multiple lines across the needle shape voids.The distance between 2 sides of the needle-shaped voids in the X-ray and those in the microscopic images was considered as corre-sponding to the original ice crystal width in all conditions.

Statistical analysisStatistical analysisStatistical analysisStatistical analysisStatistical analysisStatistical analysis was performed using Microsoft Excel 2000.

Student t tests were applied on mean values with a significancelevel of P < 0.05. The significance of differences between X-ray andSEM measurements of mean ice crystal width in paste at differentaxial positions was evaluated.

Results and Discussion

Ice crystal validation procedureIce crystal validation procedureIce crystal validation procedureIce crystal validation procedureIce crystal validation procedurePreliminary experiments showed that there was an increase in

the porosity of freeze-dried mycoprotein as opposed to the freshmaterial. However, it was not clear if the needle-shaped voids seenin X-ray scanning of freeze-dried materials corresponded to thespace left by ice crystals or whether they were artifacts. Figure 3compares the microstructure determined by the SEM and X-raymethods for fresh and freeze-dried paste frozen using the unidi-rectional slow-freezing method. The microstructure of fresh myco-protein paste was seen to be similar for the 2 methods, that is, no

ice crystal–related pore structure could be seen in either method forthe fresh sample. However, observation of the freeze-dried materialshowed parallel needle-shaped voids in the X-ray and ridges ofcompacted material in the SEM caused by distortion of microstruc-ture by ice crystals. The SEM in both cases shows more details: thestrands of mycoprotein, compressed by the needle-shaped icecrystals, can be clearly seen as they are in slow frozen sample. TheX-ray image shows the structure in less detail, but parallel voidscan clearly be seen. The microstructure of fresh and freeze-drieddough frozen under unidirectional slow freezing is seen in Figure 4.The microstructure of the fresh dough was similar to the freshpaste; X-ray analysis showed a smooth tomographic image. Again,needle shapes were seen in both SEM and on the X-ray. In contrastto both dough and paste, the steamed dough did not produce acompletely parallel needle shape (details not shown). This can berelated to its microstructural differences, produced by the egg al-bumin being heat set before freezing.

To validate whether the dendrite spacing seen by X-ray method isthe space that was originally filled by ice crystals, SEM and X-rayimages taken from different axial locations of the sample were ana-lyzed. To analyze the X-ray image at different axial positions fromfreezing plate, around 10 to 20 slices obtained by the related Sky scansoftware (2-D image obtained after multiple-slicing of shadow imageat right angle to heat flux) were randomly chosen from related slicesfor each axial position. Slices were taken from 3 parts of the sample (1)sample base, from 500 to 3900 �m from freezing plate; (2) samplecenter, from 6800 �m to 10000 �m from freezing plate, and (3) sam-ple top, from 12000 �m to 15000 �m from the freezing plate. For theSEM technique, the images were taken from the same sample loca-tion as used for the X-ray technique for each axial position. Bothtypes of images were processed by the corresponding image process-ing system. At least 300 measurements of ice crystal width were ob-tained from micrographs for each technique at each condition.

Tomographic images and ice crystal distributions for mycopro-tein frozen at –5 °C at different axial position are shown in Figure 5.For the same axial positions, microscopic images and ice crystaldistributions are given in Figure 6. At each axial position, the icecrystal width distributions of the paste were similar for both tech-niques as seen in Figure 5di-iii and 6bi-iii. However, the peak andshape at different axial positions along the sample were not thesame. The variation in peak value at different position might be

Figure 3—Microstructure of fresh (a, c) and slow-frozenand freeze-dried (b, d) paste by images made by (a) ESEMin wet mode, (b) SEM in dry mode, and (c, d) by X-ray to-mography.

Figure 4—Microstructure of fresh (a, c) and slow-frozenand freeze-dried (b, d) dough by images made by (a) ESEMin wet mode, (b) SEM in dry mode, and (c, d) by X-ray to-mography.

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Ice crystal visualization by x-ray . . .

due to differences in the number and width of ice crystals at differ-ent locations from the freezing surface. Both tomographic and mi-croscopic images show an increase in the void width with increas-ing distance from the cooling surface (Figure 5ci-iii and 6ai-iii). Themean width values obtained by the 2 techniques differed by lessthan 10 �m at each axial location (Table 1). These differences couldbe most likely attributed to the different measurement methods,that is, how the images were treated, rather than the differencebetween ice crystal widths seen by the techniques. Thus, it shouldbe concluded that the X-ray technique can detect ice crystal infor-mation in the material. Table 1 compares the mean ice crystal widthof paste frozen at –5 °C for different axial position by both tech-niques. The mean ice crystal width of the studied sample, as deter-mined by both techniques at the same axial position, was statisti-cally the same in all cases (P < 0.05). However, there was asignificant increase in ice crystal width as the distance from coolingsurface increased (P < 0.05). These results agree with Woinet andothers (1998b) and suggest that (1) X-ray micro-computed tomog-raphy is accurate for ice crystal measurement, and (2) the methodis capable of measuring the effect of different process conditions.

Effect of freezing rateEffect of freezing rateEffect of freezing rateEffect of freezing rateEffect of freezing rateScanning of freeze-dried paste frozen at –5 °C produced the

shadow image shown in Figures 5a. The ice crystal dendrite spacingis even apparent in the shadow images obtained. X-ray scanning

produced around 220 shadow images of the material, which werethen transformed using the 2-D reconstruction software and byslicing the shadow images from the top to the bottom to form animage at right angles to the heat flux direction. To get parallel imag-es to heat flux, the right angles slices were taken to T.view softwareand were cut at the same direction as heat flux.

Figure 5 shows images for the ice crystal distributions at differentpositions in a block of material frozen at –5 °C, where the coolingrates are those shown in Figure 2a and slices are right angles onesto heat flux. These cooling rates lead to the development of a den-dritic ice crystal structure that changes from the top to the bottomof the sample: this is clearly seen by the X-ray. Rapid freezing ex-periments were also conducted. Figure 7 compares the structureseen for freezing paste slowly (at –5 °C: profiles of Figure 2a) andrapidly (at –196 °C: profiles of Figure 2b). Figures 7ai and 7aii showtypical slices taken parallel to the heat flux for slow and fast frozen,respectively. For the slow-frozen material, the changes in ice crys-tal within the same sample can clearly be seen. The ice crystalsgenerally form in the direction of the heat flux, although some dis-tortion is seen. However, for the fast frozen sample, there are nodendrite/voids visible in the figure; here, the freezing rate is sorapid that no visible crystal can be seen. Similar effect are seen inslices taken at right angles to the heat flux; even at the top of thefast frozen sample, no crystal can be seen (Figure bii).

In addition to the paste, freezing by liquid nitrogen did not pro-duce needle-shaped voids in dough and steamed dough (detailsnot shown). This might be attributed to its high rate of freezing cre-ating even and tiny ice crystals throughout the products.

The data shown in Figure 5 is summarized in Figure 8, where icecrystal distributions are superimposed. Figure 8 compares clearly theshift in the shape of the measured ice crystal width distribution offrozen paste at –5 °C from the sample base to its top. As seen, icecrystal dendrite width increased from around 15 to 20 �m at bottomof the sample to more than 200 �m at the top. The differences can bedirectly attributed to the rate of cooling decreasing with increasingdistance from cooling surface, as shown in Figure 2a, leading to larger

Figure 5—Tomographic image andice crystal distribution ofmycoprotein frozen at –5 °C: (a)Typical side view of X-ray image;(b) typical reconstructed cross-sectional distance 7200 �m fromcooling surface at right angles toheat flux; (ci-iii) illustrate magnifiedimages of crystal structure: (i)2500 �m from cooling surface, (ii)7200 �m from cooling surface,and (iii)12000 �m from coolingsurface; (d) width distributions ofthe voids measured (i) 500 to 3900�m from cooling surface, (ii) 6800�m to 10000 �m from coolingsurface, and (iii) 12000 �m to15000 �m from cooling surface.

Table 1—Mean ice crystal width in �m (± standard devia-tion) for paste frozen at –5 °Ca

Bottom Center Top

SEM 18 ± 4.43 103 ± 32.30 201 ± 45.79X-ray 20 ± 4.49 111 ± 39.90 203 ± 45.76aMeasurements were made from 3 different axial positions by X-ray and SEMtechniques. The standard deviations are for the distribution of ice crystalsizes and not a measure of reproducibility of analysis. The consistency ofreplications for each measurement was also checked

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Ice crystal visualization by x-ray . . .

ice crystals. It is clearly possible to influence the microstructure bychanging the freezing conditions.

The information presented here clearly shows the capability ofthe X-ray technique to observe ice crystal structures inside solids.This is not possible by other ice crystal measurement methods atpresent. Therefore, the methods could be helpful in obtaining in-formation about ice crystals in other food materials as well as inother industrial applications. The main advantage of the proposedmethod is that ice crystal sizes can be determined directly in thesolid sample, without any microstructural change caused by sec-tioning the specimen, and the problems associated with loss of orig-inal ice crystal shape during long preparation methods. The obvi-ous disadvantage is that the sample must be freeze-dried beforescanning and thus materials can not be observed in their frozenstate unless a special freezing accessory is included.

Conclusions

X-ray micro-CT technique was successful in imaging the ice crys-tal microstructure of a variety of mycoprotein products. The

technique yields the full 3-D ice structure of the material. This hassignificant advantage over leading microscopic techniques. Thesystem can display the internal structure of material at any obser-vation angle. Rendered 3-D models can be sliced at any level, andat any angle to show the inner structure of the material, that is, nophysical slicing of sample or special sample preparation is required.Experiments were carried out to validate the method. The widthdistribution measured using the tomography method was thesame as that found for electron microscopy. The effect of cooling

Figure 6—Microscopic image and ice crystal distributionof mycoprotein frozen at –5 °C: (a) SEM images of struc-ture: (i) 2500 �m from cooling surface, (ii) 7200 �m fromcooling surface, and (iii)12000 �m from cooling surface;(b) width distributions of the voids measured: (i) 500 to 3900�m from cooling surface, (ii) 6800 �m to 10000 �m fromcooling surface, and a(iii) 12000 �m to 15000 �m fromcooling surface.

Figure 8—Ice crystal width distribution of paste frozen at–5 °C from sample base to its top using the X-ray tech-nique.

rate on the ice crystal structure can clearly be identified in terms ofchanges in the widths of the crystals formed.

The system provides a new tool to investigate the effects of freez-ing conditions on the structure, size, and morphology of ice crystals.The technique can offer benefits for the design, analysis, and pro-cessing of porous food products or microstructured materials. Itgives the possibility to examine the internal structure of thick sam-ples in 3 dimensions. The possibility of combining data obtainedfrom this system with rheological measurements, light scattering,and other physical techniques in the same experiments with speciallydesigned stages offers the possibility of obtaining detailed structuralinformation of complex food systems.

AcknowledgmentsRM and TM acknowledge the financial support of Ministry of Sci-ence and Technology of Iran. PWC acknowledges the financial

Figure 7—Tomographic images of freeze-dried paste af-ter unidirectional freezing at slow (–5 °C) and fast freezing(–196 °C) rates (temperature profiles given in Figure 2).Typi-cal reconstructed cross-sections parallel to the directionof heat flux (ai) for frozen sample at –5 °C and (aii) for fro-zen material at –196 °C; typical reconstructed cross-sec-tional images at right angles to direction of heat flux at12000 �m from cooling plate for (bi) frozen sample at –5 °C and (bii) frozen material at –196 °C.

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Ice crystal visualization by x-ray . . .

support of DEFRA, U.K. We are grateful to Dr Graham Rodger andMarlow Food Co. for providing the samples that made this workpossible.

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