Lebensm.-Wiss. u.-Technol., 34, 11}17 (2001)

Visualization of Food Stru

-F

g

Introduction giving an overview of the application of CLSM for struc-Confocal Laser Scanning Microscopy (CLSM) is arelatively new optical tool which is increasingly beingapplied in the food area (Blonk & van Aalst, 1993;Vodovotz et al., 1996). The development of CLSM goesback to an invention in the nineteenth century. In 1884,Paul Nipkow designed a rotating disk with holes ar-ranged in a spiral o!ering the possibility to scan imagesand transmit them by telegraphic wire. This principle ledto technical developments in television technology, butthe Nipkow disk itself fell into oblivion as televisiontechnology advanced. Around 1980 the Czech scientistM. Petran adapted the principle of the Nipkov disk todevelop a confocal microscope. This progress eliminatedthe blurring caused by non focal information from im-ages of thick objects. After a visit by Allan Boyde, a U.S.scientist, Petran was invited to continue his research inthe labs of Allan Boyde. The result was the "rst commer-cially available confocal microscope (from NORAN In-struments) which has since found wide application inbiological and medical sciences (Boyde, 1994; Pawley,1990; White et al., 1987). The present publication aims at

tural characterization of complex food systems. In the"rst part, the principle but also the potential and limita-tions of CLSM are described. In the second part, applica-tions of CLSM are presented based on experimentalresults. Yam (Dioscorea cayenensis rotundata) paren-chyma and cereal foods such as bread and pasta havebeen investigated by CLSM to illustrate the potential ofthis technique.

The principle

The primary value of the CLSM to research is its abilityto produce optical sections through a three-dimensional(3-D) specimen, for example a piece of tissue or otherthick objects (Fig. 1). An optical section contains in-formation from one focal plane only. Therefore, by mov-ing the focal plane of the instrument by steps of de"neddistance (km-range) through the depth of the specimen,a stack of optical sections can be recorded (Lichtman,1994). This property of the CLSM is fundamental forsolving 3-D problems where information from regionsdistant from the plane of focus can blur the image of suchobjects. For imaging in the CLSM, either the epi-#uores-Scanning MicroMarkus B. DuK rrenberger, Stephan Handsc

M. B. DuK rrenberger: Biocenter of the University oKlingelbergstrasse 70, CH

S. Handschin, B. Conde-Petit, F. Escher: SwissInstitute of Food Science, CH

(Received September 19, 2000;

Confocal Laser Scanning Microscopy is a rather new technique forconventional light microscopy the light source is replaced by laser, athe limited depth of focus. The potential and limitations of CLSM areproducts (wheat dough, bread and pasta) were imaged by CLSM usinbe a useful tool for obtaining three-dimensional information on the ceand starch networks in wheat products. Furthermore, CLSM allowsproperties of pasta in the reyection mode.

( 2001 Academic Press

Keywords:*Corresponding author.

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

11cture by Confocal Laserscopy (CLSM)hin, BeH atrice Conde-Petit*, Felix Escher

f Basel, Interdepartmental Electron Microscopy,4056 Basel (Switzerland)ederal Institute of Technology (ETH) Zurich,-8092 Zurich (Switzerland)

accepted December 14, 2000)

structural analysis of biological and food material. In contrast toscanning unit and a pinhole in the back focal plane, which improvesdiscussed based on experimental work. Yam parenchyma and wheat

Acid Fuchsin and Safranin O as yuorescent dyes. CLSM proved tollular structure of yam parenchyma and on the properties of proteinthe study of bread after baking in situ and the analysis of surfacecence or the epi-re#ection mode is generally used. As

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

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

Potential and Limitations

The CLSM detects in-focus regions only, the out-of-focusparts appearing black. Therefore, the application is notlimited to thin samples. For instance, rather thick foodsamples can be analyzed by CLSM to obtain structuralinformation. A precise view of the spatial arrangements

lwt/vol. 34 (2001) No. 1Fig. 1 Stack of optical sections from focal planes at di!erentdepths through a specimen which provides a three-dimensionalimage of the object

a valuable by-product, the computer-controlled CLSMproduces digital images which are amenable to imageanalysis and processing, and can also be used to computesurface- or volume-rendered 3-D reconstructions of thespecimen.Optical sections in a CLSM are composed from a repeat-ed point experiment to get a scanned image (Pawley,1990; Wilson & Sheppard 1984). To image a specimenpoint by point (scanned), a collimated polarized laserbeam is de#ected stepwise in the x- and y-direction bya scanning unit before it is re#ected by a dichroic mirror(beam splitter) so as to pass through the objective lens ofthe microscope, and focused onto the specimen. Theemitted longer-wavelength #uorescent light (or re#ectedlight in re#ection mode) from the components of intereststained with #uorescent dyes is collected by the objectivelens, passes through the dichroic mirror (transparent forthe longer wavelength, or semi-transparent for re#ectionmode) and is focused into a small pinhole (the confocalaperture) to eliminate out-of-focus light. Therefore, theCLSM not only provides excellent resolution within theplane of the section (50.25 km in x- and y-direction),but also yields good resolution between section planes(50.25 km in z-direction). A light-sensitive detector, aspresented in Fig. 2, which is positioned behind the con-focal aperture, records the in-focus information of eachspecimen point, and the analogue output signal isdigitized and fed into a computer to generate an imageon a monitor. A stack of serial optical sections, each

consisting of a pixel-matrix in digital form through thespecimen, o!ers the possibility to compute either a com-posite projection, or a volume-rendered 3-D representa-tion of the specimen.The confocal part of a CLSM is comparable with anelaborate, highly folded optical bench on which the laser,all optical elements and the detector are mounted. Whenworking in the epi-#uorescence mode, the laser beam is"ltered to select distinct monochromatic wavelengthsfrom one or several lasers (360 nm, 458 nm, 488 nm,543 nm, 568 nm, 633 nm, 647 nm). Furthermore, a multidichroic mirror that re#ects excitation and transmitsemission wavelengths is used to generate #uorochromespeci"c signals on up to four detectors. For the epi-re#ection mode no wavelength "lters are needed. Instead,a semi-transparent mirror re#ects 50% of the incident

12Fig. 2 Principle of confocal imaging: The numbered elementsare: (1) laser, (2) dichroic mirror, (3) objective lens, (4) thickspecimen, (5) confocal pinhole, (6) photomultiplierof structural elements may be obtained by collectinga 3-D data set of the sample. Visualization of the sampleoccurs at ambient conditions which allows the observa-tion of the sample in the hydrated state. For instance, themicrostructure of aqueous phase separated protein-poly-saccharide mixtures can be assessed without changingthe solvent conditions (Bourriot et al., 1999). A furtheradvantage of CLSM is the possibility to follow in situ thedynamics of processes such as phase separation, coales-cence, aggregation, coagulation, solubilization, etc.Specially designed stages, which allow heating, cooling ormixing of the sample, give the possibility to simulate foodprocessing under the microscope (Thorvaldsson et al.,1998).In some cases, only a few preparatory steps are necessaryfor viewing a specimen by CLSM. This often applies to

lwt/vol. 34 (2001) No. 1native plant tissue where the naturally occurring #uores-cence (auto#uorescence) may be su$cient to generatea contrast. Further examples are biopolymer emulsionscontaining gelatin, where the auto#uorescence of gelatinallows visualization of the microstructure of the emulsionin situ (Foster et al., 1997). Alternatively, the componentof interest in the sample may be labelled. Fluorescentdyes are labelling agents which contain excitable struc-tures that emit #uorescence after illumination by light ofa speci"c wavelength. The photons emitted by the #uor-escent dyes are visible even below the resolution limit ofthe microscope as these structures appear as spots witha diameter of the resolution limit of the microscopeoptics. CSLM may be combined with #uorescence inten-sity to quantify the labelled component. In aqueous bi-opolymer mixtures, for instance, this technique allowsthe quanti"cation of biopolymer concentration andphase volume (Blonk et al., 1995). Furthermore, pHgradients in a sample may be detected by using a #uor-escent compound that is sensitive to pH (Hassan et al.,1995).CLSM o!ers the possibility to analyze the topographyand surface of samples in the epi-re#ection mode. In thismode the laser light which is re#ected from the sample iscollected as signal. To increase the signal intensity thesample can be covered with a thin metal "lm by sputter-coating as for the preparation for scanning electronmicroscopy.Conventional transmission light imaging is limited whenapplied to thick sections, because the images can beblurred by out-of-focus information. Recently, with in-creasing power of image processing computers, an imagereconstruction tool called deconvolution was developedto treat focal series of transmitted light images. Theresults are deblurred 3-D representations similar to con-focal stacks of images. Drastic alterations in the design ofCLSMs could lead to the possibility of recording trans-mitted light confocal images in the future. Such methodswould imply fewer preparation steps, (e.g. the omission ofstaining), to be ready for confocal 3-D imaging.A major limitation of CLSM is the fact that most samplesrequire some treatment to be visible. All steps such asstaining or quenching of auto#uorescence, which have tobe performed in liquids at room temperature can result inartefacts like swelling and solubilization of components.Many #uorochromes are sensitive to laser-illuminationand can bleach within the time necessary for searchingand acquiring an image. Objective lenses with a highnumerical aperture (e.g. 63]NA 1.4, 40]NA 1.0) arenormally used to provide good resolving power in x-, y-and z-direction. By physical laws their working distance,measured between the front lens and the top of thesample (cover glass), is restricted to about 100 km. Theuse of objective lenses with longer working distancesresults in lower resolution of the image. Furthermore, thepenetration of the laser beam into samples is restrictedand is in a maximum range of 100 to 150 km in z-direction. Short-pulsed double photon illumination,where the energy of two photons comes to addition in thefocus plain and can be absorbed by a #uorochrome, willcause emission of higher energy (shorter wavelength)13photons. This can result in extended penetration of thebeam into the sample and in reduced bleaching of#uorochromes, but the lasers required are extremely ex-pensive and must be tuned before use. In addition onlyone #uorescence channel is available, since the principleof double photon shooting does not allow multiplewavelength illumination. The resolving power accordingto the rule of thumb (half the wavelength of illumination)is not restricting because a shorter wavelength of illu-mination results. This can be done by interference ofa double pulse of the longer wavelength.

Experimental

Sample preparation

Yam. Fresh parenchyma tissue of yam (Dioscorea cay-enensis rotundata cultivar Lopka from the Ivory Coast)was cut into sections of 150 km thickness with a manualmicrotome (Leica microsystems, CH-Glattbrugg) equip-ped with a knife-holder for conventional razor-blades.The sections were stained by immersion into aqueousSafranin O solution 0.04% (Sigma-Aldrich, CH-Buchs)for 30 min followed by rinsing in deionized water for30 min.

Wheat dough and bread. Two procedures were de-veloped for the preparation of wheat dough and breadsamples. In one case, wheat dough and bread were pre-pared and cryo-sectioned (50 km thickness) as describedby Hug-Iten et al. (1999). Prior to sectioning the auto-#uorescence of dough and bread was removed by soak-ing small pieces of samples in 10 mL aqueous solution ofHeparin (500 i.U. per mL Liquemin, Roche, CH-Basel)for 30 min, followed by rinsing in deionized water for30 min. Alternatively, a wheat dough without yeast wasprepared from low extraction wheat #our, which hadpreviously been bleached with Heparin. For this purpose,200 g wheat #our was treated with 500 mL aqueousHeparin 500 i.U. for 30 min, followed by rinsing withdeionized water (30 min). The dough samples wereheated directly under the microscope in a specially de-signed stage. Baking was simulated by heating fromambient temperature to 80 3C within 35 min.Wheat dough and bread were stained either with AcidFuchsin (Sigma-Aldrich, CH-Buchs) or Safranin O.Staining with Safranin O was carried out as described foryam. Staining with Acid Fuchsin was carried out asdescribed by Fardet et al. (1998). The cryosections whereimmersed in Acid Fuchsin solution (0.01 g Acid Fuchsinin 1% acetic acid) for 10 min followed by rinsing indeionized water for 60 min.

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

lwt/vol. 34 (2001) No. 1Spaghetti samples were cryosectioned (50 km) as de-scribed by Cunin et al. (1995). Staining with Acid Fuchsinwas performed as described for bread. For recording ofimages of the surface in the re#ection mode the spaghettisamples were sputtered with a 30 nm gold layer in a sput-tering device (Baltec, FL-Balzers).

Microscopy

Images were recorded with a Leica TCS SP (spectrom-eter type) CLSM mounted on an upright DM RXE#uorescence light microscope (research type, LeicaLasertechnik GmbH, D-Heidelberg). The samples weremounted on glass slides and covered with deionizedwater and a cover glass. The samples stained with AcidFuchsin were illuminated with the krypton laser at568 nm. Samples with Safranin O with the argon-ionlaser at 488 nm. Initially, the maximum of the emissionpeak was determined with the spectrometer of the micro-scope. The emission maxima were 540 and 620 for sam-ples stained with Safranin O and Acid Fuchsin,respectively. Therefore, the bandwidth for recording the#uorescence images was set from 530 to 550 nm forSafranin O and from 600 to 620 for Acid Fuchsin. Sam-ples were observed in the epi-#uorescence mode exceptspaghetti, where re#ection mode was also used. Approx-imately 40 images were recorded per stack coveringa depth of about 40 km in the sample. Surface projectionswere carried out using Imaris software (Bitplane AG,CH-ZuK rich). For conversion from stack to projectionpictures were processed with the shareware-programNIH Image (National Institutes of Health, Bethesda,Maryland, U.S.A.).

Results and Discussion

Preliminary experiments with fresh yam tissue showedthat yam does not exhibit auto#uorescence at thewavelengths used. Therefore staining was necessary togenerate contrast. Fig. 3 presents the microstructure offresh yam parenchyma tissue after staining with SafraninO. The cellular structure of yam is clearly visible, as wellas the native starch granules within the cells. The triangu-lar form of the starch granules, which is typical forDioscorea cayenensis rotundata, can be recognized. Notethat Safranin O stains starch, but also the polysacchar-ides in the cell wall. Similar studies on the structure ofapples (Lapsley et al., 1992), grapes (Gray et al., 1999),and strawberries (Suutarinen et al., 2000) show thatstructural features of plants in particular cell size andshape, cell adhesion and internal air spaces can be visual-ized by CSLM. Regarding the starch fraction, details oftheir morphology have been revealed by this techniquesuch as the pores on the surface of maize and sorghumstarch (Huber & BeMiller, 2000).In contrast to yam, wheat products exhibited auto-#uorescence. Control experiments con"rmed that bleach-ing with Heparin completely removes auto#uorescence(micrographs not shown). Fig. 4a and 4b show micro-14graphs of wheat dough and bread stained with AcidFuchsin. The latter dye is a well known staining agent forprotein. The bright areas on the micrograph of dough(Fig. 4a) correspond to the coarse network of wheatprotein, also termed gluten. The starch granules are alsoclearly recognizable. It is likely that proteins associatedwith the starch surface (Seguchi & Yoshino, 1999) con-tribute to the visualization of starch. In bread, a "nestranded gluten network is recognizable (Fig. 4b). Thebright spots distributed throughout the micrograph canbe attributed to yeast labelled with Acid Fuchsin. Thedark areas correspond to swollen (gelatinized) starch. Themorphology of starch in bread is less recognizable than indough, most probably due to the swelling of the starchgranules. The micrographs of both dough and bread showthat protein and starch are not evenly distributed. Indough, regions are found where starch granules are seg-regated from protein. Likewise, in dough and bread theprotein network is interrupted by accumulations of starch.Spaghetti is another wheat based product where CLSMcan be used to assess the continuity of the protein phase.Fig. 5 shows a cross section of cooked spaghetti stainedwith Acid Fuchsin. The micrograph reveals that proteinforms a dense continuous network towards the centre ofthe spaghetti strand. In contrast, in the outer layer ofcooked spaghetti the continuity of the protein network isalmost lost. This result con"rms earlier studies on themicrostructure of spaghetti using light microscopy,where it was shown that the centre of cooked pasta isdominated by the protein fraction, whereas in the outerlayer the strand swelling of starch contributes to a dis-ruption of the protein network (Cunin et al., 1995; Fardetet al., 1998). CSLM was also successfully applied tovisualize other protein networks such as the gelation ofmilk proteins as induced by milk acidi"cation (Hassanet al., 1995).Contrary to protein, visualization of starch by CLSM ismore di$cult, since there is little information on speci"c#uorescent dyes for starch in the literature except formethods based on Concanavalin A (Gibson et al., 1997).In the present investigation the suitability of SafraninO for staining starch has been studied. Fig. 6a and 6bshow bread directly after baking and after 7 d of ageing.In fresh bread swollen and mostly elongated starch gran-ules, which are not intensively stained, are recognizable.The bright zones within the starch granules and in theintergranular space most probably correspond to phaseseparated amylose (Fig. 6a). Phase separation of the twostarch polymers, amylose and amylopectin, is the resultof polymer incompatibility. Phase separation of starch inbread was previously shown by Hug-Iten et al. (1999)using light microscopy. In aged bread, an intensivelystained intergranular phase is still visible, but notthroughout the sample. Furthermore, the bright areaswithin the starch granules are no longer visible. Thisobservation suggests that the increase in molecular orderof starch, which is known to occur during ageing ofbread, has an in#uence on the staining intensity of Saf-ranin O. It is conceivable that densely packed starch, forinstance crystallites, are not as easily penetrated by thedye as the plasticized amorphous zones of starch. It

lwt/vol. 34 (2001) No. 1Fig. 3 CLSM of freshly cut yam (Dioscorea cayenensis rotundshadowed by image-processing to provide a three-dimensional vFig. 4 (a) CLSM of a cryosection of wheat dough stained with Astained with Acid Fuchsin for proteinFig. 5 CLSM of a cryosection of cooked spaghetti stained with

should be added, that Safranin O does not show #uores-cence in the absence of starch. The nature of the starch-Safranin O interaction is not fully understood and needsfurther investigation.In order to follow the structural changes of starch duringbaking of bread, dough without yeast stained with Saf-ranin O was heated to 80 3C under the microscope.

15ata, var. Lokpa), stained with Safranin O. The micrograph isiew of the cells

cid Fuchsin for protein (b) CLSM of a cryosection of fresh bread

Acid Fuchsin for protein

A micrograph of the resulting &bread is presented inFig. 7. The microstructure of bread baked under themicroscope is very similar to regular bread (Fig. 6a) withthe di!erence, that no pores can be seen. The gelatinizedstarch granules are less intensively stained since doughwas stained before heating when starch was in the nativestate. It is, therefore, concluded that the baking process

lwt/vol. 34 (2001) No. 1Fig. 6 (a) CLSM of a cryosection of fresh bread stained with Safra(b) CLSM of a cryosection of bread stored for 7 days at 20 3C,Fig. 7 CLSM of bread baked on the microscope stage stained wFig. 8 Topographic image of dried spaghetti, generated by CLSMin the protein phase

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

16nin O for starch. Arrow indicates a pore limited by the pore wall.stained with Safranin Oith Safranin Oin the epi-re#ection mode. Arrows indicate starch granules hold

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

Microscopy (SEM) in that the thickness of the gold layeris below the resolving power of light microscopy andtherefore does not in#uence the imaging of structuraldetails.

Conclusions

Based on the above presented examples it can beconcluded that CLSM broadens the application of con-ventional light microscopy. It gives the possibility toexamine the internal structure of rather thick samples in

pasta protein networks to determine in#uence of technolo-gical processes. Cereal Chemistry 75, 699}704 (1998)

FOSTER, T., UNDERDOWN, J., BROWN, T., FERDINANDO, D. P.AND NORLON, I. T. Emulsion behaviour of nongelled bi-opolymer mixtures. In: E. DICKINSON, B. BERGENSTAG HL(Eds). Food colloids, proteins, lipids and polysaccharides. Cam-bridge: The Royal Society of Chemistry (1997)

GIBSON, T. S., SOLAH, V. A. AND MCCLEARY, B. V. A pro-cedure to measure amylose in cereal starches and #ourswith 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 andshape 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.

lwt/vol. 34 (2001) No. 1three dimensions. Samples can be viewed in the hydratedstate and, therefore, food transformations during pro-cessing may be followed in situ using specially designedstages. The power of CLSM will further increase as morespeci"c labelling agents for food components becomeavailable. Conventional labelling with #uorescent dyes aswell as more sophisticated techniques such as immu-nolabelling need further investigation. Finally the simul-taneous labelling of two or more components of foodswith probes which are speci"c for each component isnecessary for an even more detailed analysis of foodstructure. The possibility to combine CLSM withrheological measurements, light scattering and otherphysical analytical techniques in the same experimentswith specially designed stages o!ers the possibility toobtain detailed structural information of complex foodsystems.

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IntroductionThe principleFigure 1Figure 2

Potential and LimitationsExperimentalResults and DiscussionFigure 3Figure 4Figure 5Figure 6Figure 7Figure 8

ConclusionsReferences