Electron Microscopy and Imaging Techniques in Food Science

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Workshop proceedings Electron microscopy and imaging techniques in food science

June 19, 2015 - University of Verona

THE SCANNING ELECTRON MICROSCOPE specimen vacuum must be produced, to prevent IN FOOD INDUSTRY: APPLICATIONS AND air from stopping or just scattering the electron EXAMPLES beam off the electron path axis. This is why vacu- S. G. Alberici1, V. Kostal2 um is needed in this microscope setup. There are different ways to manage the food 1Surface Analysis Division, Gambetti Kenologia Srl, Via Volta 27, 20082, Binasco, Italy samples for SEM analysis: from freezing the sam- 2SEM Demo Labs, TESCAN ORSAY HOLDING, a. s., ple down to low temperatures, to prevent itself Libusina trida. 21, 62300, Brno, Czech Rep. from degaussing (i.e.: changing its morphology E-mail: [email protected] and structure) when in vacuum, to coating it by conductive and impermeable films that prevent Scanning Electron Microscope (SEM) usage has water from being dispersed into vacuum, to de- strongly increased over the past decades, dealing hydrate the sample if loss of water does not imply with a number of interest fields that has constant- a change in the imaging expectations, and so on. ly grown, from semiconductors, to Metallurgy, to This presentation deals with an overview about Chemistry, to Life Science, just to cite a few. sample preparation for Food Science related sam- The technological improvement, together with ples that are then supposed to be measured by the development of user friendly interface with SEM, a discussion on the challenges and opportu- the user, made the SEM much more appealing nity that an SEM offers in the field of analysis of even to scientist who were not familiar with this the Food Science, and a session dedicated to the technique, extending their research capability to application and examples of what can be done by boundaries that were not in their focus just a few using an SEM in food applications. years ago. It is discussed the sample issues for a good SEM Here, the great step ahead with respect to the analysis, how to properly manage the sample classical microscopy, lies on being able to go itself, the usage of the instrument and the results, much further than the optical microscope resolu- with a special focus toward the ones who are not tion limit (i.e.: a few hundreds of nm) down to expert in SEM practise, though they are used to even less than one nm. analyse Food related samples for their Research In Life Science the complication arises from the areas. nature of the sample, that usually contains water, it is not stable under vacuum, is very often non electrically-conductive and, therefore, not easily OPTICAL IMAGING IN FOOD ASSESSMENT: suitable for a SEM analysis where vacuum is need- TECHNIQUES AND INSTRUMENTS ed as well as a conductive specimen would be the desirable condition. F. Boschi Instead of making use of a light beam as a probe Department of Computer Science, University of Verona, medium as it happens in the optical microscope, Strada le Grazie 15, 37134 Verona, Italy the SEM uses an electron beam as a probe, that is E-mail: [email protected] regularly scanned by a computer onto the specimen surface to be analysed, the beam being cou- The visual perception is one of the most ancient pled to a detector and the computer itself that and useful approach for food assessment. reproduces the specimen topography. Nowadays the extension of the visual perception In order to have enough mean free path for the is represented by the optical imaging which is electrons, from the electron source down to the based on the detection of ultraviolet and near

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infrared photons beyond visible photons. This ture of a food affects how it breaks down in our extension is possible thanks to the development mouths and our perception of its flavor but also of new instruments, light sources (lasers and the release and bioavailability of minerals, vita- LEDs) and very sensitive detectors (photomulti- mins and polyphenols in our digestive system. pliers and charge coupled devices – CCDs). Here Thus food structures are increasingly being recog- the attention is focused on the detection of light nized as important in technology innovation for with optical imagers and confocal microscopes. the development of healthier foods.2 Generally, the light processes detected with these instruments are: fluorescence, phosphorescence and bioluminescence. When a beam of light References passes through the matter the photons can excite 1. Rincon Cardona JA, in Biochemistry, Genetics and a molecule to a higher vibrational energy level. Molecular Biology “Confocal Laser Microscopy - The excited molecule rapidly losses part of the Principles and Applications in Medicine, Biology, energy as heat and relaxes to the previous state and the Food Sciences”, book edited by Neil Lagali , with the emission of a photon with lower energy. ISBN 978-953-51-1056-9, 2013 The process, called fluorescence, is almost imme- 2. http://csironewsblog.com/tag/food/ diate (10-8 s). Sometimes the excited molecule can stay in an intermediate energy level and returns at the ground level at significantly slower time OPTICAL PROPERTIES OF FOODS: scales. The emission, called phosphorescence, is POTENTIALITIES AND PROSPECTS thus retarded with respect the excitation. The L. Calderan third process is the bioluminescence which is a form of chemiluminescence which happens in bio- Department of Neurological, Biomedical logical systems. and Movement Sciences, University of Verona, Strada Le Grazie 8, 37134 Verona, Italy The optical imagers are composed by a dark chamber to prevent the contamination of light E-mail: [email protected] from outside and a very sensitive CCD camera Food analysis is the discipline dealing with the with very low noise level. They are also equipped development, application and study of analytical with a light lamp and many excitation and emis- procedures for characterizing the properties of sion filters which are used in fluorescence modal- foods and their constituents. Many investigative ity. The autofluorescence of cells and tissues can procedures are used to provide information about be used as endogenous fluorescent markers, a wide variety of different characteristics of foods, instead commercially available fluorescent mole- including their composition, structure, physico- cules as exogenous markers. They can be selected chemical properties and sensory attributes. among a great variety and with the required opti- Optical properties of food are a good instrument cal properties. The contemporary use of two or to evaluate many parameters as presence of bacte- three markers are also very interesting. In case of ria, product quality, freshness or state of preserva- bioluminescence modality, the lamp is off and no tion or conservation type. filters are used to detect the light. The optical Normally in the food and raw materials industry imaging technique is cheap, repeatable, allowing methods of quality and sterility control are used very simple samples preparation and it offers the (which detect the presence of bacteria, fungi or advantages of fast acquisitions (1 sec to 5 min, yeasts) through luminometric quantitative meth- depending of the modalities). ods that relate the presence of living organisms The confocal microscopes are an improvement with the emission of photons. But in this case opti- of the classic optical microscopes designed for cal properties of luciferase - luciferine system are increasing optical resolution and contrast by used to measure presence and quantity of ATP. means of adding a spatial pinhole placed at the Intrinsic optical properties of food could be confocal plane of the lens to eliminate out-of- assessed with no difficulty in case of vegetable focus light. They permit high resolution in thin foods. The fluorescence emission of chlorophyll section using autofluorescence, exogenous fluo- (680 and 700 nm) is measurable in a non invasive rescence and allowing the 3D reconstruction of and relative easy way and it is indicative of the the foods’ properties and structures.1 The struc- changes in the morphology, anatomy and physiol-

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ogy of the plant itself. Chlorophyll fluorescence POTENTIAL OF MAGNETIC RESONANCE could be used as an early stress indicator. In par- IMAGING IN FOOD SCIENCE ticular, the fluorescence could provide details on P. Marzola the ability of a plant to tolerate environmental stresses and their capacity to damage the struc- Department of Computer Science, University of Verona, ture of the plant. So it is possible to detect cultiva- Strada Le Grazie 15, 37134 Verona, Italy tion mode, mode and timing of harvest and post- E-mail: [email protected] harvest preservation methods. Magnetic Resonance Imaging (MRI) is a power- Also, food of animal origin contains molecules, ful diagnostic modality providing high resolution, as peptides or proteins or others components, that non-invasive and multi-parametric images of the have intrinsic optical properties of photon emis- human body. Dedicated equipment, mainly devel- sion. All these components could be affected by oped for imaging of small laboratory animals, can how they are to be processed, handled, stored, reach the limit of MRI microscopy that generally is and conserved. In this case the cause of photon assumed to be 100 microns.1 NMR, and particular- emission is unknown so only with the use of a ly its microscopy version, also known as MRI, has multimodal approach could it be possible to char- been applied to investigate different kind of foods: acterize the structure and identity of the source of fruit, vegetables, meat, dairy products, cereals.2 this emission. The non-invasiveness of MRI acquisitions, which The measurement of the photon emission could be is one of the most relevant features of diagnostic also useful to highlight the presence of food additives MRI, is advantageous also in food science since it or ingredients if they have, as it is often the case, allows dynamic studies to be performed during intrinsic optical characteristics of photons emission the preparation process of food or during fruit with fluorescence or phosphorescence modality. ripening. For example, MRI has been applied in Besides thanks to basic research studies it could be dynamic studies to investigate de-hydration, also monitored the presence of these components in cheese brining, fish salting, thermal processing of living organism, their biodistribution and tissue accu- cereals.2 Moreover, it is worthwhile to mention the mulation characteristics through experimental studies performed to unveil the geographical ori- model where they are viewable. gin of food growth,3 or to measure fat content in Food safety and quality control could be investi- ham.2 Interestingly, most of MRI modalities used gate through optical properties of the food but in the clinics can be translated in food science. also the study of optical properties of food is a non For example T2-weighted images can provide high invasive way to monitor the impact on human resolution images of vegetables with superb mor- health of the use of additive, preservative and food phological detail and clear discrimination of processing mode. regions containing free water. Localized spectroscopy is used in humans to detect and quantify lipids or metabolites and can be applied in food References science as well to quantify lipids or sugar concen- Fan F. and Wood K.V. Technology Review: tration. Diffusion tensor imaging (DTI) allows to Bioluminescent Assays for High-Throughput detect axonal fiber in the brain, but can also be Screening. Assay and Drug Development used in several fiber-rich vegetables as celery4 or Technologies, Vol 5, N° 1, pp.127-136. 2007 fennel to detect the organization of vegetal fiber. Karoui R. and Blecker C. Fluorescence Spectroscopy Finally dynamic contrast enhanced MRI (DCE- Measurement for Quality Assessment of Food MRI), used in humans to study in vivo tissue per- Systems - a Review. Food Bioprocess Technol 2011; fusion, has been recently used to study fluid trans- 4:364-86 portation in tomatoes.5 In conclusion, MRI at microscopic resolution has a great potential in food science that has not been fully exploited at the present, mainly due to the high costs of the equipment and the require- ment of specialized personnel.

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tration within muscles and that semi-quantitative References analysis of infiltration degree can be achieved. 1. Badea A, Johnson A, Magnetic Resonance These data were further supported by the chemi- Microscopy, Biophotonics in Pathology. 2013;185: cal analysis of meat samples corresponding to the 153. same region of interest observed in both CT and 2. Mariette F et al., Quantitative MRI in Food Science histologic investigations; dry matter, crude pro- & Food Engineering, Encyclopedia of Magnetic teins, crude fat and ash contents, calculated fol- Resonance, Online 2007-2012, John Wiley & Sons, lowing standard international methods,4 varied in Ltd. fact depending on the fat infiltrated extent, 3. Sequi P et al., Journal of the Science of Food and Agriculture. 2007;87:127. according with CT images. We finally observed 4. Gaggl W et al., Magn Reson in Medicine. 2014;72: that CT could be used in the evaluation of the 1668. same products at the end of ripening, without 5. Kenouche S, et al., Magn Reson Imaging. 2014;32: removing the outer envelope. 1418 These results are important for beef and meat industrial processing sector, suggesting that CT could be employed as an on-line instrument in MEAT COMPOSITION, FROM MICROSCOPY abattoir and dry-cured meat industry in classifying TO CHEMISTRY BY WAY OF DIAGNOSTIC the products at the beginning of the manufactur- IMAGING ing even they are frozen. Moreover, it might represent a rapid and non-invasive technique for quality S.C. Modina1, E. Trevisi2, C. Bernardi1, M. Di Giancamillo3 check at the end of the production line and for the 1Department of Health, Animal Science and Food assignment of the most appropriate nutritional Safety, Università degli Studi di Milano, via Celoria 10, 20133 Milano, Italy and commercial value to different products. 2Institute of Zootechnics, Faculty of Agriculture, Università Cattolica del Sacro Cuore, Via Emilia Parmense 84, 29122 Piacenza, Italy References 3Department of Veterinary Science and Public Health, Università degli Studi di Milano, via Celoria 10, 20133 1. Scholz AM et al. Animal. 2015; 9: 1250 Milano, Italy 2. Frisullo P et al. Journal of Food Engineering. 2009; E-mail: [email protected] 94: 283 3. Manzocco L et al. Food Chem. 2013;141(3):2246 4. AOAC International 2012. Official Methods of The increasing demand of a real-time monitor- Analysis. 19th ed. AOAC International Gaithersburg, ing of food products has encouraged the applica- MD tion of non-invasive techniques. Computed Tomography (CT) and Magnetic Resonance Imaging (MRI) proved to be very accurate and MRI (AND NOT ONLY) IN FOOD SCIENCE valuable tools in estimating body and carcass F. Moneta1, C. Vailati2 composition in farm animals.1 CT has been suc- cessfully used for the characterization of food 1Bruker Italia Srl Unipersonale, Bruker BioSpin Preclinical Italian products such as salami, providing a pre- Imaging Division, V.le Lancetti 43, 20158 Italy 2Bruker Italia Srl Unipersonale, Bruker Nano Analytics, cise evaluation of fat percentage, also assessing its V.le Lancetti 43, 20158 Italy spatial distribution.2 Manzocco and colleagues demonstrated that MRI has great potential in mon- E-mail: [email protected] [email protected] itoring the evolution of dry curing in S. Daniele hams.3 Food is any substance consumed to provide In our experience, helical CT proved to be a fast nutritional support for the body. It is usually of tool in the classification of different meat cuts, plant or animal origin, and contains essential deriving from adult cow and destined for the nutrients, such as carbohydrates, fats, proteins, preparation of aircured products as “lean meat” or vitamins, or minerals. The whole food industry “fat meat”, both in fresh and frozen samples. covers several areas from farming and food pro- Histological studies confirmed that CT clearly duction, packaging and distribution, to retail and distinguishes adipose and connective tissue infil- catering including subareas like regulation, educa-

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tion, R&D, manufacturing, agriculture, processing, with a resolution down to a nanometer. Examples marketing, wholesale and distribution, and for of uCT application in the food industry are the dis- Europe it represents the second largest industry tribution of coffee powder in coffee chowders, (after metal industry) with Euro 917 billions potato chips distribution of air/oil bubbles, maple 310.000 companies and 4.8 millions employees, seeds fine structure, determination of fat content equal to 14% of the total manufacturing sector in fish food, degradation of apples, studies on ice (EU-27 - 2013 http://ec.europa.eu/enterprise/sec- cream homogeneity. tors/food/eu-market/index_en.htm). In conclusion imaging techniques represent an With a wide range of products, Bruker innovative tool for the food industry and bridge (www.bruker.com) offers solutions in most of the gap between macroscopic observation and these fields. In particular Bruker offers a wide microscopy techniques. portfolio of imaging techniques like magnetic resonance imaging (MRI), high resolution microto- mography (microCT), PET, SPECT, CT, Optical TECHNIQUES FOR THE ULTRASTRUCTURAL fluorescence and bioluminescence, Magnetic ANALYSIS OF FOOD Particle Imaging (MPI). A. Sbarbati These techniques are routinely used in the fields of food quality and safety, food technology, Department of Neurological, Biomedical and raising, seafood, preparation of fresh products, Movement Sciences, University of Verona, Strada Le Grazie 8, 37134 Verona, Italy manufacture of prepared food products and ware- housing. E-mail: [email protected] MRI has the advantage of being a non-destruc- Ultrastructural techniques are powerful tools tive, non-invasive, molecule specific (fat, water, by which a wide variety of data can be obtained 1 31 13 23 19 alcohol, ...), nucleus specific ( H, P, C, Na, F, on the structure and composition of food. In par- etc.) technique mainly based on the detection ticular, transmission electron microscopy allows water (or generally speaking 1H) content in tis- to gain evidence on the preservation state of sues/materials, under several contrast parameters food, to investigate the processing-induced as density, mobility, relaxation, diffusion and flow changes, and to distinguish food typicalities, with a typical image resolution down to tens of while scanning electron microscopy provides microns. detailed information on the outer and cutting sur- It allows 2D/3D spatial localization, resolution faces of food products. X-ray microanalysis at of the information in the temporal dimension electron microscopy can be used to detect the even with dynamic and real time capabilities. As a occurrence of specific chemical elements which non-destructive technique it’s very useful in longi- can be either naturally present or may contami- tudinal studies. A non-exhaustive list of examples nate food during processing or packaging. These of MRI applications in the food industry are the techniques are often used to examine meat or cooking process of pasta, single onion cell imag- fruit, but they could also be applied to more spe- ing, leaves structure, melon infection studies, cialized investigations such as the analysis of plants roots growing, water uptake followed by microbial components with probiotic activity or drying of a barley corn, bread dough leavening and of nanoparticulates. Ultrastructural techniques yoghurt homogeneity determination. should therefore integrate the conventional his- A good alternative to MRI is represented by tological analyses and be extensively applied, in micro-tomography (microCT). It allows a com- future, for food characterization and safety con- plete quantitative 3D volume insight of any food trol, thus being a helpful support for the protec- during its preparation as well as in the packaging tion of public health.

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IMAGING TECHNIQUES APPLIED TO THE DISCOVERY THE MICROORGANISMS OF STUDY OF GRAPEVINE BERRY RIPENING FOODS AND BEVERAGES BY ELECTRON AND POSTHARVEST DEHYDRATION MICROSCOPY G.B. Tornielli S. Torriani, G.E. Felis Department of Biotechnology, University of Verona, Department of Biotechnology, University of Verona, Strada le Grazie 15, 37134 Verona, Italy Strada le Grazie 15, 37134 Verona, Italy E-mail: [email protected] E-mail: [email protected] Grapevine berry development is characterized The study of microorganisms has gone hand in by many metabolic and structural changes that hand with microscopy since Antoni van determine the final quality of the fruit at harvest. Leeuwenhoek observed and described single- These include the pericarp tissue softening, the celled organisms in 1684 using handcrafted simple flesh cell volume increase due to water and sugar microscopes. However, the ability to resolve import, and the accumulation of anthocyanis and structures using light microscopes is though limit- other flavonoids in the epidermal and hypodermal ed to around 200 nm. With the development of cells of the skin. For particular enological purpos- electron microscopy (EM) techniques, the resolu- es (i.e. the production of wines like Recioto and tion limits of light optical microscopy was Amarone), in the Verona area grapes are placed in breached, and a dramatic enhancement of the ventilated rooms, for a period of about three level of detail in cell morphology has been months after harvest. During this time berries achieved, as well as the possibility to reveal the undergo several physical and biochemical cellular ultrastructure. changes in part due to a slow but constant water Since the 1950s, EM technologies have been loss. We performed analyses using several differ- used in microbiological research and remain ent imaging and microscopy techniques to explore essential tools for the users to explore the micro- their potential application in revealing structural bial world and provide novel insights into a wide and composition features of grape berries through variety of aspects of modern microbiology. development and during the postharvest dehydra- This presentation demonstrated some application phases. The preliminary results showed that tion of EM related to the microorganisms associat- NMR is a powerful non-destructive technique to ed with foods and beverages, with special atten- study the structural changes occurring in berry tis- tion to the pro-technological and the probiotic sues along development. On the other hand, SEM organisms. microscopy was particularly helpful in revealing Selected scanning EM (SEM) micrographs show- differences in the outermost cell layers of berry ing pure cultures of single bacteria, yeasts and skin in different grapevine varieties. Finally, using moulds were presented to visualize the different optical imaging spectroscopy we were able to shapes, sizes and arrangements of cells. This basic select combination of excitation/emission wave- information on morphological features is fundamen- lengths able to discriminate whole grape berries tal in the taxonomic characterization of new cultur- (i) at different dehydration levels, (ii) subjected to able food-borne isolates. In addition, the description different dehydration condition and (iii) of differ- of novel genera and species should include morphol- ent varieties. Overall these preliminary data indi- ogy to ensure a rich polyphasic characterization. As cate that the microscopy and imaging approaches an example, in the proposal of Zygosaccharomyces used in this study represent promising tools for gambellarensis, a novel yeast isolated from an the detailed description of the changes that char- Italian “passito” style wine, we have reported a acterize grape berries during the on-vine growth detailed description of the cell shape and morpholo- and ripening stages and the off-vine dehydration gy as observed in photomicrographs. process. SEM also plays a paramount role for assessing

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the three-dimensional structure of microbial pop- are important for a proper drying. Development of ulations present in complex food samples, and can Botrytis cinerea in its noble form in grapevine unveil food-microbe and microbe-microbe interac- berries during withering can be followed by EM tions thought to be crucial for obtaining the examination and linked to peculiar sensory traits desired product quality. Examples of such obser- of the obtained wine. vations were reported: SEM images of yogurt and Finally, in this post-genomic era, the identification cheese revealed the presence of micro-colonies of of novel genome features coding for specific morpho- bacilli and cocci in micro-holes of the matrices. A logical characters, such as presence of pilus gene carpet of yeasts and bacteria colonized the sur- clusters in a probiotic bacterium, needs validation face of Kefir grains, and fibrillar material was also through appropriate EM observations. Thus the close observed among cells. During the first months of link between microbiology and microscopy is contin- ripening, the San Daniele ham was covered by an uing to increase our understanding of the beneficial agglomerate of moulds, yeasts and bacteria, that effects of microbes in food.

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